CA2082804A1 - Methods and compositions for treatment of angiogenic diseases - Google Patents

Abstract

The subject invention pertains to novel compositions and methods for the treatment of tumors and angiogenic diseases.
Specifically, these novel compositions and methods comprise combination therapies where FR4, or biologically-active mutants or fragments of PF4, are combined with various angiostatic, antitumor, or immunomodulatory compounds to provide enhanced levels of therapy.

Description

WO 92/02240 PCr/US91~52 ' , '' ' ', DESCE~IPrION

NOVEL METHODS AND COMPOSITIONS
FOR TREAT~NT OF ANGIOGENIC DISEASES
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oss-Reference to a Related Application This is a continuation~ -part of co-pending application Serial No. 451,021, filed December 27, 1989; which is a continuation~ part of co-pending application Serial No. 295,955, filed January 10, 1989.
Background of the Invention Angiogenesis, the developmen~ of new capillary blood vessels9 is an important process in the developing fetus and growing human. However, in healthy adults, angiogenesis occurs significantly only dunng wound healing and in the menstrual cycle.
It is now wideh~r reco~ed ~hat much of ~he angioger~ic ac~vity occurnng in adults is pathological in nature. For example, proliferation of vascular endothelial cells and formation of new capillaries is essential for grou~h of wlid tumors beyond a few cubic rnillimeters in volume (Fol~an et al. ~1983~ ~a Found. Symp. 100:132-149). We now understand that developing tumors se~e~e growth factors whieh stimulate neighboring endothelial cells to divide ~d migrate toward the tumor.
In addition to growth of solid tumors, other conditions involving angiogenic dysfunctions include diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas"mmune and non-immune inilammation (including rheumatoid arthritis), capillary proli~eration within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma have also recently been recognized as diseases possessing characteristics of dysregulated eDdothelial cell division and capillaIy growth. These conditions along with growth of solid tumors are collectively referred to as "angiogenic diseases" (FolkmaIl, J., and M.
Klagsbrun [19871 Science 235:442 447).

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WO 92/0224~ PCI'/US91/05246 2~ $~ 2 ~' In addition to angiogenic diseases, there are other conditions where endothelial cell proliferation is pathological or, at least, unwanted. For example9 endometriosis is characterized by the abnormal proliferation and positioning of certain endothelial cells which nonnally line the inner wall of the uterus. Control of the angiogenic process could help to prevent or alleviate endometriosis. Also, prevention of endotheIial cell growth in the uterus could be a means of birth control.
Endothelial cell growth is associated with wound healing. This grovvth is undesirable duling extended surgical proceedings and where excessive scar formation may occur. Therefore, a means of controlling endothelial cell proliferation would help prevent or reduce unwanted scar formation.
The mechanism of angiogenesis and endothelial cell proliferation has not been completely characterized. It has been established that mast cells accumulate at a tumor site before new capillary growth occurs; however, mast cells alone cannot initiate angiogenesis. Heparin, a mast cell product, has beenshown to significantly stimulate the capillaIy endothelial cell migration which is necessary for angiogenesis (Folhnan, J. [1984] Angiogenesis: Initiation and Modulation. In ncer Invasion and Metastasis: Biolo~c and Therapeutic Aspects. G.L. Nicolson and L ~las, eds. Raven Press, New York, pp. 201-208).
Several substances are known to have the capabili~ of inhl~iting endothelial cell ~owth in vitro. One of the most extensively studied inhibitors of endotheli~l cell growth is protarnine, which is a protein fouIld only in sperm.
Protamine has been shown to inhibit tumor angiogenesis and subsequent tumor growth (Taylor, S. and J. Folkman [1982] Nature ~97:307-312). Protamine's anti-angiogenesis activity has been attributed to its well-known capacity to bind heparin (Taylor and Folkman [198~], supra). Clinical experiments with protamine have not been pursued because of the toxicity associated with protarnine injection. Protamine, which is usually isolated from salmon sperm, isknown to be antigenic in humans, and anaphylac~c reactions ~o this protein have been observed with secondary exposures.

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At least two other compounds have been studied in regard to their heparin-binding activity: platelet factor 4 (PF4) and major basic protein. Majorbasic protein has demonstrated heparin-binding activity but is of little practical utility because of its high to~i~ty.
S Platelet factor 4 is a well-known protein which has been completely sequenced (Deuel, T.F., lR.M. Senior, D. Chang, G.L ~ R.L. Heinrikson, and E.T. Kaiser [1981] Proe. Natl. Acad. Sci. USA 78:4585~587). It is a 7~
residue secretable platelet protein with a molecular weight of approximately 7.8Kd which is released duling platelet aggregatiorL Although there is evidence of hepalin binding ac~ y and some indications of anti-an~ogenesis activi~
(Folkman [1984], supra), PF4 has never been shown to have clinical u~lity.
A compound which has been described as "oncostatin A," and which appears to be the same, or similar to, na~ive PF4, has been implicated as effecting the growth of tumors ~U.S. Patent Nos. 4,645,828 and 4,737,580; both issued to Twardzik et al.). However, the effects reported in these patents pertaln to slowly ~rowing human cancer ~ells in immunodeficient mice. The results of these e~periments cannot be reliably extrapolated to predict the effect of rapidly growing tumors which are na~ve to the host animal. F~hermore, the expenments reported in these patents in no way predict or disclsse any angiostatic properties.
~Various peptides from PF4 have been purified and their properties studied. None has been shown to have any role in the inh~ition of angiogenesis.
It is known that the ~13 peptide of PF4 is chemotactic for neutrophils and monocytes (Osterman, D.G., G.L. Griffin, RM. Senior, E.T. Kaiser, and T.H.
Deuel [198y Biochem. and Biophys. Res. Comm. 107(1):13~135). It is significant to note that the infil~ation of monocytes ~vould be expected to stimula~e the proliferation and migration of local endothelial cells by the secretion of angiogenic factors. Thus, peptides of PF~ could be expected to sdmulate, rather than inl~l~it, angiogenesis.
In addition to angiostatic properties, PF4 possesses characteristic structural features of the pro-inflammatory proteins interleukin-8 and ,B-thromboglobulin and has been shown to be chemotactic for neutrophils and SUE~S~IT~E SHE~ET

