Canadian Patents Database / Patent 2541869 Summary

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(12) Patent: (11) CA 2541869
(54) English Title: COMPOSITIONS AND METHODS FOR USE IN TARGETING VASCULAR DESTRUCTION
(54) French Title: COMPOSITIONS ET PROCEDES SERVANT AU CIBLAGE DE LA DESTRUCTION VASCULAIRE
(51) International Patent Classification (IPC):
  • C07F 9/6509 (2006.01)
  • A61K 31/661 (2006.01)
  • A61K 31/6615 (2006.01)
  • C07F 9/12 (2006.01)
(72) Inventors :
  • SHERRIS, DAVID (United States of America)
  • PERO, RONALD W. (United States of America)
  • PETTIT, GEORGE R. (United States of America)
(73) Owners :
  • ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF ARIZONA, ACTING FOR AND ON BEHALF OF ARIZONA STATE UNIVERSITY (United States of America)
(71) Applicants :
  • OXIGENE, INC. (United States of America)
  • ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF ARIZONA, ACTING FOR AND ON BEHALF OF ARIZONA STATE UNIVERSITY (United States of America)
(74) Agent: BORDEN LADNER GERVAIS LLP
(74) Associate agent:
(45) Issued: 2013-06-11
(22) Filed Date: 2000-02-16
(41) Open to Public Inspection: 2000-08-24
Examination requested: 2006-04-19
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
60/120,478 United States of America 1999-02-18

English Abstract

A compound of Formula I below, a composition comprising the compound for treating a warm-blooded animal having a vascular proliferative disorder and use of an effective neoplastic disease inhibitory amount of the compound for inhibiting neoplastic disease in a patient. (see formula I) wherein: X represents a cis- or trans- alkenyl or alkanyl group represented by -(CH=CH)1- or -(CH2-CH2)1-; R1, R2, R3, and R4 are either H or Q+, and at least one of -OR1, -OR2, -OR3, or -OR4 is -O-Q+; and,wherein Q+ is piperazine or nicotinamide cation.


French Abstract

Composé de formule I ci-dessous, composition comprenant le composé pour le traitement d'un animal à sang chaud présentant un trouble vasculaire à évolution chronique et utilisation d'une quantité inhibitrice de maladie néoplasique efficace de composé pour inhiber une maladie néoplasique chez un patient. (voir formule I) dans laquelle : X représente un groupe cis ou trans alkényle ou alkanyle représenté par (CH=CH)1- ou -(CH2-CH2)1-; R1, R2, R3, et R4 sont H ou Q+, et au moins un parmi -OR1, -OR2, -OR3, ou -OR4 est -O-Q+; et dans laquelle Q+ est pipérazine ou ion métallique de nicotinamide.


Note: Claims are shown in the official language in which they were submitted.



CLAIMS:

1. A compound of Formula I,
Image
wherein:
each of R2 and R3 independently is an alkyl group, H, a mono- or divalent
cationic salt, or
an ammonium cationic salt, and R2 and R3 may be the same or different.
2. The compound of claim 1, wherein the cationic salt is selected from Li,
Na,
K, Ca, Cs, Mg, Mn, Zn, piperazine or nicotinamide.
3. The compound of claim 1, wherein the cationic salt is Na, piperazine or
nicotinamide.
4. A composition for treating a warm-blooded animal having a vascular
proliferative disorder, said composition comprising a compound of any one of
claims 1 to
3 and a pharmaceutically acceptable carrier.
5. Use of an effective amount of a compound according to any one of claims
1
to 3 for treating a vascular proliferative disorder in a patient suffering
therefrom.
6. The use of claim 5, wherein the vascular proliferative disorder is a
tumor or
non-malignant hypervascularation.
21

Note: Descriptions are shown in the official language in which they were submitted.

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COMPOSITIONS AND METHODS FOR USE IN
TARGETING VASCULAR DESTRUCTION
This is a divisional of application Serial No. 2,358,925 filed February 16,
2000.
BACKGROUND OF THE INVENTION
This invention relates to methods of and compositions for
effecting targeted vascular destruction in warm-blooded
animals, including humans, and to procedures for identifying
drugs capable of such use.
= The
importance of vasculature to the growth of tumors is
an unquestioned scientific reality. Because one blood vessel
nourishes thousands of tumor cells, targeting tumor vasculature
as a molecular approach to cancer chemotherapies is becoming
one of the highest scientific priorities. Two drug models are
emerging, i.e., one that prevents the formation of new blood
vessels in the tumor (antiangiogenesis) and one that targets
vascular destruction as a means of limiting tumor nourishment
and/or the impermeability of the luminal surface of vessel
endothelial cells to cancer drugs such as immunotherapies (New
England Journal of Medicine 339:473-474, 1998). The antiangi-
,
ogenic model is basically a cytostatic approach where angiogen-
ic factors generally produced by tumors such as vascular
endothelial growth factor (VEGF) and platelet derived endothe-
lial cell growth factor, are blocked by antiangiogenic com-
pounds such as the natural polypeptides angiostatin and
endostatin to
new blood vessel growth (The Cancer
Journal Scientific American 4(4):209-216, 1998; Cell 88:277-
285, 1997). On the other hand, the vascular destruction model
is a cytotoxic approach where tumor vessels are targeted for

