Note: Descriptions are shown in the official language in which they were submitted.
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Treatment of Carcinomas using Squalamine in Combination
with other Anti-Cancer Agents or Modalities
BACKGROUND OF THE INVENTION
s I. INFORMATION RELATING TO PREVIOUS SQUALAMINE APPLICATIONS
This application is a continuation-in-part of 09/150,724, filed Sept. 10,
1998, which
is a continuation-in-part of 08/840,706, filed April 25, 1997, which claims
the benefit of
provisional application, 60/016,387, filed April 26, 1996, under 35 U.S.C. ~
119. All of
these applications are herein incorporated by reference in their entirety.
io This invention relates to various methods for using squalamine. Squalamine,
having the structure illustrated in Fig. 1, is an aminosterol which has been
isolated from the
liver of the dogfish shark, Squalus acanthias. This aminosterol is the subject
of U.S. Patent
No. 5,192,756 to Zasloff, et al., which patent is entirely incorporated herein
by reference.
Methods for synthesizing squalamine have been devised, such as the methods
described in
is WO 94/19366 (published September 1, 1994). This PCT publication is entirely
incorporated herein by reference. This PCT application also relates to U.S.
Patent Appln.
No. 08/023,347 (filed February 26, 1993), which application also is entirely
incorporated
herein by reference. Additional methods for synthesizing squalamine also are
described in
U.S. Patent Appln. No. 08/985,876 filed December S, 1997, which application
also is
zo entirely incorporated herein by reference.
U.S. Patent Nos. 5,733,899 and 5,721,226 describe the use of squalamine as an
antiangiogenic agent. These U.S. patents are entirely incorporated herein by
reference.
Additional uses of squalamine (e.g., as a sodium/proton exchanger (isoform 3),
or NHE3,
inhibiting agent and as an agent for inhibiting the growth of endothelial
cells) and
zs squalamine synthesis techniques are disclosed in U.S. Patent No. 5,792,635.
This U.S.
patent is also entirely incorporated herein by reference.
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II. INFORMATION RELATING TO THIS INVENTION
About 50,000 new cases of CNS (central nervous system) tumors are diagnosed
each year. Of these, about 35,000 are metastatic tumors (e.g., lung, breast,
melanomas) and
about 15,000 are primary tumors (mostly astrocytomas). Astrocytomas, along
with other
s malignant gliomas (i.e., cancers of the brain), are the third leading cause
of death from
cancer in persons between the ages of 15 and 34.
Treatment options for a patient with a CNS tumor are very limited. Currently,
surgery is the treatment of choice. Surgery provides a definite diagnosis,
relieves the mass
bulkiness of the tumor, and extends survival of the patient. The only post-
surgery adjuvant
io treatment which is known to work on CNS tumors is radiation, and it can
prolong survival.
Radiation treatment, however, has many undesirable side effects. It can damage
the normal
tissue of the patient, including the brain tissue. Radiation also can cause
the patient to be
sick (e.g., nausea) and/or to temporarily lose their hair.
The other common post-surgery adjuvant cancer treatment, chemotherapy, is
is relatively ineffective against CNS tumors. Specifically, chemotherapy
against CNS tumors
with nitrosoureas is not curative. Many other cancer treating agents have been
studied and
tested, but generally they have a minimal effect on extending survival.
In view of these limited treatment options, the current prognosis for persons
with
CNS tumors is not good. The median survival term for patients with malignant
zo astrocytomas having surgery and no adjuvant treatment is about 14 weeks.
Radiation
therapy after surgery extends the median to about 36 weeks. The current two
year survival
rate for all forms of treatment is less than 10%.
To maximize survival, it is critical to begin treatment in the early stages of
CNS
tumor development. Typically, the extent of tumor angiogenesis (i.e., blood
vessel
zs formation) correlates with survival in the patient. CNS tumors are among
the most
angiogenic of all human tumors. When the tumor is small, however, it is in an
"avascular"
phase, and its growth is restricted by a diffusion mechanism (i.e., the cells
receive their
nutrition, etc. by diffusion into the cell). In this phase, the tumor is
viable, but not growing,
and it is unable to spread. Over time, however, angiogenesis begins and the
tumor converts
so to a "vascular" phase. In this phase, perfusion replaces diffusion as the
growth mechanism,
and tumor growth is exponential (i.e., the tumor has its own blood vessels to
provide
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nutrients, etc.). Mitotic cells cluster around new blood vessels and
metastases occur in the
vascular phase (i.e., the tumor can spread to other areas in the body).
Therefore, by treating
the tumor early (before it reaches the vascular phase;), one can hope to
inhibit metastatic
spread as well as control the primary tumor.
Other types of cancer also are difficult to combat by known cancer treatments.
Lung cancer kills more Americans annually than the next four most frequently
diagnosed
neoplasms combined. Estimates for 1994 indicate more than 170,000 new cases of
lung
cancer and approximately 150,000 deaths (Boring et al.; CA Cancer J. Clin.
1994, 44: 7-
26). Approximately 80% of primary lung tumors are of the non-small cell
variety, which
io includes squamous cell and large cell carcinomas, as well as
adenocarcinomas.
Single-modality therapy is considered appropriate for most cases of early and
late
stage non-small cell lung cancer (NSCLC). Early stage tumors are potentially
curable with
surgery, chemotherapy, or radiotherapy, and late stage patients usually
receive
chemotherapy or best supportive care. Intermediate stage or locally advanced
NSCLC,
is which comprises 25% to 30% of all cases of NSCLC, is more typically treated
with
multimodality therapy. This is a stage of tumor development when angiogenesis
is a very
important factor. New blood vessels are needed to support further tumor growth
and for
the development of metastases. Therefore, this stage is amenable to treatment
with
antiangiogenic agents to prevent the development of new blood vessels. The
efficacy of
zo this therapy can be further improved by the combination of the
antiangiogenic therapy with
cytotoxic chemotherapy or radiation therapy to eliminate existing tumor.
Breast cancer also presents treatment difficulties using known agents. The
incidence of breast cancer in the United States has been rising at a rate of
about 2%/year
since 1980, and the American Cancer Society estimated that 182,000 cases of
invasive
zs breast cancer were diagnosed in 1995. Breast cancer is usually treated with
surgery,
radiotherapy, chemotherapy, hormone therapy, or combinations of the various
methods.
Like other solid tumors, breast cancer requires the development of new blood
vessels to
support its growth beyond a certain size, and at that stage in its
development, it will be
amenable to treatment with antiangiogenic agents.
~o A major reason for the failure of cancer chemotherapy in breast cancer is
the
development of resistance to the cytotoxic drugs. Combination therapy using
drugs with
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different mechanisms of action is an accepted method of treatment which
prevents
development of resistance by the treated tumor. Antiangiogenic agents are
particularly
useful in combination therapy because they are not likely to cause resistance
development
since they do not act on the tumor, but on normal host tissue.
Prostate cancer is another cancer for which new therapies are needed. Despite
the
prevalence of prostate cancer as the most frequently diagnosed malignancy
among
American men, mechanisms of prostate carcinogenesis are poorly understood. The
multiplicity of factors involved in the development, proliferation, and
dissemination of
human prostate cancer, as well as their relationships and interaction with one
another,
io magnify the difficulty of treatment.
Both prostate tumor cell growth and metastasis require adequate metabolic
support
as well as vascular access and thus rely on angiogeneis. The prostate cancer
cell-
extracellular matrix (ECM)/stromal relationship is also significant to the
growth and spread
of human prostate cancer. Of the numerous growth factors present in the ECM, b-
FGF
is (basic fibroblast growth factor, also known as FGF-2) and VEGF (vascular
endothelial
growth factor) stand out as having been implicated in both inducing a
malignant phenotype
and in promoting and maintaining angiogenic processes. Ultimately, the outcome
of a
patient with prostate cancer largely depends upon the tumor's capacity for
unhindered
growth, local invasion, and the establishment of distant metastasis. Thus,
anti-angiogenic
ao agents may effectively inhibit the growth and metastasis of such tumors.
Current therapies for prostate cancer focus on inhibiting the androgen agent.
Radiation is, as with many cancers, an initial line of treatment, often
followed by hormonal
therapy. Such hormonal therapy seeks to specifically inhibit the androgen
agent.
Inhibition can result from either surgical castration or chemical castration
with agents such
is as LHRH (luteinizing hormone releasing hormone) agonists and anti-androgens
(such as
flutamide, biclutamide, nilutamide, and luprolide). However, these therapies
fail in most
patients who thereafter present with hormone-refractory lesions. Currently,
few therapeutic
options exist for men with hormone-refractory prostate cancer, and none offer
much
durability. At this point, tumor growth can be so accelerated that life
expectancy rarely
so exceeds six months to one year. Indeed, 70% of these patients will
eventually die of their
hormonal refractory disease.
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In such patients, who have undergone anti-hormone therapies, the remaining
prostate tumor cells are likely undergoing rejuvenated proliferation.
Accordingly, anti-
angiogenic agents rnay be most effective at this stage, particularly agents
that are most
potent on freshly sprouting, young blood vessels, thereby preventing
neovascularity and
s repressing further tumor growth and metastasis.
Ovarian cancer is the most serious gynecologic tumor type. Over 50% of all
cancer-related gynecologic deaths are attributable to ovarian cancers, of
which 80-90% are
epithelial-derived tumors. In 1997 there were 26,700 new cases of ovarian
cancer and
more than 14,000 deaths. There is a clear genetic component to ovarian cancer.