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. monocytes ~n vivo (Wolpe and Cerarni [1989] the FASEB Joumal, 3:2565-2573).
This similarity of the structure and activities of P~4 to well characte~ized pro-inflammatory proteins along with the ubiquitous aggregation of platelets at sites of in1arnmation suggest that PF4 may be an endogenous mediator of S inJ lammation. l hus, it is anticipated that swelling could accompany the adl~nistration of PF4 in vivo.
There is a significant and very long-standing need to locate an effective and non-toxic inhl~itor of angiogenesis and endothelial cell proliferation.
Angiogenesis plays a major role in the initiation and progression of widespread catastrophic illnesses, including cancer. An efEective, non toxic agent which can be administered- locally and/or systernically to treat these illnesses would be highly advantageous and has long eluded identification.
The following table may be helpful in identif~ing the amiIIo acids of the subject invention:
Three-letter One-letter nino acid symbol s~rmbol Alanine Ala A
Arginine Arg R
Asparagine Asn Aspartic acid Asp D
Asn and/orAsp Asx B
Cysteine ~ys C
Glutamine Gln Q
Glutamic acid Glu E
2~ Gln and/or Glu Glx Z
Glycine Gly G
Histidine His H
Isoleucine Ile Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F

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Proline Pro P
Serine Ser S
Threonine Thr T
~yptophan Trp W
Tyrosine l~r Valine Val V

Brief Surnmarv of the Invention This invention concerns the discoveIy that recombinant PF4 ~rPF4) has clinical u~ in the treatment of diseases ~ivhich involve angiogenesis and endothelial cell proliferation. Furthelmore, PF4 fragments are demonstra~ed to be inhl~itors of angiogenesis. The ability to in~u~it angiogenesis has been found in synthetic peptides corresponding to sequences in PF4 as small as the carbo~yterminal 13 arnino acids.
A further aspect of the invention is the identifica~on of PF4 analogs (mutants) and fragments which may possess enhanced capab~i~des to inhfbi~
angiogenesis and endothelial cell proliferation.
A filrther aspect of the invention is the trea~nent of angioger~ic diseases with a combination of PF4 and an anti inilammatory agent. Anti-ini la~natory agents help to alleviate unwanted swelling, pain, or tissue damage which could accompany the administration of pro-inilammatory compounds.

Bnef Description of the Drawings gure 1 shows DNA and arnino acid sequence of native rPF4.
re 2 shows the inhibition of angiogenesis resulting ~om the treatment of rPF4 and various related peptides.
Figure 3 depicts the inhibition oE endothelial cell proliferation by rPF4.
Figurs 4 depicts the alpha-helical configurations of rPF4 and rPF4-241.
Figure S compares the inhibition of angiogenesis resulting ~om treatment with rPF4 and rPF4-241.
Figure 6 compares the inhibition of human umbiLical vein endothelial cell proliferation resulting from treatment with rPF4 or rPF~241.

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Fig~re 7 shows the ability of rPF4 and rPF~241 to inhl~it turnor growth.
Figure 8 shows ~ootpad swelling in mice as a function of time after injection with either rPF4, rPF4 and indomethacin, or a buffer solution.
Figure 9 shows quantification of ir~lammatoIy cell infiltrate after treatrnent with rPF4 or rPF4 with indomethacin.
Figure 10 shows turnor growth after administration of rPF4 alone, indomethacin alone, buffer alone, or rPF4 and indomethacin.

Detailed Description of the Invention The subject invention pertains to i-n vivo inhl~ition of angiogenesis by rPF4 and certain analogs and peptide fra~nents of PF4. rnese analogs and peptide fragments of PF4 can be used to treat angiogenic diseases. As used in this application, the term "angiogenic disease" refers to growth of solid tumors, and other conditions involving angiogemc dysfunctions including diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psonasis, angiofibromas, imrnune and non-immune inilamma~on (including rheumatoid arthritis), cap~lary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma. The subject invention also concerns the use of rPF4 and PF4 fragments for treatment of diseases of dysregulated endothelial cell proliferation.
The subject invention arises ~om the u~expected discovery that rPF4 inhibits in vivo capillaIy formation and embryonic neovascularization. It was also discovered that full length recombinant PF4 inhl~its growth factor-dependent human endothelial cell proliferation in vitro.
Significantly, it was also determined that the angiogenesis-inl~ibiting activity of PF4 was retained by synthetic peptides corresponding to sequences ofPF4 as small as 13 amino acids in length. In particular, it was found that a synthetic peptide of 13 arnino acids corresponding to the carboxyl terrninal portion of PF4 (C-13) displayed potent angiostatic activity.
The finding that PF4 directly inhibits growth of pure cultures of endothelial cells indicates that, advantageously, its effects are not mediated by some other cell ~pe. The finding that PF4 and related peptides i~ubit SUBSTlTlJTE S~ T