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cytotoxicity in order to enhance tumor cell cytotoxicity by
hypoxia or direct acting chemotherapy.
One of the most potent classes of cancer therapeutic drugs
is the antimitotic tubulin polymerization inhibitors (Biochem.
Molecular Biology Int. 25(6):1153-1159, 1995; Br. Journal
Cancer 71(4):705-711, 1995; Journal Med. Chem. 34(8):2579-2588,
1991; Biochemistry 28(17):6904-6991, 1989). They characteris-
tically have ICs, in vitro cell cytotoxicities in the nM-AM
range, but often show poor specificity for killing tumor over
normal tissues in vivo, examples of such drugs including
combretastatins, taxol (and other taxanes), vinblastine (and
other vinca alkaloids), colchicinoids, dolastatins, podo-
phyllotoxins, steganacins, amphethiniles, flavanoids, rhiz-
oxins, curacins A, epothilones A and B, welwistatins, phensta-
tins, 2-strylquinazolin-4(3H)-ones, stilbenes, 2-ary1-1,8-
naphthyridin-4(1H)-ones, 5,6-dihydroindolo (2,1-a)isoquino-
lines, 2,3-benzo(b)thiophenes, 2,3-substituted benzo(b)furans
and 2,3-substituted indoles (Journal of Med. Chem. 41(16):3022-
3032, 1998; Journal Med. Chem. 34(8):2579-2588, 1991; Antican-
cer Drugs 4(1):19-25, 1993; Pharm. Res. 8(6):776-781, 1991;
Experimentia 45(2):209-211, 1989; Med. Res. Rev. 16:2067, 1996;
Tetrahedron Lett. 34:1035, 1993; Mol. Pharmacol. 49:288, 1996;
J. Med. Chem. 41:1688-1695, 1998; J. Med. Chem. 33:1721, 1990;
J. Med. Chem. 34:2579, 1991; J. Md. Chem. 40:3049, 1997; J.
Med. Chem. 40:3525, 1997; Bioorg. Med. Chem. Lett. 9:1081-1086,
1999; International (PCT) Application No. US 98/04380; U.S.
Provisional Patent Application No. 60/154,639).
Although
tubulin binding agents in general can mediate effects on tumor
blood flow, doses that are effective are often also toxic to
other normal tissues and not particularly toxic to tumors (Br.
J. Cancer 74(Supp1. 27):586-88, 1996).
Many tubulin binding agents such as the combretastatins
and taxol analogs are water insoluble and require formulation
before evaluation in the clinic. One approach which has been
used successfully to overcome this clinical development problem
is the formulation of biolabile water soluble prodrugs, such as
the phosphate salt derivatives of combretastatin A4 and taxol,
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that allow metabolic conversion back into the water insoluble
form (Anticancer Drug Des. 13(3):183-191, 1998; U.S. patent No.
5,561,122; Bioorganic Med. Chem. Lett. 3:1766, 1993; Bioorganic
Med. Chem. Lett. 3:1357, 1993). A prodrug is a precursor which
will undergo metabolic activation in vivo to the active drug.
Stated with further reference to the aforementioned phosphate
salt derivatives, the concept here is that non-specific
phosphatases such as alkaline phosphatases in mammals are
capable of dephosphorylating phosphate prodrugs into the
original biologically active.forms. This prior art teaches how
to administer a water insoluble drug to warm blooded animals
for therapeutic purposes under conditions of more maximum
absorption and bioavailability by formulation into a water
soluble biolabile form (Krogsgaard-Larsen, P. and Bundegaard,
H., eds., A textbook of Drug Design and Drug Development,
Harvard Academic Publishers, p. 148, 1991).
When the combretastatin A4 phosphate prodrug was used in
in vitro and in vivo cell and animal models, it displayed a
remarkable specificity for vascular toxicity (Int. J. Radiat.
Oncol. Biol. Phys. 42(4):895-903, 1998; Cancer Res. 57(10):
1829-1834, 1997). It was not obvious from this to one skilled
in the art that phosphate prodrugs in general, which serve as
substrates for alkaline phosphatase, had anything to do
whatsoever with vascular targeting. However, the reported data
on the combretastatin A4 phosphate prodrug did disclose the
principle of preferential vascular toxicity, even though there
was no understanding or appreciation of the fact that the
prodrug itself was responsible for vascular targeting. In
other words, the prior art teaches that AA and not AA prodrug
was responsible for vascular toxicity by assuming that there
was no difference in vascular toxicity between the two forms.
The nonobviousness noted above is exemplified by the fact that,
although AA phosphate prodrug and other taxol phosphate
prodrugs were promoted as susceptible to phosphatase conversion
to the cytotoxic tubulin binding forms, it was never recognized
that this enzyme was elevated in microvessels thus targeting
them to cytotoxicity.
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SUMMARY OF THE INVENTION
An object of the invention is to provide compositions and
methods useful in targeting the microvessel destruction model
for the treatment, in warm-blooded animals including (but not
limited to) humans, of cancer, Kaposi's sarcoma, and other,
non-malignant vascular proliferative disorders such as macular
degeneration, psoriasis and restenosis, and, in general,
inflammatory diseases characterized by vascular proliferation.
Another object is to provide procedures for identifying
drugs that are capable of use in producing such compositions
and performing such methods.
To these and other ends, the present invention in a first
aspect broadly contemplates the provision of a method of
treating a warm-blooded animal having a vascular proliferative
disorder, comprising administering, to the animal, an amount of
a prodrug other than combretastatin A4 disodium phosphate
effective to achieve targeted vascular destruction at a
locality of proliferating vasculature, wherein the prodrug is
substantially noncytotoxic but is convertible to a substantial-
ly cytotoxic drug by action of an endothelial enzyme selective-
ly induced at enhanced levels at sites of vascular prolifera-
tion.
In a second aspect, the invention contemplates the
provision of a method of treating a warm-blooded animal having
a nonmalignant vascular proliferative disorder, comprising
administering, to the animal, an amount of a prodrug effective
to achieve targeted vascular destruction at a locality of
proliferating vasculature, wherein the prodrug is substantially
noncytotoxic but is convertible to a substantially cytotoxic
drug by action of an endothelial enzyme selectively induced at
enhanced levels at sites of vascular proliferation.
In a further aspect, the invention contemplates the
provision of compositions for treating a warm-blooded animal
having a vascular proliferative disorder to achieve targeted
vascular destruction at a locality of proliferating vascula-
ture, comprising a prodrug, other than combretastatin A4,
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pancratistatin and taxol phosphate prodrugs, which is substan-
tially noncytotoxic but is convertible to a substantially
cytotoxic drug by action of an endothelial enzyme selectively
induced at enhanced levels at sites of vascular proliferation.
In yet another aspect, the invention provides a procedure
for identifying prodrugs suitable for use in the above methods
and compositions, such procedure comprising the steps of
culturing proliferating endothelial cells, and other cells, in
the presence of a prodrug other than combretastatin A4 disodium
phosphate for a limited time period; comparing the respective
cultures thereafter to determine whether the culture of
proliferating endothelial cells exhibits a significantly
greater cytotoxic effect than the culture of other cells; and,
if so, culturing the aforesaid other cells in the presence of
the prodrug and an endothelial enzyme selectively induced at
enhanced levels at sites of vascular proliferation, enhanced
cytotoxic effect with respect to the other cells in the
presence of the enzyme as compared to the cytotoxic effect in
the initial culture of the other cells indicating suitability
of the prodrug for such methods and compositions. Conveniently
or preferably, the "other cells" may be nonmalignant fibro-
blasts, e.g., normal human fibroblasts.
In an important specific sense, to which however the
invention is in its broadest aspects not limited, the prodrug
in the foregoing methods, compositions and procedures may be a
phosphate within the class of compounds having the general
formula
11
R]....x_p_y R2
Y R3
wherein
X is 0, NH, or S;
Y is 0, NH, S, 0-, NH- or S-;
Z is 0 or S;
5