Newly
io developed detection methods have shown strong correlations between breast
cancer,
ovarian cancer and expression of genetic markers that include BRCAI or mutant
oncogenes
such as aberrant forms of erbB-2 or c-Myc. Another good marker for ovarian
cancer of
clinical utility is the circulating analyte CA125, for which serum levels
generally reflect the
state of cancer progression. CA125 is often monitored in ovarian cancer,
although it is not
is a validated marker. There is also a hormonal influence on cancer risk in
ovarian cancer, as
is seen in breast cancer
About one-third of ovarian cancer patients present with localized disease.
Early
stage ovarian cancer is treatable with some combination of surgery, radiation
and
chemotherapy; the 5 year survival rate for localized ovarian cancer is greater
than 80%.
ao However, the 5 year survival rate for metastatic stage III or IV ovarian
cancer is less than
20%. It is these advanced patients, those for whom the ovarian tumors escape
the ovarian
capsule and invade intraperitoneal surfaces, who require the most aggressive
therapy yet
benefit only marginally with chemotherapy. The first line chemotherapeutic
treatment for
ovarian cancers has been the use of platinum-based regimens for over a decade.
With the
as advent of newer agents such as the taxanes (paclitaxel, docetaxel),
gemcitabine, newer
vinca alkaloids (vinorelbine), and topoisomerase inhibitors (topotecan,
irinotecan),
combination chemotherapy has become more widely explored in advanced ovarian
cancer
patients. The combination (sequential or concurrent) of a taxane and a
platinum agent is
the present standard first line treatment for advanced ovarian cancer
patients. However, the
3o poor prognosis for these patients despite the aggressive use of
chemotherapy and surgery
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(with or without radiotherapy) suggests the further addition of non-cytotoxic
agents such as
an angiogenesis inhibitor would be beneficial.
There are recent estimates that the incidence of deaths due to liver cancer
worldwide could be as high as 106 or more per year, Hepatocellular carcinoma
(HCC) or
s hepatoma is the most common liver cancer tumor and is a tumor type that is
closely
associated with chronic hepatitis B or hepatitis C infection. The high
frequency of hepatitis
viral infections observed in Asia and Africa make these regions the sites of
the largest
number of liver cancers, most of which are HCC tumors. Although viral
infection is
considered essential for predisposition to HCC, infection alone is not the
only contributing
~o factor to hepatocellular transformation and proliferation. By comparison,
there were 13,500
deaths of liver cancer in 1992 in the United States, of which two-thirds were
HCC.
The prognosis for liver cancers is universally poor with the minor exception
of
those tumors which are only locally invasive and are located favorably for
curative surgical
removal. For locally advanced disease, limited efficacy has been seen with
interferon
is therapy, polyprenoic acid (vitamin A derivative), S-fluorouracil or
cryoablation. No
meaningful combination chemotherapy has proven itself in human trials for HCC
to date,
but the urgent clinical need continues to drive experimental evaluation of
various
therapeutic approaches. A recent report (LX Qin et al., Ann Acad Med Singapore
28,147-
51 (1999)) suggests that angiogenesis inhibitors can inhibit hepatoma tumor
growth in
zo mouse xenograft tumor models and may be candidates for the control of
recurrence and
metastasis after HCC resection. It therefore is a reasonable approach to
consider
combining treatment of HCC patients not eligible for curative surgery with an
angiogenesis
inhibitor and an active second modality such as a-interferon, a cytotoxic
agent such at 5-
fluorouracil, or a vitamin A derivative.
zs Pediatric tumors are among the less common tumors seen in the oncology
clinic.
There were only 7700 children under the age of 15 reported with cancer in
1994.
Although this only represents about 1 % of the entire cancer population, much
attention is
given by oncologists to the special problems and to the social benefit
associated with
treating cancer among children. The most common solid tumor among affected
children is
3o neuroblastoma, which is of neuroendocrine origin.Other solid tumors among
children are
Wilms' tumor, rhabdomyosarcoma and retinoblastoma. Neuroblastomas represent 9%
of
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all childhood cancers, but 15% of pediatric cancer deaths. Many pediatric
tumors have a
genetic basis; for example, it is estimated that perhaps 20% of neuroblastomas
are genetic
in nature.
Neuroblastomas most commonly are seen in the abdomen, many of these being
s found in the adrenal gland. The median age at diagnosis for neuroblastoma
patients is 2
years. Surgery can be curative in early stage neuroblastomas, but most
children present
with metastatic disease. Even in children with minimal residual disease
following surgery,
recurrences are seen in more than half of all patients. Clinical trials have
emphasized the
positive value of using post-surgical chemotherapy with or without radiation
therapy.
~o Neoadjuvant chemotherapy is also commonly used with neuroblastoma patients,
but it is
more common to use chemotherapy in the adjuvant setting. Neuroblastoma is a
particularly difficult tumor to treat, and platinum-based regimens are
frequently used in
first or second-line treatments. The chemotherapeutic regimens for pediatric
patients are
highly aggressive, often involving megatherapy with 4-6 cytotoxic agents.
Neuroblastoma
is tumors represent an interesting opportunity for antiangiogenic therapy as
neuroblastomas
are highly vascular, grow quickly and metastasize rapidly. Antiangiogenic
therapy for
neuroblastoma may allow new aggressive combination chemotherapy treatments
involving
cytostatic agents since they are anticipated to provide minimal additional
toxic side effects
and should not diminish the efficacy of the cytotoxic agents to which they are
matched.
ao SUMMARY OF THE INVENTION
It is an object of this invention to provide a method for treating malignant
and
cancerous tumors using squalamine, in combination with other, conventional
cancer
treating agents. Although the invention can be applied to any responsive
tumor, in
particularly preferred aspects of the invention, the tumors treated are found
in the CNS,
as lung, breast, ovary, liver, neuroendocrine and prostate tissues.
In one method according to the invention, squalamine is used in combination
with
conventional cancer treatments to treat tumors. Such conventional cancer
treatments
include the use of cytotoxic agents as well as anti-hormonal agents. In one
embodiment,
the tumor is treated by administering an effective amount of a cytotoxic
chemical
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_g_
compound or a combination of cytotoxic compounds in a first treatment
procedure, and an
effective amount of squalamine is administered in a second treatment
procedure.
In this method, the cytotoxic chemical compound used in the first treatment
procedure is a conventional cancer treating agent. Preferable agents include a
nitrosourea,
s cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan and
irinotecan,
cannustine, estramustine, paclitaxel and its derivatives, and cisplatin,
carboplatin,
iproplatin and related platinum compounds. These conventional cancer treating
agents are
well known to those skilled in this art. Note, M.C. Wiemann and Paul
Calabresi,
"Pharmacology of Antineoplastic Agents," Medical Oncology, Chapter 10, edited
by Paul
io Calabresi, et. al., McMillan Publishing (1985). Medical Oncology is
entirely incorporated
herein by reference. One particularly preferred nitrosourea is BCNU, which
also is known
as carmustine. Another preferred cytotoxic agent is a platinum compound such
as
carboplatin, iproplatin or cisplatin, and yet another is cyclophosphamide.
Other
conventional cytotoxic chemical compounds, such as those disclosed in Medical
Oncoloev,
is supra., can be used without departing from the invention.
In another embodiment the tumor is treated by first inhibiting hormones that
affect
the tumor and then administering an effective amount of squalamine in a second
treatment
procedure. In one aspect, the hormones may be specifically inhibited. When the
tumor is
of the prostate, the hormone inhibition may result from orchiectomy, i.e., the
removal of
Zo one or both testes. Orchiectomy may result from surgery or from the
administration of
chemical agents such as LHRH (luteinizing hormone releasing hormone) agonists
and/or
anti-androgens (such as flutamide, biclutamide, nilutamide or luprolide).
The cytotoxic and anti-hormonal chemical compounds administered in the first
treatment step may be administered by any conventional technique used in the
art (i.e., oral,
zs subcutaneously, intralyrnphatically, intraperitoneally, intravenously, or
intramuscularly).
In one embodiment of the invention, the cytotoxic chemical compound
(preferably BCNU,
cisplatin, or cyclophosphamide) is administered intravenously. Likewise,
squalamine can
be administered by any conventional administration method known in the art,
such as those
mentioned above. Subcutaneous injections of squalamine one or two times a day
are used
3o in one embodiment of this invention. Intravenous administration of
squalamine one or two
times a day are used in another embodiment of the present invention.
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The first treatment procedure with the cytotoxic chemical or anti-hormonal
compound may take place prior to the second treatment procedure (using
squalamine), after
the second treatment procedure, or at the same time as the second treatment
procedure.
Furthermore, the f rst treatment procedure may be completed before the second
treatment
s procedure is initiated (or vice versa). In one embodiment of the invention,
the first
treatment procedure is a one time intravenous administration of a cytotoxic
chemical or
anti-hormonal compound (i.e., BCNL1, cisplatin, or cyclophosphamide), and the
second
treatment procedure involves daily subcutaneous injections of squalarnine.
In addition, the invention encompasses the use of squalamine together with
io cytotoxic compounds or antihormonal compounds or the use of two or more of
these
compounds with squalamine. The invention also encompasses the use of
squalamine
together with a cytostatic agent or the use of these two treatment modalities
with a
cytotoxic compound. A cytostatic agent is any chemical compound which is
capable of
arresting the growth of tumor cells or normal stromal cells in a tumor but
which is not toxic
is at pharmacologically active concentrations. A pharmacologically active
concentration of a
cytostatic agent used in the first treatment procedure may be any know cell
growth
modulator, but it is preferably the calcium pump inhibitor
carboxyamidotriazole.
In a second method for treating a tumor according to the invention, the first
treatment procedure is a radiation treatment, which may be one or more
conventional
zo radiation modalities, using a conventional radiation treatment regimen
known to those
skilled in the art. The tumor is exposed to radiation in this first treatment
procedure. In a
second treatment procedure, an effective amount of squalamine is administered
to treat the
tumor. Appropriate timing of the radiation treatment procedure with respect to
the
squalamine treatment regimen can be determined by those skilled in the art
through routine
zs experimentation in order to provide effective tumor treatment.