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angiogenesis in vivo (CAM assay) and in vitro (endothelial cell proliferation assay) is particularly unexpected in view of PF4's chemotactic activity for monocytes.
The activity of the ~13 peptide is especially surprising in light of its S inability to affect the anticoagulant ac~vity of hepalin. The use of the ~13 peptide offers several advanta~es over whole rPF4 such as reduced dosage (weight basis), reduced likelihood of antigenicity, and grea~er likelihood of effectheness in novel dosage forms.
The ~13 peptide of PF4 also retains the ability to prevent Con-~
induced immunosuppression in mice9 an activity which is unaffected by heparin and probably independent of the abi]ity of the peptide to inhl~it angiogenesisO
It is well understood that angiogenesis is required for solid tumors to grow beyond a few cubic millimeters. Thus for the treatrnent of solid tumors, use of rPF4, or a fragment thereof, to cause hlmor rejection by inhibiting angiogenesispresents a noYel and highly advantageous means of therapy. The fact that the C-13 peptide inhibits angiogenesis without affecting the anticoagulant actiYity of heparin demonstrates that this small peptide would also have the benefit of not interfering with concurrent anticoagulant therapy. Additionally, small peptides are generally less antigenic than larger proteins, and, thus, the PF4 fragments can be used advantageously for oral and transdermal administration. These types of delivery are particularly useful in the treatment of gastrointestinal capillary proliferation (e.g., Kaposi's Sarcoma) and skin lesions, respectivelyOIntralesional, as well as systemic, administration of PF4 fragments are also appropriate for treatment of these conditions.
2S Analogs of PF4 were created which lack heparin binding activity but retain ability to inhibit angiogenesis. One such analog, known as rPF4-241, was created by cassette mutagenesis of a s~nthetic PF4 gene whereby the DNA
sequence encoding the four Iysine residues near the carboxy telminus of PF4 were converted to a sequence encoding two GlII-Glu couplets. If rPF4-241 is administered intralesionally, it can be applied such that the dosage isi betweenabout 1,~ esion and about 4 mg/lesion. For systemic administratio~ the dosage of rPF~241 can be between 0.5 mg/kg of body weight and about 100 .
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WO 92/02240 PCr/US91/05246 2~ ~ 8 ~' mg/kg of body weight. Similar and higher dosages can be used for the administration of native sequence rPF4 as well as peptide fragments. For example, dssages of rPF4 and ~agments thereof may be twice that of rPF4-241 or higher.
As discussed above, PF4 has been shown to be chemotactic for neutrophils and monocytes in vitro~ suggesting that it may mediate an inflammatory response. To assess whether these observations have in vivo relevance, the ability of PF4 to induce acute and chronic dermal ini lammation in the mouse was tested. When injected into the murine dermis, recombinant human PF4 (rPF4) induces acute inflammation within two hours, which peaks at about 12 to 18 hours and which resol~es by about 36 hours. lnjection of an equivalent amount of cytochrome c, buffer alone, or an amino terminal PF4 peptide failed to elicit a significant inflammatory response, however, the carbo~y terminal PF4 peptide was pro-inflammatory. The ini lamrnatory infiltrate inducedby both rPF4 and the 41 amino acid COOH terminal peptide was composed of neutrophils and to a lesser degree mononuclear cells. Although rela~vehJ high concentrations of rPF4 are required to elicit an in~lammatoIy response, these concen~ations may be locally obtainable during platele~ aggregation or at sites of administration of rPF4 or related compounds.
Advantageously, it was found that the rPF4 pro-inflammatory e~ect was significantly suppressed by systemic administration of aII anti-inflammatory agent without reducing the angiostatic activit~.

Materials and Methods Chicken Chorioallantoic Membrane (CAM~ Assay. Fertile eggs were incubated in a stationary position for 3 days at 37C and 7~80~o relative humidity. During this time, the embryo rose to the upper surface of the egg contents. At the beginning of the 4th day, the eggs were cracked without inversion and carefully deposited into sterile plastic petri dishes such that the embryo remained on the upper surface. The shell-free eggs were incubated for an additional 72 hours at 37C, under an atmosphere containing 2.5~3.5% CO2 after which the growing embryos developed a recognizable CAM. Discs, made Sl IBSTITUTE SHEET

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by mixing test samples with 1% (w/v~ methylcellulose, weré dried and placed on the CAM between major veins aIld approximately 0.5 cm f~om the embryo.
Following another 48 hour incubation at 37C (2.5-3.5% CO2), the samples were scored for their ability to inhibit angiogenesis. Inhibition appears as an avascul~
S zone surrounding the implant and can often include elbows formed by ve~n$avoiding the disc and a reduced number of capillaries in the re~ion of the implant.
Endothelial Cell Proliferation Assav. Human umbilical vein endothelfal cells (HUVEC~ were cultured in Medium 1~9 (Gl~bco) containing 10% (vh) fet~
bovine serum (FBS), 150 mcg/rnl endothelial cell grow~h supplement (ECGS) and S units/ml heparin at 37C and ~5% CO2. Every 3~ days, the cultures were harvested by tlypsin treatment, diluted, replated, and grown to coni luence~ ~orto the start of an expenment, the cells were centrifuged and resuspended in heparin-free media and incubated with the test substance (PF4) for 3 days under standard culture conditions. At the end of the incubation period, the cells wereremoved by tlypsin treatrnent and counted with a Par~icle Data Elzone 180 Cell Counter. Statistical significance between means was determined by a standard Student t-test for unpaired data.
In~ubition of DNA synthesis was measured by plating the cells ~
described, then incubating with the test substance for 24 hours. 3H-Thyrnidine (1 ,c~Ci/well) was added ~or an additional 6 hours and the plates were frozen ae--70C. Following 2 freeze/thaw cycles, the cells were aspirated onto a fiber filter, washed with distilled water, fixed with MeOH, and counted for incorporation of radioactivity into DNA.
~n vivo Tumor Growth Assav. Normal C57BL~6J female mice (~8 weeks old) were inoculated subl~utaneously with S x 105 log phase cells of a B1~F10 melanoma tumor line~ This protocol led to progressive tumor growth resulting in large (300 rnm3) necrotic tumors after approximately 10 days, followed by death of untreated animals usually within three weeks of tumor inoculation.
To test the efficacy of rPF4 in preven~ng in vivo tumor growth and angiogenesis, tumor bearing animals were injected daily, directly into the nascent tumor, with either rPF4 or with buf~er lacking rPF4, beginning one day after SUBSTITIJ~E SHEET