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each of R2 and R3 is an alkyl group, H, a mono- or divalent
cationic salt, or an ammonium cationic salt, and R2 and R3 may
be the same or different; and
R3 is defined by the formula 12.1-Ra representing a compound
that contains at least one group (designated Ra) which is a
group, containing X, that can form a phosphate or other salt
that serves as a substrate for non-specific vascular endotheli-
al phosphatases, and is thereby converted from a relatively
non-cytotoxic phosphate form to a cytotoxic Ri-Ra form.
Currently preferred prodrugs for the practice of the
invention are those having the following formulas:
0 0 0
11 11 11
R3-0-P-0- Na* R1-N-P-0 Na R3-N-P-OCH2CH3
1 1 1 1 1
0- Na + H 0- Na* H OCH2CH3
More particularly, the compound with formula Ri-Ra may be a
tubulin binder. In specific aspects it may be selected from
the known tubulin binding agents already previously listed such
as the combretastatins, taxanes, vinblastine (vinca alkaloids),
colchicinoids, dolastatins, podophyllotoxins, steganacins,
amphethiniles, flavanoids, rhizoxins, curacins A, epothilones A
and B, welwistatins, phenstatins, 2-strylquinazolin-4(3H)-ones,
stilbenes, 2-aryl-1,8-naphthyridin-4(1H)-ones, 5,6-dihydroindo-
lo(2,1-a)isoquinolines, 2,3-benzo(b)thiophenes, 2,3-substituted
benzo(b)furans and 2,3-substituted indoles. In a
still more
specific sense, this tubulin binder may be a compound selected
from the group consisting of combretastatins (other than
combretastatin A4), colchicine, and 2-methoxy estradiol.
Stated with reference to phosphate prodrugs, for an under-
standing of the invention it may be explained that vascular
endothelial cells have high levels of phosphatase activity
because of (i) stress injury response of microvessels due to
blood circulation (J. Invest. Dermatol. 109(4):597-603, 1997)
and (ii) the induction of phosphatase in vascular endothelial
cells by IL-6 produced by inflammatory cells during wound
6