In addition to radiation and squalamine treatments, the tumor also may be
treated
with one or more cytoxic chemical or anti-hormonal compounds in a third
treatment
procedure. Further, in addition to radiation and squalamine treatments, the
tumor also may
be treated with one or more cytostatic chemical compounds in a third treatment
procedure.
3o The cytostatic agent used in the third threatment procedure may be any
known cell growth
modulator which is not cytotoxic, but it is preferably the calcium pump
inhibitor
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carboxymidotriazole. It is additionally envisioned in the present embodiment
of the
invention that tumors treated with radiation, squalarnine and a cell growth
modulator may
also be treated with one or more cytotoxic chemical compounds in a fourth
treatment
procedure.
s Of course, the invention also relates to the use of both cytotoxic chemical
and/or
anti-hormonal compounds in addition to radiation, or any other combination of
treatment
methods.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other advantageous features of the invention will be more fully
io appreciated when considered based on the following detailed description and
the attached
drawings, wherein:
Fig. 1 shows the general structural formula of squalamine;
Fig. 2 shows a general overview of the angiogenesis process;
Fig. 3 is a drawing used to illustrate the sodium hydrogen exchanger (NHE)
is process;
Fig. 4 illustrates the effects of conventional amilorides on inhibiting
various
isoforms of mammalian NHEs;
Figs. Sa and Sb illustrate the effect of squalamine on NHE isoform 3 (NHE3)
and
NHEI inhibition, respectively;
Zo Figs. 6a, 6b and 6c show the results of a pharmacokinetic study relating to
squalamine;
Fig. 7 illustrates squalamine distribution in various tissues after i.v.
administration;
Fig. 8 shows an angiogenesis index using squalamine as determined in the
rabbit
corneal micropocket assay;
is Fig. 9 shows the inhibitory effect of squalamine on growth of endothelial
cells as
compared to tumor cell lines;
Fig. 10 illustrates survival test results using squalamine in a glioma
lethality study
with a rat 9L glioma introduced into the brains of healthy rats;
Fig. 11 shows the survival of mice carrying human MX-I breast tumor xenografts
3o and treated with squalamine subsequent to cyclophosphamide treatment;
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Fig. 12 depicts the inhibition of a human lung adenocarcinoma (H460) in a
mouse
xenograft-combination therapy study with squalamine and cisplatin;
Fig. 13 illustrates the number of lung metastases following various
chemotherapeutic treatment procedures in mice with subcutaneous implanted
Lewis lung
s carcinomas.
Fig.l4a and 14 b illustrate the effect of squalamine, VEGF, and a combination
of
VEGF and squalamine treatment on LNCaP human prostate cell growth; Fig. 14b
illustrates the effects of squalamine, VEGF, and a combination of VEGF and
squalamine
treatment an C4-2 human prostate cell growth. In both situations, ['H]-
thymidine
io incorporation assays were utilized.
Figs. 15a and 15b illustrate the effect of squalamine on PSA levels (a) and
tumor
growth (b) in control athymic mice post-orchiectomy.
Figs. 16a and 16b illustrate the effect of squalamine on PSA levels (a) and
tumor
growth (b) in athymic mice that underwent orchiectomy and subsequent treatment
with
is squalamine.
DETAILED DESCRIPTION OF THE INVENTION
Squalamine has been recognized to have angiogenesis inhibiting activity, i.e.,
it
inhibits the formation of blood vessels. Therefore, it is believed that
squalamine, as an
antiangiogenic agent, will be effective in treating certain diseases or
ailments which depend
ao on neovascularization. For example, squalamine may be used for treating
such disparate
conditions as solid tumor cancers, macular degeneration, diabetic retinopathy,
psoriasis, or
rheumatoid arthritis, all of which require a separate and new blood flow.
In addition, squalamine can selectively inhibit certain sodium/proton
exchangers
(also called "NHEs" or "proton pumps" in this application). Several different
isoforms of
zs NHE are known to exist in mammals (i.e., NHE1, NHE2, NHE3, NHE4, and NHES).
Squalamine has been found to specifically inhibit NHE3 and not NHE 1 or NHE2.
Accordingly, squalamine may be used for treating proliferation or activation
dependent
conditions which rely on the function of NHE3, such as cancer, viral diseases,
and ischemic
reprofusion injury.
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Further studies with squalamine and NHE have demonstrated that squalamine acts
on a very specific portion of the NHE3, namely the 76 carboxyl-terminal amino
acids of the
molecule. If this portion of the NHE3 molecule is removed, squalamine has
virtually no
effect on the activity of the molecule, even though the molecule is still
active as a
s sodium/proton exchanger.
Applicants have discovered still further uses of squalamine. Specifically,
applicants
have found that squalamine in combination with conventional cancer treating
agents, i.e.,
cytotoxic chemical and anti-hormonal compounds and radiation treatments, will
decrease
the size and growth of tumors. Even more significantly, applicants have found
that the
io combination decreases the growth rate of highly proliferative CNS tumors,
lung tumors,
breast tumors, ovarian tumors, liver tumors, neuroendocrine tumors and
prostate tumors
and can confer survival advantages.
In the practice of this aspect of the invention, either a cytotoxic chemical
compound
or an anti-hormonal agent is used in a first tumor treatment procedure, and
squalamine is
is used in a second tumor treatment procedure. The first and second treatments
may be
administered in any time sequence or even simultaneously. In another
embodiment, two or
more cytotoxic chemical and/or anti-hormonal agents may be administered
simultaneously
or sequentially in the first treatment process.
The cytotoxic chemical compounds) used in the first treatment procedure may be
ao any conventional agent, but it is preferably one of the following agents: a
nitrosourea,
cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan,
irinotecan,
carmustine, estramustine, paclitaxel and its derivatives, and cisplatin
carboplatin, iproplatin
and related platinum compounds. These materials are conventional cancer
treating agents
which are known to those skilled in this art, as set forth in Medical
Oncolo~v, su~r_a. One
as particularly preferred nitrosourea is BCNU, which is also known as
"carmustine" or "1,3-
Bis(2-chloroethyl)-1-nitrosourea." Cyclophosphamide also is known as N,N-Bis-
(2-
chloroethyl)-N'-(3-hydroxypropyl)phosphordiamidic acid cyclic ester
monohydrate.
Doxorubicin also is known as adriamycin.
Well known topoisomerase inhibitors include irinotecan [7-ethyl-10-[4-(1-
3o piperidino)-1-piperidino]carbonyloxycamptothecin], also known as CPT 11,
and topotecan
[9-dimethylaminomethyl-10-hydroxy-camptothecin].
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Paclitaxel is available under the tradename "Taxol." Various derivatives of
paclitaxel may be used in accordance with the invention, such as taxotere or
other related
taxanes. Cisplatin, another of the cytotoxic chemical compounds which may be
used in
accordance with the invention, also is known as cis-Diamminedichloroplatinum.
WeII
known analogues of cisplatin are carboplatin and iproplatin (also known as
CHIP[cis--
dichloro-trans-dihydroxo-bis[isopropylamine]platinum IV). Those of ordinary
skill in the
art would be familiar with other specific cytotoxic agents that could be used
in the process
of the invention.
The anti-hormonal agent used in the first treatment procedure may be any
io conventional agent, but it is preferably an androgen inhibiting agent.
Among the preferred
androgen inhibiting agents are LHRH (luteinizing hormone releasing hormone)
agonists
and anti-androgens such as flutamide, biclutamide, nilutamide, and luprolide.
These agents
are preferred for the treatment of prostate tumors, but other anti-hormonal
agents may be
used for other tumors, as would be recognized by those skilled in the art.
is In addition, the invention encompasses the use of cytotoxic compounds
together
with anti-hormonal compounds or the use of two or more of these compounds.
Furthermore, there are no limitations on the chemotherapeutic agent that can
be
used in this invention. Other conventional chemotherapeutic agents that can be
used with
squalamine in the process of the invention include methotrexate, melphalan,
thiotepa,
ao mitoxantrone, vincristine, vinblastine, etoposide, teniposide, ifosfamide,
bleomycin,
procarbazine, chlorambucil, fludarabine, mitomycin C, vinorelbine, and
gemcitabine.
The first and/or second treatments may be administered by any suitable
technique,
such as oral, "s.q.," "i.p.," "i.m.," "i.l.," or "i.v." In this application,
the terms "s.q.," "i.p.,"
"i.m.," "i.l.," and "i.v." will be used to refer to subcutaneous
administration of squalamine
as or other substances, intraperitoneal administration of squalamine or other
substances,
intramuscular administration of squalamine or other substances, intralymphatic
administration of squalamine or other substances, and intravenous
administration of
squalamine or other substances, respectively.
In one embodiment, BCNU is delivered to a patient first as a one time
intravenous
3o dosage, and thereafter squalamine is injected s.q. twice daily. In another
embodiment,
cyclophosphamide is the cytotoxic agent. In another embodiment, cisplatin is
the cytotoxic
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agent. In yet another embodiment carboplatin is used in combination with
paclitaxel. If
appropriate, the cytotoxic chemical compound and the squalamine may be
delivered
simultaneously by a common pharmaceutical Garner (i.e., one injection
including both
squalamine and the cytotoxic chemical compound). Other appropriate
combinations of
s administration techniques may be used without departing from the invention.
Those skilled
in the art will be able to ascertain the appropriate treatment regimens,
depending on the
cytotoxic chemicals used, the dosages, etc., through routine experimentation.