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tumor inoculation. Tumor volume was measured at regular intervals with digital calipers by laboratory personnel un~nformed of the specific treatment received by each subject animal.
Footpad Assay. 0~05 ml of PBS containing a test substance was injected S intradermally mto the right hind footpad of each mouse. An identical amount of diluent, not containing the test substance, was injected in~o the left hind footpad. At various time points, footpad thicknesses were measured with a spring loaded engineer's micrometer (Fowler Ca., Biggswald, England).
At various time points, mice were sacrificed and footpad tissue was prepared for light microscopy. This tissue was used to quantify infiltrating cell types. Biopsy specimens were hxed in 10% buffered formalin for at least 48 hours and then prepared using standard techniques of paraffln embedding and sta~ning with hematoxylin and eosin. Using an ocular grid, four cellular areas of dermis in each specimen were examined in a coded fashion at lOOOX
rnagnification and inflammatory cells were quantified. Differences between groups were assessed by Student's t test or analysis of variance, where appropriate.
rPF4 Production. Recombinant PF4 was produced in E. coli as an N-terminal fusian protein containing a unique rnethionine residue immediately preceding the PF4 portion. More specifically, expression plasmid pPF4-211 was constructed by cloning a synthetic gene encoding native sequence PF4 (Figure 1) (Poncz et al. [19871 Blood 69:219) into the multiple res~iction site region of plasmid pREi~2.2 (deposited July 30, 1986; accession ~ NRRL B-18091).
Codon usage in the synthetic gene was optimized for expression in E. ~, and synthetic DNA linkers were included on each end to facilitate the directional insertion of the PF4 gene into the vector. The restriction sites Hindm and SmaI
were chosen for insertion into pREV2.2. The resulting construct, pPF4-211, expressed a fusion protein containing 34 amino acids of E. coli ~-glucuronidase (BG) separated from the PF4 sequence by a unique methionine residue.
Cells expressing the fusion protein were subjected to Iyso~yme (1 mglg cells), DNase I (500 units/100 g celLs) and bead mill treatments. The Iysis pellet containing the fusion protein was treated with CNBr (10 g/lOOg cells) in 70%

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formic acid to cleave the fusion protein at the me~hionine between the BG and PF4 portions. Following evaporation of the CNBr/formic acid, the recombinant protein was e~ckacted with 200 ml of 50 mM Tris-CI, pH 7.6, 5 mM ~DTA, and 10 rnM Dl'r per 100 g of cell starting material. Native sequence rPF~211 was S purified by binding the protein to heparin agarose, remo~ing contamina~ng proteins with 0.6 M NaCI, and eluting with 1.2 M NaCI. The resulting matenal was dialyzed into 20 mM sodium acetate, pH 4.0, and analyzed on a 15% SDS-PA gel stained wi~h Coomassie Brilliant Blue. Minor contaminants could be removed using C4 reverse phase high pressure liquid cbromatography (HPLC~
to prepare the protein for m YiVo use.
Production of rPF~241 and other PF4 analogs. A synthetic gene encoding the mutant designated rPF~241 was constructed by changing the codons for the four lysine residues near the C-termimls of PF4 ~o sequences encoding two C;ln-Glu couplets (CAA GAA) by ~assette mutagenesis between the BbeI and SmaI sites. Linkers were included at the ends of the synthetic gene, and the gene was inserted into pREVæ2 as descn~ed above. Genes encoding other PF4 mutants or analogs were prepared in a similar manner.
The mlltant proteins (e.g., rPF~241) were cleaved and ex~racted as descnbed above. The extracts were then purified using DEAE~epharose chromatography, and ehlted with a gradient of ~1 M NaCI. The PF4 proteins generally eluted at approDmately 0.5 M NaCI and were dia}yzed into 20 mM
phosphate buffer, pH 7.5. The samples were further purified by reverse phase HPLC.
PF4 peptides. Peptides were prepared by standard solid phase synthesis procedures, cleaved fiom the solid support and deblocked, and puuified by reverse phase HPLC.
Reagents. Recombinant human IL-1 (rrL-1) was purchased from Ger~ne Corporation (Cambridge, ~iA). Cytochrome c and E. coli endotoxin were purchased from Sigma Chemical Co. (St. Louis, MO). Slow release indomethacin pellets were purchased from Innovative Research (l~ledo, OH).
Mice. C57BV6J, A/J and C3~UHeJ female rnice, ~8 weeks oldg were purchased from the Jackson Laboratory (Bar Harbor, ME).

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Following are examples which illustrate procedures, incl-ïding the best mode, for practicing ~e invention. These examples should no~ be construed as limiting. All percentages are by weight and all solvent m~ure proportions are by volume unless otherwise noted.
Exarnple 1 Chicken eggs, prepared as descnbed above, were treated with discs containing several concentrations of recombinant PF4 or peptides derived from the sequence of PF4. rPF4 and ~terminal peptides as small as 13 amino acids inhibited angiogenesis on the CAM (Figure 2). In each case, '~he inhlbition was dose-dependent and the response appro~nately equivalent (molar basis) for the inhibitors containing the C-terminal region of PF4. An N-term~nal peptide of PF4 (N-29) did not inhl~it angiogenesis even at the highest concentration tested9 suggesting that all of the anti-angiogenic activity of PF4 is probably associated with the C-terminal portion of the molecule. Since the ~terminus of PF4 is rich in Iysine, polylysine was tested in this assay system and foulld not to cause inhibition at 6.5 nmol dosages.

Example 2 The lysine rich region of PF4 (residues 61-66) is also the domain associated with the binding of heparin by PF4. Heparin is known to play a role in modulating angiogenesis, which can also be affected by protamine, another well characterized heparin-binding protein. To assess the ability of PF~based synthetic pep~des to bind heparin, we assayed the activity of coagulation-cascade enzymes which are inhibited by heparin. The Factor Xa assay used here has previously been described in Denton et al. (1983) Biochem. J. ~09:455-460.
Protamine and platelet factor 4 are able to prevent the heparin inhibition of thrombm and Factor Xa at appro~mately equimolar concentrations. The 41 amino acid C~tenninal peptide of PF4 (~41) prevented hepann inhibition less effectively, but the C-13 peptide was unable to prevent the inhibition of thrombin even at concentrations ten times that of an effec~e level of rPF4.

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This unexpected finding suggests that the C-13 peptide inhl~its angiogenesis by some method other than heparin binding.

Example 3 Many angiostatic agents act by direct in~u~ition of endothelial cell proliferation. Endothelial cell division and growth is tightly controlled and strictly dependent on the presence of growth factors. We evaluated the abil;ty of rPF4 haYing the wild 1ype sequence (rPF~211) and related peptides to inhibi~
growth factor-stimulated human endothelial cell proliferation in vitro. As shownin Figure 3, rPF4 significantly inhl~ited endothelial ~ell growth in a dose~
dependent ~ashion at a concent~ation as low as 1.3 ~M. Inl~ibition was comple~e at 3.~ f~M in the heparin-deficient medium employed here.