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healing or by invasive tumor cells (FEBS Lett. 350(1):99-103,
1994; Ann. Surg. Oncol. 5(3):279-286, 1998). High
levels of
phosphatases (e.g. alkaline) are a part of the normal physiolo-
gy of microvessels, because together with the blood clotting
mechanism, calcium deposits generated from alkaline phosphatase
activity aid in the wound healing process. The
present
invention embraces the discovery that phosphate or other
appropriate prodrug constructs, which become substrates for
phosphatases such as alkaline phosphatases, are useful in
targeting microvascular toxicity. Examples of
phosphatase
enzymes suitable for this purpose require an ectoplasmic
cellular location because of the poor absorption of phosphory-
lated molecules through the cytoplasmic membrane. Dephosphor-
ylating enzymes known to have an ectoplasmic location are non-
specific alkaline phosphatases, ATPase, ADPase, 5'-nucleotid-
ase, and purine nucleoside phosphorylase. Another property
necessary for targeting cytotoxic agents by dephosphorylation
via phosphatases is that they could utilize a broad spectrum of
phosphate prodrugs as substrates. In
this regard, alkaline
phosphatase is an attractive target for delivering selective
toxicity to vascular endothelial cells.
Further features and advantages of the invention will be
apparent from the detailed description hereinbelow set forth,
together with the accompanying drawings.
=BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C illustrate the structures of various
cytotoxic compounds and noncytotoxic prodrugs thereof as
examples of molecular diversity capable of targeting microvas-
cular cellular toxicity by formation of phosphate prodrugs;
FIGS. 2A and 2B are graphs showing the effect of exposure
time on combretastatin A4 prodrug cytotoxicity;
FIGS. 3A and 3B are graphs showing the effect of alkaline
phosphatase on cultured HMVEC and HDF;
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FIGS. 4A and 4B are graphs showing the dose response
effect of added alkaline phosphatase on the cytotoxicity of
HMVEC and HDF to A4 prodrug; and
FIG. 5 is a series of graphs showing the effects of
exposure time on the clonogenic toxicity induced by a variety
of tubulin binding drugs.
DETAILED DESCRIPTION
This invention embraces the use of phosphate prodrugs
comprising administering to warm-blooded animals having a tumor
or non-malignant hypervascularation, a sufficient amount of a
cytotoxic agent formulated into a prodrug form having substrate
specificity for microvessel phosphatases, so that microvessels
are destroyed preferentially over other normal tissues, because
the less cytotoxic prodrug form is converted to the highly
cytotoxic dephosphorylated form.
Examples of preferred
cytotoxic agents for vascular targeting are tubulin binding
agents, because they can be transformed from water insolubility
to water solubility, tubulin binding agents to non-tubulin
binding agents, and cytotoxicity to non-cytotoxicity by
phosphate prodrug formulation (Anti-Cancer Drug Design 13: 183-
191, 1998).
Examples of the molecular diversity for targeting micro-
vessel cellular toxicity by formation of phosphate prodrugs are
presented in FIGS. 1A-1C, and they were selected from the known
tubulin binding agents already previously listed such as the
combretastatins, taxanes, vinblastine (vinca alkaloids),
colchicinoids, dolastatins, podophyllotoxins, steganacins,
amphethiniles, flavanoids, rhizoxins, curacins A, epothilones A
and B, welwistatins, phenstatins, 2-
strylquinazolin-4(3H)-
ones, stilbenes, 2-aryl-1,8-naphthyridin-4(1H)-ones, 5,6-
dihydroindolo(2,1-a)isoquinolines, 2,3-benzo(b)thiophenes, 2,3-
substituted benzo(b)furans and 2,3-substituted indoles. The
compounds listed in FIGS. 1A-1C satisfy the structural require-
ments of having either aromatic hydroxyl or amino groups
8