The squalamine treatment procedure in accordance with the invention also may
be
used with radiation treatment (i.e., cobalt or X-ray treatment) as the first
treatment
io procedure. In this embodiment of the invention, the first treatment
procedure is a radiation
treatment, and the second treatment procedure is squalamine administration.
Radiation
treatments can proceed on a schedule in combination with the squalamine
treatments to
provide optimum results. Such scheduling of the treatment procedures can be
ascertained
by the skilled artisan through routine experimentation. Any conventional
radiation
is treatment, such as those described in Medical Oncology, supra., may be used
without
departing from the invention. In addition to radiation and squalamine
treatments, the tumor
also may be treated with one or more cytotoxic chemical or anti-hormonal
compounds in a
third treatment procedure.
The invention will be described below in terms of various specific examples
and
Zo preferred embodiments. These examples and embodiments should be considered
to be
illustrative of the invention, and not as limiting the same.
I. PHYSIOLOGICAL PROPERTIES OF SQUALAMINE
A. Antiangiogenic Activity
Squalamine has been demonstrated to be useful as an antiangiogenic agent, i.
e.,
Zs squalamine inhibits angiogenesis. Angiogenesis, the process of forming new
blood vessels,
occurs in many basic physiological processes, such as embryogenesis,
ovulation, and
wound healing. Angiogenesis also is essential for the progression of many
pathological
processes, such as diabetic retinopathy., inflammation, and malignancy (tumor
development}. In view of its antiangiogenic properties, squalamine may be used
for
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treating various ailments and conditions which depend on angiogenesis, such as
those
identified above.
Angiogenesis is a multiple step process which is schematically illustrated in
Fig. 2.
First, endothelial cells must become activated, for example, by attaching a
growth factor
s such as vascular endothelial growth factor ("VEGF") or basic-fibroblast
growth factor
("b-FGF"). The cells then move, divide, and digest their way into adjacent
tissue through
the extracellular matrix. The cells then come together to form capillaries and
lay down new
basement membrane. This angiogenesis process is illustrated in the upper
portion of Fig. 2.
Each of these development stages during angiogenesis is important and may be
affected by
io antiangiogenic agents.
Certain compounds which are believed to be antiangiogenic compounds (i.e.,
matrix
metalloproteinase inhibitors, such as minocycline, SU101 or marimistat) act at
later stages
in this multistep angiogenesis process. These compounds will be referred to as
"downstream" angiogenesis inhibitors. For a discussion of matrix
metalloproteinase
is inhibitors, please refer to Teicher, Critical Reviews in
Oncologv/Hematology, Vol. 20
(1995), pp. 9-39. This document is entirely incorporated herein by reference.
In contrast to
these known antiangiogenic compounds, squalamine acts at a very early stage in
the
process by inhibiting the cell activation action of growth factors, i.e., it
is an "upstream"
angiogenesis inhibitor. As shown in Fig. 2 (toward the bottom), squalamine
inhibits the
ao sodium-proton pumps that are normally active and activated by the growth
factors.
Inhibition of the proton pump places the cell in a quiescent state, and, in
this way, capillary
formation and angiogenesis is impeded. In effect, the growth factor signal is
aborted in the
presence of squalamine.
B. Capillary Regression Activity
zs In addition to antiangiogenic characteristics, squalamine has been shown to
have a
capillary regression effect in newly formed capillaries. A one time dose (100
ng) of
squalamine was applied to capillary beds of young chick embryos that were 2-3
days old.
After five minutes, this dose of squalamine appeared to have little effect on
the capillary
beds. In twenty minutes, however, the capillary bed appeared to be
disappearing (i.e., the
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vessels appeared to be closed off). After forty minutes, additional capillary
regression was
observed.
The capillary bed also was observed after sixty minutes. At this time, it was
noted
that some of the capillary vessels were beginning to re-appear, but only the
more major
s vessels were re-appearing. The small vessels were not re-appearing at that
time. Four to
five days after the one time squalamine treatment, the effect of the
squalamine dose was no
longer apparent, but newly formed capillaries in the embryos remained
susceptible to
squalamine induced regression for a limited time while they were newly formed.
From this test, applicants concluded that squalamine-induced capillary
regression is
io reversible, at least with respect to certain capillaries. It also was
concluded that squalamine
is more effective against small microcapillary blood vessels (i.e., the
microvascular bed) as
compared to the major blood vessels. Close histological examination of chick
microvessels
exposed to squalamine revealed vessel occlusion was due to shrinkage of
endothelial cell
volumes in cells wrapped around the vessel lumen. The applicants postulate
that occlusion
is or regression of small blood vessels by squalamine significantly
contributes to the ability of
squalamine to impede the flow of nutrients and growth factors into tumors and
thereby
slows or blocks the rate of growth of the tumors.
C. NHE Inhibitory Activity Of Squalamine
Cell growth and division is necessary for blood vessel and capillary growth
and
ao formation. Capillary formation requires a specific extracellular matrix.
The NHE
antiporter system of a cell is connected to the extracellular matrix.
Activation of the NHE
antiporter is necessary to induce cell growth, and interference with the NHE
antiporter
interrupts the matrix signal and interferes with cell growth. When endothelial
cell growth
is interrupted, capillary growth is impeded.
is The NHE antiporter of cells may be activated in different ways. For
example,
insoluble fibronectin activates the NHE antiporter by clustering and
immobilizing Integrin
a"~3,, independent of the cell shape (the growth of anchorage-dependent cells
requires both
soluble mitogens and insoluble matrix molecules). In addition, the attachment
of stimuli to
the extracellular matrix or cell attachment events involving viruses also
activate the NHE
3o antiporter.
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When activated, the NHE antiporter induces cell growth by regulating the pH of
the
cell. As shown in Fig. 3, the chloride-bicarbonate exchanger and NHE are
complementary
pH regulators in cells. The chloride-bicarbonate exchanger makes the cell
become mare
alkaline, while NHE contributes to the control of hydrogen ion concentration
in the cell.
s When the NHE is inhibited, the cells become acidic (lower pH) and growth
stops. This
does not mean that the cell dies; it means only that the cell enters a
quiescent state (i. e., it
does not divide). If the cell returns to a normal pH, growth rnay resume. When
the NHE is
activated, the cell becomes more alkaline (higher pH), it pumps out protons,
and growth
proceeds. Interaction of various modulatory factors (i.e., serum components,
secondary
io messengers, etc.) with one portion of the cytoplasmic region of NHE
activates the
antiporter, while interaction with another portion inhibits the antiporter.
These portions of
NHE are described in Tse, et al., "The Mammalian Na+/H+ Exchanger Gene Family -
Initial
Structure/Function Studies," J. Am. Soc. Nephr., Vol. 4 (1993), pg. 969, et
seq. This article
is entirely incorporated herein by reference.
is Sodium-proton pumps (NHEs) are responsive to different growth stimuli which
activate the pump. As noted above in connection with Fig. 2, the proton pump
may be
activated by attachment of growth factors (e.g., VEGF and b-FGF) to the cell.
Additionally, as shown in Fig. 3, other stimuli, such as virus attachment,
addition of
various mitogens, sperm attachment to an egg, etc, also can cause NHE
activation and
Zo alkalinization of the cell. Attachment of these stimuli to the
extracellular matrix activates
the NHE antiporter of the cell and induces cell growth.
At least five different mammalian isoforms of NHE exist, and each has a
distinct
tissue distribution. Nonetheless, all act in the same manner. NHE1 is the
antiporter found
in all tissues. NHE2 and NHE3 are more restrictive in their tissue
distribution.
as The effect of squalamine on NHE activity was measured to determine which
isoforms of NHE were affected by squalamine. NHE activity can be measured
under
various different cellular conditions. Acid loading a cell activates all of
the antiporters and
permits measurement of NHE. NHE activity also can be measured after growth
factor
stimulation of the cell. Additionally, the NHE activity can be measured when
the cell is in
3o an unstimulated state, because the antiporters, even if unstimulated,
continue to function at
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a slow, but non-zero rate. In each of these cellular conditions, NHE activity
usually is
measured in the absence of bicarbonate.
Amilorides, which are the classic inhibitors of activated NHE antiporters and
which
act as direct competitive inhibitors of Na+ ion binding to NHE, do not turn
off the
s antiporter activity in unstimulated cells. As illustrated in Fig. 4,
amiloride and amiloride
analogues specifically act against NHE1 over NHE2 or NHE3. NHE3 in particular
is
relatively resistant to inhibition by the amilorides. In contrast to the
amilorides, when
NHE1 activity was measured in unstimulated melanoma cells, applicants found
that
squalarnine substantially down regulates the activity of the antiporter.
io The following describes the test used to determine that squalamine inhibits
NHE3,
but not NHE1 or NHE2. NHE deficient fibroblast cells (PS120) transfected with
an
individual human NHE gene were loaded with a pH sensitive dye 2'T-bis(2-
carboxyethyl)-
5,6-carboxyfluorescein (BCECF). NHE activity was measured by
spectrofluorometric
methods using this dye and by amiloride sensitive isotopic zZNa+ cellular
uptake. The cells
is were acidified by exposure to ammonium chloride in the absence of sodium to
eliminate
sodium and deactivate the proton pumps. The ammonium chloride was washed out
by
exposing the cells to tetramethyl ammonium chloride in bicarbonate free
medium. The
cells were consequently acidified, but in the absence of sodium, the NHE ion
pumps did
not activate. For this test, as shown in Figs. Sa and Sb, 7 pg/ml of
squalamine was added to
zo the cells in each case. Sodium then was added back at various
concentrations (see the
abscissa of Figs. Sa and Sb) to drive the antiporters (human NHE3 in Fig. Sa
and human
NHE1 in Fig. Sb). The antiporters were driven at different rates, as evidenced
by the
cellular pH change rate, depending on the amount of sodium added. As shown in
Fig. Sa,
when measuring the effect of squalamine on the human NHE3 antiporter, the pH
change
Zs rate was lower in the squalamine treated cells than the pH change rate in
the control group
(without squalamine). This indicates that squalamine inhibits human NHE3. In
Fig. Sb,
however, there is no effective difference in the pH change rate between the
squalamine
treated samples and the control when measuring the human NHE1 antiporter. From
these
tests, applicants concluded that squalamine inhibits human NHE3, but not human
NHE 1.
so Additionally, in similar tests, it was found that rabbit NHE1 and NHE2 are
not affected by
squalamine, but rabbit NHE3 is inhibited by squalamine treatment.