Example 4 To assess the importance of the hepa~in binding activity of PF4 in 'Lhe inhibition of endothe]ial cell proliferation, cells were incubated in media containing or lacking S uI~its/ml heparin. The presence of hepa~in s~mulated proliferation of these cells during the three day incubation of this experimentDrPF4 significantly ir~bited both control (100%) and heparin st~mulated (45%) endothelial cell growth (Table 1).

Table 1. Attenuation of rPF4 inhl~itiorl of endo~elial cell growth by heparinO
rPF4 %
25 Addition -- 50mcg/ml Inlu~ition8 --- 14.4 1 2.5 b6.0 i 0.6 ~ 100 S u/ml hepann 18.9 + 1.2 bl4.0 + 0.4 45 8Based on seeding of 8 x 104 ceDshvell bSignificantly different from appropriate control (p< 0.005) SUBSTITUTE SHEET

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wo 92/02240 ~ Pcr/US9l/OS246 z~ 14 Example S - Construction of rPF~241 ,r' ' A niutant of PF4 was created by converting the four Iysine residues at the -carboxy termmus of PF4 to two Gln-Glu couplets as disclosed above. This protein apparently retains the alpha-helical secondary skucture (Figure 4) for S this region of the molecule with the cnncurrent loss of heparin bindirlg activity.
The protein was reactive with polyclonal antibodies to native PF4 a~d was determined to possess the appropnate modifications by amino acid analysis.
Significantly, the purified mutant protein lacked heparin-binding activity in the Factor Xa inhl~ition assay.
The substitutions described here can be made with the peptide fragments as well as with the full length PF4 molecule. For example, ~13-241 has the ~ollowing sequence:
Pro-Leu-Tyr-Gln-C31u-Ile-Ile-Gln-Glu-Leu-Leu-Glu-Ser E~ample 6 - lnhl~ition of Angiogenesis bY rPF~241 Purified rPF~241 was tested for its ability to inhibit capillary growth in the chicken chorioallantoic membrane (CAM) assay. Even at the lowest concentrations tested (1.25 nmoVdisc) rPF~241 extensively inhllbited angiogenesis in the CAM system (Figure 5). This in~u~ition was even more effective than that caused by equal concentrations of native rPF4 as suggested by larger avascular zones on the membrane. The in~itoIy effect of rPF~241 was not reversed by hepann.

Example 7 - Inhibition of Human Endothelial Cell Proliferation bv rPF~241 In a test of inhibition of human umbilical vein endothelial cell proliferation by native rPF4 and mutant rPF4-241, both were shown to be effective at inhibiting the proliferation of these cells. The results of this test are shown in Figure 6.
These results are remarkable in that previous theories of PF4 in~ubition of angiogenesis assumed that the PF4 effects were due to heparin binding. We have designed a protein, retaining most of the structural features of native PF4but lacking detectable heparin binding activi~, which m~y be more active than SUBSTI~lJTE SHEET

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native PF4 in inlubiting angiogenesis in vivo and endothelial cell proliferation in vitro. Additionally, the mutant we have designed would not be expected to interfere with heparin anticoagulant therapy.

S Example 8 ~ ~ition of In Vivo Tumor Growth The efflcacy of rPF~211 or rPF~241 in preventing tumor growth and angiogenesis was tested. The inhibition of in vivo tumor growth was assayed after injection of either rPF~211 (in 20 rnM NaOAc, pH 4.0) or rPF~241 (in 50 mM sodium phosphate, pH 6.5, 50 rnM NaCI) directly into the nascent tumorD
as described in the materials and methods section above. ~lthin seven days of tumor inoculation, ar~imals injected with buffer possessed obvious three dimensional tumors, while rPF~211-treated animals were essentially tumor-free (Figure 7). Continued treatment with rPF4 completely suppressed tumor growth under these conditions where control animal tumors became necrotic and large as seen previously with untreated mice. The sarne efEect was observed when rPF4-241 was used as the inhibitory agent.
This finding supports the proposition that rPF4, as an inhl~itor of angiogenesis, will possess clinical usefulness in the management of malignant melanoma and other cancers. Progressive growth of tumors requires new blood vessel formation which, if inhibited, may not only restrict tumor growth, but stimulate regression of existing vessels, as well as enhance other responses to malignant invasion~
The finding that rPF4 inhibition of in vivo tumor growth was apparent within three days of the initial inoculation (of rPF4) indicates that rPF4 acts to ~5 modulate tumor growth by local mechanisms rather than by immunomodulationwhich would require a longer time course. Additionally, rPF4 did not direc~ly inl~ibit tumor cell growth in vitro. It appears9 therefore, that rPF4 modulated the host's angiogenic response to the growing tumor.

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WO 92io2~ ` PC~/US91/05246 Z~>8~ 16 sigr~ificantly alter the protein secondary structure (Kaiser, E.T., and FJ. Kezdy [1984] Science 223:249-255). The subject invention includes other mutants or fragments of the PF4 sequences depicted herein which lack affinity for heparin and exhibit substantially the same or higher angiostatic activity. A preferred S region for modification is the Iysine rich region near the carboxy terminus corresponding to the heparin binding domain (residues 60-70). As a general rule, amino acids 60 through 70 cannot be eliminated. Also, as a general rule, it is necessaIy to have at least one charged residue behveeII positions 60 and 70.
Maintenance of an amphipathic a-hel~ in this region does not seem to be necessa~y, however, an amphipathic structure may be preferable. Thus, the subject invention includes mutants of the arnino acid sequences depic~ed herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained. In particular it should be understood that conservative substitutions of amino acids may be made. For example, arniIlo acids may be placed in the following classes: basic, hydrophobic, acidic, polar,aIId amide. Substitutions whereby an amino acid of one class is replaced with another amillo acid of the same type fall within the scope of the subject invention so long as the substitution does not materialb alter the biological activity of the compound. Table 2 provides a listing of examples of amino acids belonging to each class.