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present capable of chemical reaction to produce a phosphate
salt, and the further conversion of a cytotoxic agent into a
non-cytotoxic phosphate prodrug construct. Other
criteria
necessary for targeting vascular toxicity are:
1. Tubulin binding agents or other cytotoxic agents (e.g.
pancratistatin has not been reported to bind to tubulin
polymers) must induce similar levels of toxicity to both
human microvessel cells and other normal human cells such
as fibroblasts when in the cytotoxic (tubulin binding)
form, or, alternatively, the tubulin binding form must be
much less inherently cytotoxic to normal cells than to
microvessel cells. If this were not the case and fibro-
blasts (i.e. normal cells) were much more sensitive than
microvessels to the cytotoxic form, then when in the non-
cytotoxic prodrug form, even though fibroblasts had much
less phosphatase to activate the cytotoxic form, much less
would in turn be needed to induce cytotoxicity in fibro-
blasts. The net result would be that prodrugs could still
be more toxic to microvessels instead of normal cells,
because of their enhanced alkaline phosphatase activity
producing the cytotoxic form.
2. The tubulin binding or cytotoxic forms of potential
phosphate prodrugs must not be cytotoxic in the prodrug
form, which in turn needs to be converted into the
cytotoxic form within 1-3 hours, preferably within 1-2
hours.
Tubulin binding agents clear from peripheral
circulation within a few hours. So in
order to be
effective in targeting vascular destruction in vivo, the
phosphate prodrug constructs must be converted to the
cytotoxic forms within 1-3 hours by phosphatase in the
microvessels in order to elicit a preferential toxicity of
the cells. Hence, the kinetics of binding to tubulin must
be nearly complete within 1-3 hours.
Although high levels of alkaline phosphatase are useful for
targeting vascular destruction of tubulin binding agents, this
invention also embraces, in a broader sense, that any enzyme or
protein specifically amplified in microvessels, and that is
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capable of converting metabolically a nontoxic prodrug into a
cytotoxic drug, would be equally useful in targeting vascular
destruction.
Compositions in accordance with the invention having use
in targeting vascular destruction are illustratively exempli-
fied, without limitation, by compounds embraced within the
class of compounds having the general formula
0 0
1111
R'-0-P-0 R2 = or R1-N-P-0 R2
1 1 1
0 112 H 0 R3
=
wherein R1 is defined by the formula 111-Ra representing a
compound that contains at least one group (designated R.') which
is a phenolic hydroxyl group, or an aromatic amino group, or
any other appropriate hydroxyl or amino group, that can form R2-
R3 phosphate metal or amine salts or phosphate esters that serve
as substrates for non-specific vascular endothelial phosphat-
ases, and are thereby converted from a relatively non-cytotoxic
phosphate form to a cytotoxic hydroxyl or amino form.
Thus, in illustrative embodiments, R' is defined by the
formula R."-Ra representing a compound that contains at least one
phenolic hydroxyl group (designated Ra) that can form a sodium
phosphate or other appropriate salt (e.g., R?, 123 may be Li, Na,
K, Ca, Cs, Mg, Mn, Zn, piperazine, nicotinamide and other
examples as found in International (PCT) patent application No.
99/US/5368 that serves as a substrate for non-specific vascular
endothelial phosphatases, and that is thereby converted from a
relatively non-cytotoxic phosphate form to the cytotoxic phenolic
hydroxyl form.
The invention particularly embraces discoveries made in
ascertaining the heretofore unknown explanation for the
observed apparent selective. targeting of proliferating endothe-
lial cells by combretastatin A4 disodium phosphate, and in
recognizing the applicability of those discoveries to drugs
other than combretastatin A4 and .to the treatment of nonmalig-
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nant as well as malignant disorders involving vascular prolif-
eration.
The pertinent studies respecting combretastatin A4
disodium phosphate will now be further described.
Chemicals. GMP manufactured combretastatin A4 disodium
phosphate was purchased from OXiGENE, Inc. (Boston) and
dissolved in physiological =saline for addition to cell cul-
tures. Alkaline phosphatase was purchased from Sigma* (P-6774)
as a buffered solution and was added to cell cultures directly.
Cell culture. Four commercially
available human cell
lines were grown in the indicated media below in 5% CO2, 80%
humidity and 37 C:
1. HL60 human leukemic cells, a pro-apoptotic cell line
-- cultured in RPMI 1640 fortified with 10k fetal
calf serum.
2. K562 human leukemic cells, an apoptotic-resistant
cell line -- cultured in RPMI 1640 fortified with 10%
fetal calf serum.
3. Human neonatal microvascular endothelial cells
=(HMVEC) -- cultured in medium 131 + microvascular
growth supplement (MVGS) + attachment factor (AF) =
500 ml + 25 ml (AF is added 2-3 ml/T-25 flask; all
reagents supplied by Cascade Biologics, Inc., Port-
land, Oregon).
4. Human neonatal dermal fibroblatts (HDF) cultured
in medium 106 + low serum growth supplement (LSGS) =
500 ml + 10 ml (Cascade Biologics, Inc.).
The cells used in all experiments were first subcultured
up to 2-3 days at an initial density of 2 x 105 cells/ml prior
to use in the vitro assays. This resulted in an exponential
growth stage and the cell viability was >95% by trypan blue
exclusion.
Cell survival by clonoaenic assay. This assay is based on
a description reported by Schweitzer et al. (Expt. Haematol.
21: 573-578, 1993) with slight modifications. Briefly, HL60 and
K562, HDF, HMVEC cells at concentrations of 4.2 x 103/m1 were
cultured in 96-well flat-bottomed microculture plates in a
*Tradename 11