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In the transfected cells used in this test, it took at least 30 minutes before
the NHE3
inhibition effect induced by squalamine was observed. Thus, squalamine did not
act like
the classic NHE inhibitor amiloride or analogues of amiloride, which are
direct competitive
inhibitors for sodium and, therefore, act rapidly as NHE inhibitors.
Furthermore, it was
s observed that the NHE inhibiting effect of squalamine occurred in the
absence of lactase
dehydrogenase (LDH) leakage from the cell. Because LDH leakage is a non-
specific
marker of cytotoxicity, it was concluded that squalamine does not have a
general cytotoxic
effect.
This NHE3 inhibiting activity of squalamine has been mapped to the 76 C-
terminal
io amino acids on the NHE3 molecule. If the 76 C-terminal amino acids of
rabbit NHE3 are
removed from the molecule, squalamine has been found to have virtually no
effect on the
activity of the molecule, while the molecule remains active as a
sodium/hydrogen
exchanger. Thus, the 76 C-terminal amino acids of NHE3 are the site of
inhibition by
squalamine. It is believed that the squalamine effect on these accessory
proteins of NHE3
is is tied to an inhibitory effect on tyrosine kinase-dependent activity,
although applicants do
not wish to be bound by any specific theory of operation.
As noted above, it has been concluded that squalamine inhibits NHE3 and not
NHE1. This inhibitory effect of squalamine, however, has been found to work in
a manner
different from classical and known NHE3 inhibitors. In contrast to squalamine,
other
zo inhibitors of NHE3 (e.g., amiloride, amiloride analogues, genestein,
calmodulin, and
protein kinase C) also inhibit NHEI. Such inhibitors affect only the absolute
number of
protons that can be secreted by the cell (i.e., "Vm~"), if one looks at the
kinetic
characteristics of the inhibition. Squalamine, on the other hand, not only
inhibits Vm~, but
it also forces the cell to fall to a lower pH, as evidenced by a reduction in
the Km value.
zs Note the following Table l, which correlates to data collected in the test
of Fig. Sa.
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Table 1
Squalamine (7~ug/ml) Control
Km 0.338 0.595
n 1.88 1.22
5 Vmax 1282 2958 -'
Thus, squalamine inhibits NHE with nonallosteric kinetics (i.e., nonclassical
allosteric
inhibition). In additional tests, it also was found that squalamine (at a 1
hour pretreatment)
decreased the Vm~ of rabbit NHE3 in a concentration dependent manner (13%,
47%, and
57% with 1, 5, and 7 ~tg squalamine/ml, respectively). This observed
squalamine effect on
~o the V,"ax was time dependent, with a maximum effect occurring at one hour
exposure. The
observed effect was fully reversible within three hours after removing the
cells from the
medium.
In view of the test results relating to the effect of squalamine on NHE3,
applicants
believe that NHE3 is important in maintaining homeostasis of the unstimulated
cell. The
is applicants further believe that prevention of cellular activation by
squalamine, especially
activation of endothelial cells or precursor cells which participate in
formation of new
blood vessels during pathophysiological vascularization (such as during tumor
growth), is
the mechanism through which squalamine inhibits tumor growth.
Applicants have further observed that squalamine changes endothelial cell
shape.
zo This suggests that transport proteins which control cell volume and shape
may be a
squalamine target.
Additional testing of squalamine has indicated that squalamine inhibited brush
border membrane vesicle (BBMV) NHE only when the tissue was pretreated with
squalamine (51 % inhibition at 30 minutes exposure). Direct addition of
squalamine to
zs PS 120 fibroblasts during measurement of the exchanger activity had no
effect.
D. Pharmacokinetic Studies Of Squalamine
A pharmacokinetic study of squalamine was performed to ascertain the residence
time of squalamine in the body. Figs. 6a to 6c illustrate the test results
where squalamine
was administered subcutaneously (50 mg/kg, Fig. 6a), intraperitoneally (dose
240 p.g; 10
3o mg/kg, Fig. 6b), and intravenously (10 mg/kg, Fig. 6c). The half life of
squalamine when
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given intravenously (Fig. 6c) was acceptable (35 minutes), but it was even
higher when it
was administered intraperitoneally (Fig. 6b, half life =172 minutes) and
subcutaneously
(Fig. 6a, half life = 5.6 hours).
In addition to these squalamine half life tests, applicants have tested to
ascertain the
s distribution of squalamine in a mouse after intravenous administration. Fig.
7 illustrates
the distribution of squalamine in mouse tissue two hours after i.v.
administration. Some
squalamine is contained in most of the tissues, with most of the squalamine
concentrating
in the liver and the small intestine. The test results shown in Fig. 7
indicate good
squalamine distribution. Notably, however, not much squalamine is present in
brain tissue.
io From this, applicants conclude that squalamine probably does not cross the
brain/blood
barrier. In treating brain tumors, it is believed that the squalamine acts on
the endothelial
cells in the brain, and in this way, it need not cross the brain/blood barrier
The following examples describe more detailed experiments used to test the
antiangiogenic characteristics of squalamine in the process of the invention.
is II. THERAPEUTIC ADMINISTRATION AND COMPOSITIONS
The mode of administration of squalamine may be selected to suit the
particular
therapeutic use. Modes of administration generally include, but are not
limited to,
transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal,
inhalation, intralymphatic, intralesional, and oral routes. The squalamine
compounds may
ao be administered by any convenient route, for example, by infusion or bolus
injection, or by
absorption through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal, and
intestinal mucosa, etc.), and it may be administered together with other
biologically active
agents. Administration may be local or systemic.
The present invention also provides pharmaceutical compositions which include
zs squalamine as an active ingredient. Such compositions include a
therapeutically effective
amount of squalamine and a pharmaceutically acceptable carrier or excipient.
Examples of
such a carrier include, but are not limited to, saline, buffered saline,
dextrose, water, oil in
water microemulsions such as Intralipid, glycerol, and ethanol, and
combinations thereof.
The formulation of the pharmaceutical composition should be selected to suit
the mode of
so administration.
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The pharmaceutical composition, if desired, also may contain effective amounts
of
wetting or emulsifying agents, or pH buffering agents. The pharmaceutical
composition
may be in any suitable form, such as a liquid solution, suspension, emulsion,
tablet, pill,
capsule, sustained release formulation, or powder. The composition also may be
s formulated as a suppository, with traditional binders and carriers, such as
triglycerides.
Oral formulations may include standard carriers, such as pharmaceutical grades
of
mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium
carbonate, etc.
Various delivery systems are known and may be used to administer a therapeutic
ro compound of the invention, e.g., encapsulation in liposomes,
microparticles, enteric coated
systems, microcapsules, and the like.
In one embodiment, the pharmaceutical composition is formulated in accordance
with routine procedures to provide a composition adapted for intravenous
administration to
humans. Typically, compositions for intravenous administration are solutions
in 5%
is dextrose and sterile water or Interlipid. Where necessary, the
pharmaceutical composition
also may include a solubilizing agent and a local anesthetic to ameliorate
pain at the site of
an injection. Generally, the ingredients of the pharmaceutical composition are
supplied
either separately or mixed together in unit dosage form, for example, as a dry
lyophilized
powder or water-free concentrate in a hermetically sealed container such as an
ampoule or
zo sachette indicating the quantity of active agent. Where the pharmaceutical
composition is
to be administered by infusion, it may be dispensed with an infusion bottle
containing
sterile pharmaceutical grade water, dextrose, saline, or other
pharmaceutically acceptable
earners. Where the pharmaceutical composition is administered by injection, an
ampoule
of sterile water or saline for injection may be provided so that the
ingredients may be
zs mixed prior to administration.
The amount of the therapeutic compound (i.e., active ingredient) which will be
effective in the treatment of a particular disorder or condition will depend
on the nature of
the disorder or condition, and can be determined by standard clinical
techniques known to
those skilled in the art. The precise dose to be employed in the formulation
also will
3o depend on the route of administration and the seriousness of the disease or
disorder, and
should be decided according to the judgement of the practitioner and each
patient's
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circumstances. Effective therapeutical doses may be estimated from
extrapolations of
dose-response curves derived from in vitro or animal-model test systems.
Suitable dosages for intravenous administration are generally about 1
microgram to
40 milligrams of active compound per kilogram body weight. Suitable dosage
ranges for
s intranasal administration are generally about O.Oi mg/kg body weight to 20
mg/kg body
weight. Suitable dosages for oral administration are generally about 500
micrograms to
800 milligrams per kilogram body weight, and preferably about 1-200 mg/kg body
weight.
Suppositories generally contain, as the active ingredient, 0.5 to 10% by
weight of
squalamine. Oral formulations preferably contain 10% to 95% active ingredient.
io For use of squalamine as an antiangiogenic or cytotoxic agent or in cancer
therapies, exemplary dosages are from about 0.01 mg/kg body weight to about
100 mg/kg
body weight. Preferred dosages are from 0.1 to 40 mg/kg body weight.