Table 2.
Class of Amino Acid Example of Amino Acids Basic K, R, H
Hydrophobic A, L, I, V, P, F, W, Y, M
Acidic E, D
Polar S, T, N, Q, C
Amide Q, N

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WO 92/02240 PCr/US91/0524~
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! 17 In some instances, non-conservative substitu~ions can also be made. For example, a lysine residue near the C-terminus of PF4 may be replaced with any of the following amino acids: E, Q, D, N, M, A, L, and I. The clitical factor istha~ these substitutions must not significantly detract ~om the biological activi1 S of the rPF4 or the rPF4 fragment.
~ e have conducted experiments whereby amino add substitutiorls have been made, and the reulting rPF4 mutants have been tested for biolo~cal activity. Various mutants which have been constructed are shown in l~ble 3.

Table 3.
Designation Sequence rPF4-211 [PF4AA1-571-PLYKKIIKKLLES
rPF4 231 [PF4 AA 1-571 - P L Y
rPF4 241 [PF4 AA 1-571 - P L ~Y Q E I I Q E L L E S
rPF4302 [PF4AA1571--PLYQQIIQQLLES
rPF4303 [PF4AA1-571-PLYKKQEKKQEES
rPF4307 1PF4AA1-571-PLYQIEIQLELES
rPF4308 [PF4AA1-571-PLYNDIINDLLES
rPF4315 Lrl?F4AA1-571--PLYGEIIGELLES

Results from experiments testing the biological activity of these peptides are shown in Table 4.
.

2S ble 4.
-CAM ~IUVEC
rPF~211 + +
rPF~231 +/-rPF4-241 + + +
rPF4-302 +/-rPF4-303 + NA

rPF4-307 + + + +
rPF4-308 + NA
3S rPF~315 + NA

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WO 92/022~0 ~ PCI/llS91/05246 .~ . . ..
, ~ ~ 18 The results shown in Table 4 clearly demonstrate that it is possible to make rPF4 mutant which retain the biolog~cal activity of rPF4 with respect to inhibition of cell growth in the CAM assay and the HllVEC assay. Two of these peptides (rPF4~241 and rPF~307) e~bited enhanced activity in these assays.
S The mutants described here are amino acid sequences which are largely homologous with wild ~pe rPF4 (rPF~211), but which have certain amino acid substitutions. These substitutions were made between amino acids 60 and 70.
Although most of the resulting compounds still e~ubit biological activi~
in the C~M and HUVEC assays, they do not bind hepalin. rPF~3029 which does not e~ibit significant activity in either ~he CAM or the HUVEC assay, has no charged amino aeid residues between residues 60 and 70. rPF4-231, which also does not exhibit sigr~ficant biological activity, terminates at amino acid number 60. If a person sl~lled in the art wished to investigate the biological activity of other rPF4 mutants, it would now be a straightforward procedure to make the desired mutations and test the resulting peptides for activity. Using the teachings of this document, the researcher could prepare and readib test peptides which could be expected to haYe the desi~ed properties. For example, the amino acid substitutions just described for the full length rPF4 molecule can also be made with the C-13 and C 41 fragments which are described above.
Example 10 - Inflammatorv Properties of rPF4 and Related Compounds The inilammatory properties of rPF4 and related compounds were assessed using the footpad assay as descrl~ed above. At 8 hours, local injectionof 25 f~g of rPF4-211 into the murLlle derrnis resulted in a brisk inilammatory response as measured by footpad swelling (Figure 8) and quantification of inflammatoly cell inEiltrate (Figure 9). At higher doses the tissue edema does not increase further ~nd rnay even drop off slightly. It has been found that relatively high local concentrations of PF4 are required to exert a pro-in~lammato~y efEect. Although a brisk ini lammatory response occurs ~nth 25 f~g of PF4 injected into the muline derrnis, at 0.25 ,ug, the inflamma~ory response is minimal. The time course of rPF4 induced acute inflammation is broad and resolves by about 36 hours (Figure 8).

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WO 92/02240 PCI'/US91/05246 The time course of rPF4 induced intlammation shows a rapid increase from baseline and peaks at between 6 and 12 hours and almost completely reso~ves by 36 hours.

Example 11 - EfEects of Ant-Inflammatory Agent wi~h rPF4 For each mouse, 0.05 mg, slow release indomethacin pellets (Innovative Research, Toledo9 OH) were implan~ed subcutaneously under light ether anesthesia 48 hours prior to an expeliment. These pellets continuously release their contents over 14 days.
Systemic treatment of animals with indolmethacin significantly blunts the rPF4 pro-inflammatory response (Fi~ure 8). The area under the culve of footpad swelling in the rPF4 plus indomethacin treated mice is 45.7% of the areaunder the cune of the rPF4 alone treated mice. The inflammatory cell in~ïltrate is also partially abrogated with indomethacin treatment. The results of these experiments are summarized in Table 5.

Table 5.
Pro-inflamma~o~y response Treatment ~g Infiltrating rPF4 ~ + + -~
C 41 +~ ~+

rPF4-241 ~ ~
rPF4/indomethacin +/-- ~/--Thus, indomethacin can be used to decrease the swelling which could accompany the administration of PF4 or PF4-related substances. Other non-steroidal anti-inflammatory agents could also be used. The anti-inflammatory agents useful in the combinations and methods of this invention include steroidal and non-steroidal anti-inflamrnatory agents. The non-steroidal anti-inflammatoryagents include, but are not limited to, acetyl salicylic acid ~aspirin), methyl salicylate, sodium salicylate, phenylbutazone, oq~yphenbutazone, apazone, indomethacin, sulindac, tolmetin, mefenamic acid, ibuprofen, naproxen, Sl )BS~ITl~TE SHEET

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wo 92/02240 PCr/US91tO5246 2~i8~:` 20 fenoprofen, flurbiprofen, ketoprofen, and other compourlds. Other anti-inflarnmatory agents useful in the combinations and methods of this invention are lipocortins derived from natural sources or lipocortins and lipocortin-like polypeptides produced by recombinant techniques (see UrLited States patent S applications SeAal Nos. 690,146; 712,376; 765,877 and 772,892; Wallner, B. et al.
[1986] Nature 320:77-81) and uromodulin (Muchmore, A.V., and J.M. Decker [1985] Science 229:479-481)? or cyclosporin and its deAvatives. Steroidal anti-inflammatory agent~ which could be used according to the subject invention include, but are not limited to, hydrocortisones. Suitable anti-inflammatory agents also inchlde inhl~itors of leukotriene or pros~glandin synthesis.