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volume of 190 Al per well plus different concentrations of
combretastatin PA disodium phosphate or other tubulin binding
agents and their prodrugs or units of alkaline phosphatase
added in a 10 gl volume. After 5 days of incubation under the
standard culture conditions stated above, colonies (>40 cells)
were counted by an inverted light microscope or estimated by
MTT assay. ICõ, values were obtained from the fitted curves of
percentage of the control versus the drug concentrations.
Alkaline phosphatase metabolism of combretastatin A4
disodium phosphate to the highlv cvtotoxic combretastatin A4.
There were three types of experiments designed to demonstrate
the importance to convert A4 prodrug to A4 in order to target
toxicity to vascular endothelial cells.
Experiment 1. HL60, K562, HDF, and HMVEC cells were
either cultured in 96-well plates at the indicated concentra-
tions (FIGS. 2A and 2B) for 5 days in the presence of A4
prodrug, or after 2 hours exposure the drug-containing media
was removed, fresh media added, and the cells cultured for an
additional 5 days. Clonogenic growth was recorded after 5 days
incubation for all treatments.
Experiment 2. HMVEC
and HDF were cultured in 96-well
microtiter plates initially containing 800 cells/well. The
cells were cultured for 1 hour in the presence of the indicated
concentrations of A4 prodrug + 1 unit of alkaline phosphatase.
The medium was removed, the cells washed, and fresh medium
added, and the cells were incubated for an additional 5 days.
Clonogenic growth was then established by the MTT assay.
Experiment 3. HMVEC were cultured in 96-well microtiter
plates initially containing 800 cells/well. The
cells were
cultured for 1 hour in the presence of the indicated concentra-
tions of A4-prodrug + the indicated units of alkaline phospha-
tase. The medium was removed, the cells were washed in medium,
and the cultures were further incubated in fresh medium for an
additional 5 days. Clonogenic growth was then established by
the MTT assay.
Referring to the drawings, FIGS. 2A and 2B are graphs
showing the effect of exposure time on A4 prodrug cytotoxicity.
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HMVEC, HDF, HL60, and K562 cells were exposed for 2 hours (FIG.
2A) or 5 days (FIG. 2B) to combretastatin A4 disodium phosphate
before clonogenic cytotoxicity was estimated at 5 days. Note
that the IC50 values were similar for all the cells after 5 days
exposure being 1.5 to 2.5 nM whereas only HMVEC showed IC50
cytotoxicity when exposure was limited to 2 hours.
FIGS. 3A and 3B are graphs showing the effect of alkaline
phosphatase on cultured HMVEC and HDF. Dose response cytotox-
icity was estimated after 1 hour exposure to various concentra-
tions of combretastatin A4 disodium phosphate in the presence
or absence of 1 unit alkaline phosphatase. Note the lack of
cytotoxicity of HDF without added alkaline phosphatase, but the
cytotoxicity of A4 prodrug was the same for HMVEC and HDF when
alkaline phosphatase was added.
FIGS. 4A and 4B are graphs showing the dose response
effect of added alkaline phosphatase on the cytotoxicity of
HMVEC and HDF to PA prodrug. HMVEC and HDF were cultured for 1
hour in the presence of the indicated concentrations of added
combretastatin A4 disodium phosphate + the indicated units of
added alkaline phosphatase. The data
clearly showed added
dependence of the alkaline phosphatase on the cytotoxicity
especially at the higher A4 prodrug concentrations.
EXAMPLE 1
Example 1 discloses the importance of time of exposure to
the preferential cytotoxicity of vascular endothelial cells to
tubulin binding agents such as combretastatin A4 prodrug. If
the clonogenic assay is set up to treat HMVEC, HDF, K562 and
HL60 cells for 5 days in the presence of increasing concentra-
tions of combretastatin A4 disodium phosphate (prodrug), all
the cell lines had similar ICõ) values of about 1.5 to 2.5 nM
(FIG. 2B). These data teach that there is no inherent differ-
ence in the toxicity of the human cell lines regardless of
their origin, if the exposure time is long enough. However, A4
prodrug, as well as other tubulin binding drugs, clear from
peripheral circulation in vivo within a few hours, and under
these conditions A4 prodrug showed a preferential toxicity to
13

CA 02541869 2000-02-16
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proliferating endothelial cells in tumors, whereas other
tubulin binding agents have not been shown to possess this
property (Cancer Res. 57(10):1829-1834, 1997). Hence, we have
limited the exposure of the various cell lines to AA prodrug
for 2-3 hours, removed the A4-containing medium and replaced it
with fresh medium, and continued culturing for an additional 5
days. These conditions showed that HMVEC were quite sensitive
to A4 prodrug-induced cytotoxicity compared to the HDF, K562
and HL60 cells (FIG. 2A). These data teach that (i) an in
vitro cell model can be used. to demonstrate selective induction
of toxicity to vascular endothelial cells by tubulin binding
agents such as A4 prodrug, (ii) this only occurs under in vitro
conditions that mimic in vivo pharmacokinetic-'regulated limita-
tions of exposure, and (iii) either tubulin binding parameters
regulating cytotoxicity or metabolic differences or both are
responsible for the selective toxicity of A4 prodrug to
vascular endothelial cells.
EXAMPLE 2
The combretastatins are a family of naturally occurring
tubulin binding agents comprising an A-,B-,C- and D- series of
structures (U.S.. patents Nos. 4,940,726; 4,996,237; 5,409,953;
and 5,569,786).
Example 2 compares the 1050 values of the
clonogenic toxicity induced by a selection of these compounds
in in vitro cultures of HDF, HMVEC and HL-60. The compounds
were added to microcultures in DMS0 (i.e <0.5%) and toxicity
was evaluated by MTT assay after 5-7 days in culture. The data
in Table 1 show that the combretastatin analogs varied consid-
erably in their overall clonogenic toxicity between the various
analogs as well as between the different human cell types being
evaluated. A4 had the most toxic mechanism of binding tubulin
in all the cell types tested, and it showed no preference for
clonogenic toxicity between the cell types.
However, the
cytotoxicity of the other combretastatins generally could be
ranked according to the clonogenic toxicity of greatest to
least toxic as:
HL-60 > HDF > HMVEC.
14