The invention also may include a pharmaceutical pack or kit including one or
more
containers filled with the pharmaceutical compositions in accordance with the
invention.
is Associated with such containers may be a notice in the form prescribed by a
government
agency regulating the manufacture, use or sale of pharmaceuticals or
biological products,
which notice reflects approval by the agency of manufacture, use or sale for
human
administration.
The conventional cytotoxic chemical and anti-hormonal compounds used in
ao accordance with the invention may be present in any suitable form known to
those skilled
in the art. These chemical compounds also may be administered by any suitable
means
also known to those skilled in this art, such as orally, subcutaneously,
intravenously,
intraperitoneally, intralymphaticly, and intramuscularly.
In describing the invention, applicants have stated certain theories in an
effort to
is disclose how and why the invention works in the manner in which it works.
These theories
are set forth for informational purposes only. Applicants are not to be bound
to any
specific chemical or physical mechanisms or theories of operation.
While the invention has been described in terms of various specific preferred
embodiments and specific examples, those skilled in the art will recognize
that various
3o changes and modifications can be made without departing from the spirit and
scope of the
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invention, as defined in the appended claims. All patents, publications and
references
referred to throughout this application are herein incorporated by reference
in their entirety.
EXAMPLE 1
Rabbit Corneal Micropocket Assay
s In determining whether a compound is antiangiogenic, the rabbit corneal
micropocket assay is an accepted standard test. In this test, an incision is
made in one
rabbit cornea, and a stimulus is placed in the incision. The stimulus is used
to induce blood
vessel formation in the normally avascular corneal region. As one example, a
solid tumor
in a polymeric matrix can be placed in the cornea as the stimulus because the
tumor will
io release a number of angiogenic growth factors to stimulate new capillary
growth. The
tumor-derived angiogenic growth factors stimulate the endothelial cells at the
scleral
junction in the eye to initiate blood vessel growth toward the stimulus. A
second polymer
pellet (e.g., an ethylene/vinyl acetate copolymer) is placed between the
scleral junction and
the stimulus. This polymer pellet is either empty (a negative control test
pellet), or it
is contains a compound whose antiangiogenic characteristics are to be tested.
The polymer
pellet is used to provide a controlled release of the material to be studied.
Because of the
avascular cornea background in the rabbit cornea, one can visually assess the
results
qualitatively. In addition, the number of blood vessels can be counted and
their length,
etc., can be measured to provide a more quantitative evaluation of the
results.
Zo The VX2 rabbit carcinoma was implanted in 26 rabbit eyes, in the normally
avascular corneal region, to act as an angiogenesis stimulus. Squalamine was
incorporated
into a controlled release ethylene/vinyl acetate copolymer (20% squalamine and
80%
polymer by weight). The loaded polymer pellets were placed in 13 of the
corneas to
provide a sustained local release of squalamine. Polymer blanks were provided
in the
as remaining 13 eyes as a control. In this manner, one eye of each rabbit
served as the
squalamine test eye and the other eye of the same rabbit served as the control
eye. The
eyes were examined weekly using a slit lamp stereomicroscope for three weeks
after tumor
implantment, and the Angiogenesis Index ("AI") was calculated (this
calculation will be
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described in more detail below with reference to Fig. 8). The squalamine
loaded polymer
was found in vitro to release active squalamine throughout the treatment
period. After the
test, the corneas were examined histologically.
Using this test, squalamine was found to be a potent inhibitor of tumor
induced
s capillary formation. Fewer blood vessels were observed in the cornea treated
with
squalamine as compared to the control cornea, and these vessels were generally
shorter
than the vessels in the control cornea.
Some of the corneas were then sectioned to observe the effect of squalamine on
the
tumor cells themselves. The untreated control corneas had many vessels in and
adjacent to
io the tumor. The tumors in the squalamine-treated corneas were still viable
(i.e., the tumors
were not dead), but there was essentially no vasculature associated with those
tumors.
Thus, the squalamine-treated tumors had greatly diminished vascularity as
compared to the
corresponding control tumor sections. These findings suggested that squalamine
works
against the blood vessels, and not against the tumor itself.
is Fig. 8 shows a graphical representation of the results of the rabbit cornea
micropocket assay test. To provide a quantitative evaluation, the Angiogenesis
Index
("AI") of each eye was determined. To determine the Angiogenesis Index, first
the vessel
density ("D~esse~") in an eye was graded on a 0-3 scale as follows:
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Table 2
Dvessel Value Determinations
D,,~sse~ Value Visual Observation
0 No vessels present
1 1-10 vessels present
2 > 10 vessels present, but
loosely packed
3 > 10 vessels present, packed
densely
The vessel length ("Lessen') was then measured in each cornea. The vessel
length is
the length of the longest vessel measured from the cornea-scleral junction to
the distal edge
io of the longest vessel growth. The Angiogenesis Index then is determined
from these
measurements by the following equation:
~ = Dvessel x Lvessel~
Figure 8 shows the mean Angiogenesis Index for each group of corneas
(squalamine
treated and untreated) in the rabbit cornea micropocket assay after 1, 2, and
3 weeks. As
is shown in the figure, squalamine was very inhibitory to the growth of new
blood vessels.
The squalamine treated eyes showed a significantly reduced AI value as
compared to the
untreated eyes (37% reduced at Day 14 (p=0.05, Wilcoxon rank sum test) and 43%
reduced
at Day 21 (p<0.01)). This data illustrates that squalamine inhibits tumor
induced growth of
new blood vessels or capillaries over a long time period. More specifically,
squalamine
zo exhibits high antiangiogenic activity even after three weeks.
EXAMPLE 2
Squalamine Does Not Cause Inflammation
In the rabbit corneal micropocket assay test, if the rabbit cornea becomes
inflamed,
this inflammation can lead to the formation of new blood vessels in the
cornea. Such
as inflammation would skew the test results. Therefore, tests were conducted
to determine
whether squalamine, in and of itself, was responsible for any inflammatory
response in the
cornea. Several non-bioresorbable ethylene/vinyl acetate copolymer pellets
were loaded
with different concentrations of squalamine, namely, 2%, 10%, and 20%
squalamine, by
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weight. These pellets were then placed in rabbit corneas which did not include
an
angiogenic stimulus. Squalamine did not induce inflammation at any of these
concentrations. Thus, squalamine does not lead to the generation of new blood
vessels by
inflaming the cornea.
EXAMPLE 3
Squalamine Use In Brain Tumor Treatment
The rabbit corneal micropocket assay test results suggested to applicants that
squalamine may be a potent antiangiogenic agent that inhibits
neovascularization.
Recognizing that the exponential growth of solid tumors in the brain is
dependent on
io neovascularization, applicants assessed the activity of squalamine in an
animal model on
the growth of solid tumors in the brain.
Of solid brain tumors, malignant gliomas are the most common form of cancerous
tumors. These tumors are the third leading cause of death from cancer in young
adults
between the ages of 15 and 34. Malignant gliomas are characterized by their
ability to
is induce the normally quiescent brain and/or CNS endothelial cells into a
highly proliferative
and invasive state. The gliomas express vascular endothelial growth factor
("VEGF") and
other growth factors which stimulate inducible receptors on CNS endothelial
cells in a
paracrine manner (i.e., the VEGF originates from the tumor cell and stimulates
the
endothelial cells). The CNS endothelial cells subsequently initiate angiogenic
invasion and
Zo thus provide nourishment of the glioma. Applicants tested the
antiangiogenic activity of
squalamine against gliomas by testing (1) its ability to selectively inhibit
VEGF-mediated
stimulation of endothelial cells and (2) its effect against experimental
marine glial tumors.
In vitro tests were first performed to determine that squalamine acts
specifically on
endothelial cells. Applicants used endothelial cells because such cells are
involved in the
zs early steps of angiogenesis, as described above in conjunction with Fig. 2.
Specifically,
tumor angiogenesis is a series of sequential and overlapping steps. First, the
endothelial
cells activate and proliferate. Then, proteolytic enzymes are produced and the
cells
migrate. New basement membranes must then be generated. In this manner, new
blood
vessels are generated and tumor size increases.
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In conducting this in vitro analysis, the following cell lines were tested:
(a) bovine
retinal endothelial cells; (b) 9L and C6 rat glioma cells; (c) human H80
glioma cells; and
(d) VX2 rabbit carcinoma cells (the same type as the tumors implanted in the
rabbit corneal
micropocket assay test described above). The endothelial mitogen which was
used in this
s analysis was VEGF at a concentration of 20 ng/ml.
The cells were allowed to attach overnight to tissue culture plates containing
an
optimized growth media. Following attachment, the cells were exposed to
solvent only or
to increasing concentrations of squalamine (0, 10, 20, 30, 60, and 90 pg
squalamine/ml).
Cell growth was counted daily for three days using a Coulter Counter. A total
of 10,000
io cells per well were plated and each experimental concentration was tested
in quadruplicate.