Example 12--Anti-Tumor Activitv of rPF4 Combined with Indomethacin Four groups of mice were used in this expenment. In two groups of rruce, slow release indomethacin pellets (50,ug) were implanted surgical}y underthe sl~n of the left flar~ The other two groups were not treated with indomethacin. l~mors were implanted subcutaneously in all four groups in the right flank As shown in Figure 10, the addition of indomethacin to PF4 did not compromise the antitumor actMty of PF4. Implanted tumors grew rapidly after day 6 when the tumor was treated ~nth either buffer alone or indomethacin alone. By contrast, the tumors grew veIy little, if at all, when treated with PF4 or a combination of PF4 and indomethacin.
From these results it is apparent that PF4 retains its antitumor activity even when combined with the anti-ini lammatory agent indomethacin.
Example 13--Administration of PF4 and Anti-Inilammator~ Agents The combinations and methods of the present invention may allow the administration of PF4, or related compounds, in higher doses in some cases than those tolerated in conventional treatment regimes based upon PF4 alone.
Accordingly, the combinations and methods of this invention advantageously reduce or eliminate the inflammatory effects of high dose treatments with PF4 alone. Thus, the use of PF4 in combiDation with an anti-inflammatory agent may SUBSTIT~E SHEET

Wo 92/02240 PCI/US91105246 21 Z~
reduce the duration of treatment which would be required by therapies based upon conventionally tolerated lower dosages of PF4 alone.
The combinations and methods of this invention are useful in treating any mammal, including humans. According to this inventionj mammals are treated S with pharmaceutically effective amounts of the two active components--PF4 and an anti-inflarnmatory agent--of the combinations of this invention for a period of ~dme sufticient to inhibit angiogenesis or endothelial ceM proliferation.
In accordance with this invention, pharmaceutically effective amounts of an anti-in~ammatory agent and the PF4 (or PF~related compounds) are administered sequentially or concurrently to the patient. The most effective mode of administration and dosage regimen of P~4 and anti-inflammatoTy agent will depend upon the type of disease to be treated, the se~erity and course of that disease, previous therapy, the patient's health status, and response to PF4and the judgment of the treating physician. PF4 may be administered to the patient at one time or over a series of treatments.
Preferably, the anti-inflammatory agent and the PF4 are administered sequentially to the patient, with the anti-inflammatory agent being administeredbefore, after, or both before and after treatment with PF4. Sequential administration involYes treatment with the anti-inflammatoTy agent at least on the same day (within 24 hours) of treatment with PF4 and may inYolve continued treatment with the anti-inflammatoTy agent on days that the PF4 is not administered. Conventional modes of administration and standard dosage regimens of anti-inflammatory agents may be used (see Gilman, A~&. et al. [eds.]The Pharmacolo~a~Basis of Therapeutics, pp. 697-713, 1482, 1489-91 [1980];
Physicians Desk Reference 1986 Edition). For example, indomethacin may be administered orally at a dosage of about 2S 50 mg, three ~mes a day. Higher doses may also be used. Alterna~ -ly, aspirin (about 150~2000 mg/day), ibuprofen (about 1200-3200 mg/day) onventional therapeutic doses of other anti-in~ammatory agents may be used. Dosages of anti-ini lammatory agents may be titrated to the individual patient.
~ccording to one embodiment of this invention, the patient may receiYe concurrent treatments with the anti-inflammatory agent and PF4. Local, ~;~BsT~TuTE S~EET

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intralesional, or intravenous injection of PF4 is preferred (see Gilman et al., supra at pp. 129~91). The anti inilammato~y agent should preferablv be administered by subcutaneous injection, subcutaneous slow-release implant, or orally.
S Alternatively, the patient may receive a composition comprising a combination of PF4 (or PF~related compounds) and an anti-inilammatory agent according to conventional modes of administra~ion of agents which e~ubit anticancer, antitumor, or anti-inflarnmatory activity. These include, for example, parenteral, subcutaneous, intravenous, or intralesional routes of administration.
The compositiorls used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusfble solutions. The preferred form depends on the intended mode of adrninistration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable calTiers and adjuvants which are known to those of skill in the ar~ Preferably, the compositions of the inven~on are in the form of a umt dose and w~l usually be administered to the patient one or more times a day.
The compounds of the subject invention may also be admi~istered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of ~me.
PF4, or related compounds, may be administered to the patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection. An effective dose may be in the range of from about 0.01 to about 1.0 mg/kg body weight, it being recognized that lower and higher doses may also be useful. More particularly, doses of PF4 higher than those typically tolerated in patients treated with PF4 alone may advantageously be used in the methods and compositions of the invention. It should, of course, be understood that the compositions and me~hods of this invention may be used in combination with other therapies.

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Once improvement of ~he patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