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These data establish the prerequisite for tubulin binding drugs
to have a property whereby toxicity to normal cells is not much
greater than that to HMVEC, if phosphate prodrugs are to be
used in vascular targeting of antimitotic toxicity.
TABLE 1
clonogenic toxicity values in nM
Combretastatin HDF HMVEC HL-60
A4 1-2 1-2 1-2
A3 8-10 >12 5
A2 25-35 30-40 15
Al 20 500 n.d.
Bl = 200-300 200-300 500
B2 1100 800-1000 125
K-228 40-90 90-120 90
K-332 800-900 >1000 500
Combretastatins were kindly supplied by Professor G.R. Pettit
of Arizona State University. HDF = human diploid fibroblasts;
HVMEC = human microvessel endothelial cells; HL-60 = human
myeloid leukemic cells
EXAMPLE 3
The effect of exposure time on the clonogenic toxicity
induced by a variety of tubulin binding drugs is presented in
FIG. 5. Taxol, taxotere, vincristine, and combretastatins Al
and A4 were added to microcultures of HMVEC and HDF for 1 and 6
hours, washed with saline and incubation continued in complete
medium for 3 more days before estimating clonogenic toxicity by
MTT assay. The data in this example show that the kinetics of
binding of various tubulin binding drugs influences their
cytotoxicity under conditions that are similar to in vivo
exposure (i.e. 1 hour). For example, taxol, taxotere and
Combretastatin Al did not induce maximum toxicity to HMVEC
after 1 hour exposure but required 6 hours, and in addition,
the degree of kinetic-regulated cytotoxic responses were also
different in HDF compared to HMVEC.
Hence, in order to target microvessel toxicity in humans
the tubulin binding cytotoxic mechanism needs to be completed

CA 02541869 2000-02-16
W000/48606 PCT/US00/03996
within a 1-3 hour period after treatment in a manner that
permits the toxicity to HMVEC to be comparable to HDF or other
normal cells. When this is the case then phosphate prodrugs
are able to target microvessel toxicity because they have
elevated alkaline phosphatase compared to normal cells to
transform the prodrug into its cytotoxic form.
EXAMPLE 4
Both stress injury and the presence of invasive tumor
cells can induce microvessels to produce up to 50-fold in-
creased levels of alkaline phosphatase (J. Invest. Dermatol.
109(4):597-603, 1997; FEBS Lett. 350(1):99-103, 1994).
Alkaline phosphatases present in cell membranes and circulation
can hydrolyze organic phosphate-containing compounds separating
or freeing the phosphate salt portion (e.g. calcium phosphate)
from the organic molecule portion. The physiological need of
microvessels to repair damage to themselves by elevating
alkaline phosphatases is a part of normal wound healing process
leading to an increased deposition of calcium deposits in the
injured area. A consequence of this metabolic specificity may
be that cytotoxic tubulin binding agents modified into a
phosphate salt (e.g. A4 prodrug) may also be a substrate for
alkaline phosphatase. This process then could in turn lead to
an increased cytotoxic sensitivity of microvessels to tubulin
binding drugs, that do not bind tubulin in a phosphorylated
form and are not cytotoxic to the dephosphorylated form which
does bind tubulin and is cytotoxic. This example shows that
indeed this is the case. HDF and HMVEC exposed to in vitro
culture for 2 hours to increasing concentrations of A4 prodrug
in the presence or absence of 1 unit of added alkaline phospha-
tase, demonstrate a high degree of selective cytotoxicity to
HMVEC without added alkaline phosphatase, but HDF become
identically cytotoxic as HMVEC to A4 prodrug in the presence of
added alkaline phosphatase (FIGS. 3A and 3B). It was concluded
that targeting vascular destruction was directly dependent on
the presence of high levels of alkaline phosphatase in HMVEC,
and the lack of it in other normal and tumor cells such as HDF.
16

CA 02541869 2000-02-16
V/000/48606 PCT/US00/03996
Hence, this example teaches a method for targeting preferential
destruction of microvessels, whereby cytotoxic agents such as
tubulin binding compounds, which when converted into a prodrug
form by for example forming a phenolic hydroxy phosphate salt
that cannot induce cytotoxicity, can be selectively metabolized
by alkaline phosphatase, that is present in high amounts only
in vascular endothelial cells, back into a cytotoxic form.
EXAMPLE 5
Example 5 further establishes and verifies the disclosure
presented in Example 4. Here,
the experimental design was
designed to demonstrate the dose dependence of alkaline
phosphatase on regulating cytotoxicity of A4 prodrug. The data
clearly show how the amount of alkaline phosphatase determines
the clonogenic cytotoxicity of combretastatin A4 disodium
phosphate to both HMVEC and HDF (FIGS. 4A and 48). The results
teach that more alkaline phosphatase must be added before HDF
can be killed by A4 prodrug, whereas HMVEC directly express
clonogenic toxicity to A4 prodrug without or after addition of
low levels of alkaline phosphatase, but at high added levels of
alkaline phosphatase the toxicities become equal for both cell
lines.
It is therefore demonstrated that in vivo targeting of
tumor vascular destruction is directly dependent on alkaline
phosphatase, and that this knowledge would be useful in
designing agents and methods for the treatment of cancer and
other, non-malignant, vascular proliferating disorders.
EXAMPLE 6
The compounds presented in Table 2 represent examples of
how toxicity can be targeted to microvessel cells by converting
the cytotoxic forms into phosphate prodrugs, which are in turn
not cytotoxic until converted back into the cytotoxic form by
cellular phosphatases such as alkaline phosphatase, which has
a50-fo1d higher concentration in proliferating microvessel
endothelial cells than other normal cells. In general, tubulin
binding drugs cannot bind tubulin in the phosphate salt form,
17