The results were then averaged. The bovine retinal endothelial cells were
grown and
treated in an identical manner to the other cell lines, except that the growth
of these cells
was measured after the addition of 20 ng/ml of human recombinant VEGF to the
cells prior
to the squalamine treatment.
is Cell proliferation by all tumor lines and by endothelial cells not treated
with VEGF
was statistically unaffected after exposure for 24 and 48 hours to squalamine
concentrations
up to 30 wg/ml. Growth of the VEGF-stimulated endothelial cells, however, was
significantly reduced by squalamine at these same times in a concentration
dependent
manner. Percentage endothelial cell growth inhibition (%I) was determined by
the
ao following equation:
(# of cells in control sample - #of cells in experimental samplel x 100 = %I
(# of cells in control sample)
The following Table shows the results at 48 hours for the VEGF-stimulated
endothelial cell
line.
as
Table 3
Percent Inhibition Data
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Squalamine Conc. % Inhibition (average)
10 Etg/ml 38% (p<0.01)
20 pg/ml 57% (p<0.001)
30 p,g/ml 83% (p<0.001)
Additional data is illustrated in Fig. 9. This figure shows the growth of the
various
cell lines as a percentage of the growth imthe control groups for in vitro
administration of
squalamine at 30 ~,g/ml after 1, 2, and 3 days. As shown in Fig. 9, growth is
reduced for
the VEGF-stimulated endothelial cells specifically, while the growth in the
other cell lines
(H80, C6, and VX2) is not dramatically affected.
io Based on this information, applicants concluded that squalamine
dramatically and
specifically inhibits VEGF-stimulated growth of endothelial cells in vitro.
Thus,
squalamine is a potent inhibitor of tumor-induced angiogenesis, and this
effect appears to
be precipitated through specific inhibition of endothelial cell proliferation
induced by
VEGF. Thus, squalamine is believed to be well suited for reducing or
diminishing the
is neovasculature induced by tumors for use in tumor specific antiangiogenic
therapy.
In addition to inhibiting VEGF-stimulated growth of endothelial cells,
squalamine
also has been found to interfere with growth stimulation in human brain
capillary
endothelial cells induced by b-FGF, PDGFbb, scatter factor (HGF or hepatocyte
growth
factor), conditioned tumor media, and human brain cyst fluid. Thus, as the
tumor puts out
zo a variety of different growth factors, squalamine has an inhibitory effect
on several.
In view of these test results, applicants tested squalamine in an animal model
for
brain cancer. To test the effect of squalamine on tumors located in the brain,
small sections
(1 mm3) of existing rat gliomas were taken from rat flanks where they were
being
maintained and were implanted into the rat brains in two groups of rats. Thus,
in this
zs model, the tumors were viable when placed in the rat brain. Three days
after implantation,
and after some vasculature had developed, treatment with 20 mg/kg/day of
squalamine
(i.p.) was initiated in one group of rats. The control animals ("vehicle
control" in Fig. 10)
were given the Garner vehicle only (no squalamine), and the other animals were
treated
with squalamine ("Squalamine" in Fig. 10). As shown in the figure, the animals
treated
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with squalamine had a 38% increase in mean survival time (~ = 24.9 days v. X
=18.0 days).
Fig. 10 further illustrates that in this animal model, the squalamine treated
rats, in general,
had an increased survival time.
A squalamine toxicity test was performed in another animal model. Conventional
s cytotoxic chemical compounds are quite toxic. For example, BCNU, which is a
conventional chemotherapy agent, has a cumulative toxicity effect. For this
reason, it is
administered only one time to a patient. The use of BCNLJ is described on
pages 304 and
305 of Calabresi in Medical Oncology, supra. In order to test the toxicity of
squalamine, a
group of rats was given a daily squalamine dose of 20 mg/kg/day (i.p.) for
more than 30
io days and maintained for up to 200 days following dosing. The animals in
this study
remained healthy. This result indicates that squalamine has little or no
toxicity.
EXAMPLE 4
Squalamine Use With Conventional Cancer Treatments
As described above, squalamine is an upstream inhibitor of the angiogenesis
is process by inhibiting the activation of endothelial cells after growth
factor interaction.
Because of its angiogenesis inhibiting properties, squalamine has been
demonstrated to be
effective in treating solid tumors which rely on neovascularization to
proliferate.
Applicants tested to determine whether beneficial results could be obtained
when treating
tumors by combining a squalamine treatment (an upstream angiogenesis
inhibitor) with a
zo conventional cancer treatment using an alkylating agent.
a. The Squalamine 9L Glioma Flank Study
Four groups of rats (twenty total Fisher 344 rats, 200 g) were given s.q.
transplants
of I mm3 9L gliosarcoma tumors (9L glioma) on Day 0. The tumors were implanted
in the
rat flanks to avoid complications relating to adequate brain levels of
squalamine.
zs Randomization and treatment began on Day 5 according to the following
scheme:
Table 4
Treatment Conditions
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Group Treatment
No.
1 Saline (control group)
2 One time dose of 14 mg/kg BCNU given i.p.
on Day 5
3 Squalamine - 20 mg/kg given s.q. B.LD.'
One time dose of 14 mg/kg :BCNU given i.p.
s 4 on Day 5 and
daily injection of squalamine - 20 mg/kg
given s.q. B.LD-
beginning on Day 5.
The term "B.LD." means that the component is administered twice a day (10
mg/kg
given at two different times each day).
On Day 25 or 26 after tumor implantation, the tumor size was measured
directly.
The tumor size (i.e., its volume "V") was estimated based on volumetric
calculations
io determined from the measured length ("L"), width ("W"), and height ("H") of
the tumor
(Vtumorspheroid ~ 0.5 x L X W x H). Table 5 summarizes the results. The tumor
volumes
shown in Table 5 represent the mean tumor volumes for each treatment group for
those
animals that survived to the end of the experiment.
Table 5
is Tumor Volumes
Grou No. No. of AnimalsMean Tumor volume
Reduction
p 3 (based on control
(mm
)
volume)
1 5 18,324 -
2 6 2,547 86.1
3 5 3,347 81.7%
204 4 38 99.8%
Table S illustrates the advantageous results achieved when treating tumors
with the
combination of squalamine and the nitrosourea BCNU (Group 4). A 99.8% reduced
mean
tumor size was observed when treating with both squalamine and BCNU in this
group.
Table 5 further shows that squalamine alone (Group 3) was effective in
treating the tumor.
is The tumor size was reduced by 81.7% in Group 3, as compared to the control
group.
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Applicants conclude that the use of squalamine in combination with
conventional
cytotoxic chemical compounds can slow or halt the spread of brain cancers. The
tumor
itself shrinks and becomes necrotic. It is expected that combined squalamine
and cytotoxic
chemical treatment will extend survival. Thus, this treatment potentially will
allow
s management of brain cancers.
b. Squalamine Use in Breast Tumor Treatment
The human MX-1 breast cancer line has previously been used to document in vivo
activity of cyclophosphamide and other cytotoxic chemotherapeutic compounds
either as
single agents or in combination (T. Kubota, et al., Gann 74, 437-444 (1983);
E. Kobayashi,
io et al., Cancer Research 54" 2404-2410 (1994); M.-C. Bissery, et al.,
Seminars in Oncoloev
22 (No. 6, Suppl. 13), 3-16 (1995)). These documents each are entirely
incorporated herein
by reference. Squalamine was examined as adjunctive therapy following a single
200
mg/kg dose of cyclophosphamide. The cyclophosphamide was injected on day 14
following implantation of the tumor, at a time when the tumors measured 65-125
pQ. The
is cyclophosphamide caused partial regression in all animals and complete
regression in a
small fraction of the animals. The animals were then randomized to three
treatment arms
(each n=27): vehicle dosing only (Intralipid); squalamine given 10 mg/kg/day
in Intralipid;
and squalamine given 20 mg/kg/day in Intralipid for five days a week. Animals
whose
tumors exceeded 2 grams at any time during the experiment were euthanized. The
zo expenment was continued for 90 days after initiating squalamine treatment
to ensure that
only mice experiencing long-term cures were still alive. The high dose
squalamine was
discontinued after five weeks of treatment because of animal weight loss and
potential
toxicity concerns, so these animals did not receive squalamine for the last
eight weeks of
the experiment. The low dose squalamine treatment produced a significant
(P<0.01 )
Zs inhibition in the rate of progression of the breast tumors at all times
examined (Fig. 11 ).
The high dose squalamine treatment produced significant (P<0.05) delay in
progression of
the breast tumors only at 30 days post-initiation (i.e., only while squalamine
was still being
given), but high dose squalamine also doubled the long-term cure rate in these
animals
compared to controls which received cyclophosphamide alone (Fig. 11).
Examination of
so the history of the long-term cure animals which received cyclophosphamide
and high dose
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squalamine revealed that the additive effects of squalamine were manifested
within two
weeks after starting squalamine treatment.
c. Squalamine Use in Lung Tumor Treatment
Studies in a nude mouse xenograft model of lung cancer have been carried out
using
s several human lung cancer lines which differ in their growth rate. The data
collected show
that squalamine has synergistic activity in combination with cisplatin (e.g.,
Fig. 12). The
experimental lung cancer model design involves subcutaneous injection of 5 x
106 tumor
cells followed by a single injection of the chemotherapeutic drug on day 3 or
4. Daily
intraperitoneal squalamine injections with 20% Intralipid as a vehicle began
the following
io day for some groups of mice and continued until the experiment was
terminated 7-14 days
later. Groups of mice receiving squalamine alone started receiving the
aminosterol on the
same day as aminosterol treatment in the combination chemotherapy groups.
Tumor
volumes were then determined at termination of the experiment and compared. It
was
found for both the aggressively growing H460 human lung adenocarcinoma line
and for the
is more slowly growing Calu-6 human lung adenocarcinoma line that squalamine
had
minimal effects on tumor growth as a monotherapeutic agent when started on day
4 or 5,
but could contribute to growth inhibition if it were started on day 1.