Example 14 As descnbed above, PF4 and related compounds can be administered in conjunction with anti-inilarnmatory agents. Also, as disclosed above, PF4 and related compounds can be used in combination with other therapies. For exarnple, PF4 and related compounds can be used with angiostatic agents, antitumor agents, irmnunomodulators, inflammatory mediators, and hematopoietic factors.
Among the angiostatic agents which can be used m combination with PF4 are steroids, sulfated polysaccharides/cyclodextrin, retinoids, cyclosporins andother angiostatic agents derived from fungal extracts, t~rombospondin (or fragments thereof), ~, ~, or y-interferons, tumor necrosis factor~, hbroblast growth factor antagonists, angiogenin antagonists, and certain antibiotics with angiostatic properties. Among the arltibiotics with angiostatic properties that can be used according to the subject invention are fumagiDin and its analogs, and herbimycin.
The PF4 compounds of the subject invention may also be used in combination with biological, chemical, and radiation turnor therapies. Biological agents include such compounds as tumor necrosis factors, interferons, and turnorselective antibodies or immunotoxins. Chemotherapeutic agents which can be used in combination with PF4 include doxorubicin, methotrexate, cisplatin, vinblastine, vincristine, and bleomycin.
Combination therapies whereby PF4 or related compounds are used in conjunction with immunomodulators, inflammatory mediators, or hematopoietic factors can also be practiced according to the subject invention. These `:
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WO 92/02240 PCr/US91/05246 2~ 24 compositions may be, for example, interferons, interleukins (1 through 8), tumornecrosis factor ct or ~, transforming growth fac~or ,B, erythropoietin, colony stimulating factors (monocyte, granulocyte, and granulocyte-monocyte), and megakaryocyte stirnulating factor.
The adminis~ation of PF4 or related compounds in the course of these combination therapies may be accomplished as described above in E;~ample 13.
The timing of the administlation of the PF4 or rela~ed compouuld in relation to the administration of a combination therapy will depend upon the nature of the combination therapy and the goal of the treatment. Where one form of treatrnent serves to facilitate a second form of treatment, then the ~acilitating treatrnent will occur before7 or simul~eously with, the second treatment.
Where the two forms of treatment result in synergistic benefits, then a concurrent application of the treatments could be utilized.
The use of combination therapies can facilitate the administration of lower dosages of the individual therapeutic agents, thereby decreasing the possibility of toxiGi~ or other side e~ects. Also, the use of appropriate combinations allows the physician to treat multiple disease symptoms and/or causes, thereby enhancing the overall value of the therapy.

It should be understood that the examples and embodiments descrl~ed herein are for illustrative purposes only and that various rnodifications or changes in light thereof wiM be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

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Claims (24)

Claims

1. A pharmaceutical composition for the treatment of angiogenic diseases, said composition comprising a first compound which is rPF4, or an angiostatic mutant or fragment thereof, and one or more additional compounds which are angiostatic agents.

2. The pharmaceutical composition, according to claim 1, wherein said additional angiostatic compound is selected from the group consisting of steroids, sulfated polysaccharides, cyclodextrim, retinoids, cyclosporins, thrombospondin,laminin, interferons, tumor necrosis factor-.alpha., fibroblast growth factor antagonists, angiogenin antagonists, fumagillin, herbimycin, and analogs or fragments of the above-named compounds.

3. The composition, according to claim 1, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

4. A pharmaceutical composition for the treatment of angiogenic diseases, said composition comprising a first compound which is rPF4, or an angiostatic mutant or fragment thereof, and one or more additional compounds which have antitumor activity.

5. The pharmaceutical composition, according to claim 4, wherein said compound with antitumor activity is selected from the group consisting of tumor necrosis factors, interferons, tumor selective antibodies or immunotoxins, doxorubicin, methotrexate, cisplatin, vinblastine, vincristine, bleomycin, and injectable radioisotopes.

6. The composition, according to claim 4, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

7. A pharmaceutical composition for the treatment of angiogenic diseases, said composition comprising a first compound which is rPF4, or an angiostatic mutant or fragment thereof, and one or more additional compounds selected from the group consisting of immunomodulators, inflammatory mediators, and hematopoietic factors.

8. The pharmaceutical composition, according to claim 7, wherein said additional compound is selected from the group consisting of interferons, interleukins, tumor necrosis factor .beta., transforming growth factor .beta., erythropoietin, colony stimulating factors, megakaryocyte stimulating factor, and analogs or fragments of the above-named compounds.

9. The composition, according to claim 7, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

10. A method for the treatment of angiogenic diseases, said method comprising the administration to a human or animal in need of said treatment effective amounts of a first compound which is rPF4, or an angiostatic mutant or fragment thereof, and one or more additional compounds which are angiostatic agents.

11. The method, according to claim 10, wherein said additional angiostatic compound is selected from the group consisting of steroids, sulfatedpolysaccharides, cyclodextrin, retinoids, cyclosporins, thrombospondin, laminin,interferons, tumor necrosis factor-.alpha., fibroblast growth factor antagonists, angiogenin antagonists, fumagillin, herbimycin, and analogs or fragments of the above-named compounds.

12. The method, according to claim 10, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

13. The method, according to claim 10, wherein said compounds are combined with an acceptable pharmaceutical carrier in a single pharmaceutical composition.

14. The method, according to claim 13, wherein said compounds are administered to said human or animal by means selected from the group consisting of tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, liposomes, slow release capsules, implantable pumps, and biodegradable containers.

15. A method for the treatment of angiogenic diseases, said method comprising the administration to a human or animal in need of said treatment effective amounts of a first compound which is rPF4, or an angiostatic mutant or fragment thereof, and one or more additional compounds which have antitumor activity.

16. The method, according to claim 15, wherein said compound with antitumor activity is selected from the group consisting of tumor necrosis factors, interferons, tumor selective antibodies or immunotoxins, doxorubicin, methotrexate, cisplatin, vinblastine, vincristine, bleomycin, and injectable radioisotopes.

17. The method, according to claim 15, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

18. The method, according to claim 15, wherein said compounds are combined with an acceptable pharmaceutical carrier in a single pharmaceutical composition.

19. The method, according to claim 18, wherein said compounds are administered to said human or animal by means selected from the group consisting of tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, liposomes, slow release capsules, implantable pumps, and biodegradable containers.

20. A method for the treatment of angiogenic diseases, said method comprising the administration to a human or animal in need of said treatment effective amounts of a first compound which is rPF4 or an angiostatic mutant or fragment thereof, and one or more additional compounds selected from the group consisting of immunomodulators, inflammatory mediators, and hematopoietic factors.

21. The method, according to claim 20, wherein said additional compound is selected from the group consisting of interferons, interleukins, tumor necrosis factor .beta., transforming growth factor .beta., erythropoietin, colony stimulating factors, megakaryocyte stimulating factor, and analogs or fragments of the above-named compounds.

22. The method, according to claim 20, wherein said first compound is rPF4-241 or an angiostatic fragment thereof.

23. The method, according to claim 20, wherein said compounds are combined with an acceptable pharmaceutical carrier in a single pharmaceutical composition.

24. The method, according to claim 23, wherein said compounds are administered to said human or animal by means selected from the group consisting of tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, liposomes, slow release capsules, implantable pumps, and biodegradable containers.