CA 02541869 2000-02-16
W000/48606 PCT/US00/03996
and so they represent a cytotoxic mechanism preferred as a
cytotoxic mechanism for vascular targeting. All
of the
compounds were evaluated for toxicity after a one-hour exposure
in microculture and assayed for cytotoxicity by MTT assay after
an additional 5 days' incubation in culture. Under these
conditions, the kinetics of tubulin binding were sufficiently
rapid to cause toxicity in both normal proliferating HDF and
HMVEC. The
data reported in Table 2 establish that (i)
phosphate prodrugs in general spare normal HDF from toxicity
while not affecting the toxicity to HMVEC as shown by higher
values for the prodrugs in HDF but not HMVEC, (ii) if the
cytotoxic agent is more toxic to HDF than to HMVEC, then even
though the prodrug spares toxicity in HDF it cannot make up for
the difference in inherent toxicities between HDF and HMVEC,
(iii) not all metal or amine salts of phosphate prodrugs are
equally effective since combretastatin Al piperazine phosphate
was only marginally effective at protecting HDF from cytotoxic-
ity, and (iv) because pancratistatin is not known to bind
tubulin, compounds having other cytotoxic mechanisms can also
be targeted by the phosphatase mechanism. In summation, these
data show that cytotoxic agents can target microvessel cellular
destruction by phosphate prodrug construction, if there is
protection for normal cells having little alkaline phosphatase
to metabolize enough of the phosphate prodrug to its cytotoxic
form within one hour of exposure (i.e., mimics in vivo condi-
tions).
18

CA 02541869 2000-02-16
W000/48606 PCT/US00/03996
TABLE 2
Evidence for targeting microvessel cellular toxicity
by converting cytotoxic compounds into non-cytotoxic
phosphate prodrugs (Note: "FIG. 1 No." in the left-
hand column refers to the structure identification
number in FIGS. 1A, 1B and 1C of the drawings.
Compounds I to VIII were supplied by Professor G.R.
Pettit of Arizona State University and compounds X to
XVI by Dr. Kevin G. Pinney of Baylor University in
Waco, TX)
FIG. 1 Cytotoxic Non-Cytotoxic IC50 values
No. form form (Drodruq) HMVEC HDF
Combretastatin A4 75-150nM 50nM
11 Combretastatin AA
Na2PO4 75-150nM >500nM
III Combretastatin Al 10-15AM
>0.5-1AM
IV Combretastatin Al
Na2PO4 10-15AM 5-10AM
V Combretastatin Al 10-15AM
>O.5-1/LM
VI Combretastatin Al
Piperazine PO4 1O-15/LM 1-2AM
VII Combretastatin Al 10-15AM
>0.5-1AM
VIII Combretastatin A2
Nicotinamide PO4 10-15AM >10AM
X Amino Combretastatin
A4 Phosphoroamidate 8-10AM 15-20AM
XI Dihydronaphthalene 0.5-1AM 0.5-
1AM
XII Dihydronaphthalene
Phosphoroamidate 5-7/LM >50AM
XIII Pancratistatin 20-25AM 20AM
XIV Pancratistatin
Na2PO4 20-25 M 60-80 M
XV Benzo(a)thiophene 5-10AM 5-
10AM
XVI Benzo(a)thiophene
Na2PO4 8-10AM 30-
40AM
19

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PCT/US00/03996
EXAMPLE 7
To simulate pathogenic ocular angiogenesis, ocular
neovascularization was induced by administration of lipid
hydroperoxide (LHP) by intra-corneal injection at a dosage of
30 g to rabbit eyes. Seven to 14 days later, ocular vessels
formed in the injected eyes due to LHP insult. The subjects
were divided into two groups; those of one group were given
combretastatin A4 disodium phosphate by intravenous administra-
tion at a dosage of 40mg/kg once a day for five days, while a
vehicle without combretastatin A4 disodium phosphate was
administered to the other group by i.v. administration as a
dosage of water for the same time period. The eyes of both
groups were examined seven days later. A reduction of vessels
of 4096 or more was observed in the group treated with combreta-
statin A4 disodium phosphate, but not in the other group.
It is to be understood that the invention is not limited
to the features and embodiments hereinabove specifically set
forth, but may be carried out in other ways without departure
from its spirit.

A single figure which represents the drawing illustrating the invention.

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Title Date
Forecasted Issue Date 2013-06-11
(22) Filed 2000-02-16
(41) Open to Public Inspection 2000-08-24
Examination Requested 2006-04-19
(45) Issued 2013-06-11

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Current owners on record shown in alphabetical order.
Current Owners on Record
ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE STATE OF ARIZONA, ACTING FOR AND ON BEHALF OF ARIZONA STATE UNIVERSITY
Past owners on record shown in alphabetical order.
Past Owners on Record
OXIGENE, INC.
PERO, RONALD W.
PETTIT, GEORGE R.
SHERRIS, DAVID
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.

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