Note: Descriptions are shown in the official language in which they were submitted.
WO 92/02240 PCr/US91~52
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DESCE~IPrION
NOVEL METHODS AND COMPOSITIONS
FOR TREAT~NT OF ANGIOGENIC DISEASES
', S
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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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
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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
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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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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
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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
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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)
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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
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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.
3n ample 9
It has been shown that proteins of identified structure and function may
be constructed by changing the amino acid sequence if such changes do not
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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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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
60 70
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
NA = Not available
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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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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 +~ ~+
N-29
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,
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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
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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,
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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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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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