However, when used
starting on day 4 or 5, in combination with cisplatin, given at or near a
maximum tolerated
dose, squalamine significantly and reproducibly improved tumor growth
inhibition over
ao cisplatin alone in a dose-dependent fashion for both the H460 and Calu-6
cell lines.
d. Squalamine Use in Metastatic Lung Cancer
The murine Lewis lung adenocarcinoma was implanted subcutaneously in the hind-
leg of male C57BL/6 mice and allowed to grow for one week. Groups of mice were
then
left untreated or treated with either squalamine (20 mg/kg/day, s.c.),
cyclophosphamide
is (125 mg/kg, i.p. on days 7, 9 and 11), cisplatin (10 mg/kg, i.p. on day 7),
the combination
of squalamine and cyclophosphamide, or the combination of squalamine and
cisplatin. On
day 20, the animals were sacrificed, and the mean number of lung metastases
were
determined for each group. All treatments reduced the number of metastases;
however, the
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most effective treatments were the combination of squalamine with either of
the cytotoxic
agents (Fig. 13).
EXAMPLE 5
In Vitro Studies of Squalamine _
s on Human Prostate Tumor Cell Growth
Prior to in vivo studies, applicants evaluated the therapeutic potency of
squalamine
on human prostate cancer cells in vitro.
The anti-proliferative effect of squalamine was evaluated in tissue culture by
both
crystal violet staining and [3H]-thymidine incorporation, on the LNCaP/C4-2
human
io prostate cancer progression model. Developed by applicants' laboratory,
this lineage-
derived cell line recapitulates the progression of human neoplastic prostate -
disease from an
androgen-dependent and minimally metastatic condition (LNCaP cells) to an
androgen-
independent (defined as being able to proliferate in castrate hosts) and
highly aggressive
state (C4-2 subline). The LNCaP/C4-2 progression model has been previously
is demonstrated to be an effective model for screening the efficacy of
therapeutic agents for
prostate cancer.
By itself squalamine had a nominal effect on the growth of either the parental
LNCaP or lineage-derived C4-2 cells. However, as demonstrated in Fig. 14, with
the
addition of exogenous VEGF (supplied by Genitech Corporation, South San
Francisco,
Zo CA), both LNCaP (Fig. 14a) and, to a greater extent, C4-2 proliferation
(Fig. 14b) was
inhibited. Visually, the outcome appeared to result from tumor cell
destruction, rather than
from decreased growth. Later studies (data not shown) suggest that this effect
is both time-
and dose- dependent, with increasing amounts of and exposure to VEGF yielding
enhanced
results. Furthermore, this synergistic effect appears to be predicated on an
initial exposure
zs to VEGF, followed by subsequent exposure to squalamine, and not the reverse
(data not
shown).
EXAMPLE 6
In Vivo Studies on the Therapeutic Effect of Squalamine
on Prostate Tumor Growth and Dissemination
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in a Human Prostate Cancer-Mouse Xenograft Model
LNCaP cells admixed with MatrigelTM were implanted subcutaneously in 52
athymic mice. Those that developed measurable tumors and sufficient elevations
in serum
prostate specific antigen ("PSA") (measured using a standard commercially
available kit
s such as from Abbott Diagnostics (St. Louis, MO)} underwent orchiectomy.
Subsequent to
castration, these animals were distributed among the four groups ( 1 -4)
outlined below:
and an additional control cohort of mice which were castrated but never
treated with
squalamine.
In the majority of animals, PSA levels fell to zero at time of castration. By
three
io weeks they began to rebound and continued to rise steadily until the
animals succumbed or
were sacrificed. In these same mice, tumor size tended to plateau at time of
orchiectomy,
before beginning to increase in size correspondingly with PSA rebound. The
serum PSA
curve and tumor volume measurements from two members of this last control
cohort are
illustrated in Fig. 15a and b, respectively.
is Ten mice began treatment with squalamine concomitant with the fall in PSA
after
castration (Group 1). Of these, three never experienced a post-orchiectomy PSA
nadir, and
one experienced an early death, of unknown etiology. Six animals, however,
were treated
with squalamine (at 20 mg/kg/day) for a range of six to ten weeks. These
animals began
squalamine treatment at the time of castration, or during the fall in PSA post-
orchiectomy.
ao All of these animals experienced a post-orchiectomy PSA nadir of less than
I ng/dI, and
maintained this negligible level for the duration of squalamine therapy. Tumor
masses
tended to diminish and, eventually, disappear, contrary to the response in
control animals.
The serum PSA levels and tumor volume measurements from two representational
members of Group 1 are illustrated in Fig. 16 a and b, respectively. The
response of
as animals that were treated with squalamine after the emergence of PSA
rebound (Groups 2-
4) was similar to the control, non-squalamine heated cohort, (Fig. 15) and is,
therefore, not
graphed.
EXAMPLE 7
Squalamine activity in combination with hormonal ablation
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Human androgen dependent LNCaP cells (2 x 106 cells/flank in both flanks) were
implanted subcutaneously with an equal volume of MatrigelTM in forty athymic
mice.
These animals were followed with periodic bleedings until serum human PSA
levels
exceeded 100 ng/mL and tumor volume measurements exceeded 1000 mm'
(approximately
s six weeks after implantation). All animals were then surgically castrated
and were
randomized into five treatment groups with 8-10 animals per group. One group
of animals
was not treated further (controls), while a second group immediately began
daily
intraperitoneal injection of 20 mg/kg/day squalamine (five days per week with
a two day
respite). A third group of animals began to receive squalamine (20 mg/kg/day
squalamine,
io five days per week with a two day respite) when their serum human PSA
levels reached
values of 4-10 ng/mL. A fourth group of mice began squalamine treatment (20
mg/kg/day
squalamine, five days per week with a two day respite)when serum human PSA
values
reached 20-40 ng/mL, and a fifth group began squalamine treatment when serum
human
PSA levels reached 100 ng/mL.
is It was found that the human tumor xenografts responded to castration by a
slowing
or cessation of tumor growth following castration for 2-4 weeks before the
tumors began
growing again. Similarly, the serum human PSA levels fell to undetectable
levels with a
nadir at two weeks after castration, but by week three the serum human PSA
levels began
to rise in all control animals. For animals that were treated with squalamine
after the
Zo emergence of PSA rebound, their response was similar to that of the
control, non-
squalamine treated animal cohort. For animals treated with squalamine
concomitant with
the fall in human PSA after castration, six of ten animals were treated for 6-
10 weeks.
Three mice in this group never experienced a post-orchiectomy PSA nadir, and
one mouse
experienced an early death of unknown etiology. The remaining six animals in
this group
is experienced a post-orchiectomy PSA nadir of less than 1 ng/mL and
maintained this
negligible level for the duration of squalamine therapy. Tumor masses diminish
with time
and, eventually disappeared in all six animals.
Tissue histology was carried out on the tumor masses (or the site of tumor
implantation, in the instance of the animals which underwent successful
squalamine
3o treatment} to examine the distribution of integrins in these tissues. One
notable finding
was that successful squalamine treatment coincided with observation of a
reduction in the
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expression of the integrin a"[i3 and an increase in the integrin a6[i4. The
integrin a,,(33 has
been associated with a greater propensity for increased angiogenesis in tumors
and for a
tumor to grow and metastasize (e.g., cf. B.P. Eliceiri and D.A. Cheresh, J.
Clin. Invest. _1Q,
1227-1230 (1999)). Thus, part of the mechanism by which squalamine
successfully alters
s LNCaP tumor growth is by interfering with cellular integrin expression that
is associated
with tumor aggressiveness.
In vitro experiments were also conducted with the LNCaP cell line in which
cell
proliferation as judged by [3HJ-thymidine uptake was determined. LNCaP cells
were
maintained as previously described (cf. J.T. Hsieh et al., Cancer Research 53,
2852-2857
io (1993)). Cells were exposed to 20 ng/mL vascular endothelial growth factor
(VEGF), 20
pg/mL squalamine or a combination of VEGF and squalamine. It was found that
VEGF or
squalamine alone had minimal effects on cell growth in media containing serum,
but that
the combination of VEGF and squalamine after 24 hours of exposure reduced [3HJ-
thymidine uptake by greater than 98% (Table 6). As a consequence, the
mechanism
is underlying the effectiveness of squalamine treatment of the LNCaP tumor in
vivo may
under certain conditions also involve direct inhibition of tumor cell growth
and/or
induction of tumor cell death.
Table 6
Endothelial Cell Treatment Percent of Control
[3H]-Thymidine Uptake
ao None (controls) 100%
20 ng/mL VEGF 84%
20 pg/mL squalamine 75%
20 ng/mL VEGF plus 20 pg/mL ~2%
squalamine
Zs EXAMPLE 8
Squalamine activity in combination with carboplatin
About 5 x 1 O6 cells from each of two human lung tumor lines, H460 (a rapidly
proliferating large cell carcinoma cell line, ATCC HTB-177) and Calu-6 (an
anaplastic
carcinoma, ATCC HTB-56), were inoculated subcutaneously in the right foreleg
of nude
so BALBc mice. Once tumors were visible (3-5 days) with a mean volume of
approximately
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50-80 mm3, mice were treated intraperitoneally with 60 mg/kg carboplatin
(single dose) or
20 mg/kg/ squalamine (once daily for 5 days) or a combination of carboplatin
and
squalamine. There were 8 mice per group. Tumor growth inhibition was scored by
quantifying the size of tumors once control tumors reached a size of 1.0 gram
and
s determining the ratio of mean treated tumor size to mean control tumor size
(T/C). Tu_mor
growth inhibition was then scored as 100% x [ 1 - (T/C)J. A separate antitumor
activity
variable, tumor growth delay, was scored for the tumors by determining the
length of time
it required for the mean tumor size in a treatment group to reach a size of
500 mm3 (B.A.
Teicher et al., Anticancer Research 18, 2567-2574 (1998)). The tumor growth
inhibition
~o seen with carboplatin plus squalamine was greater than that seen with
carboplatin or
squalamine alone. The relative inactivity of squalamine as a single agent by
this assay
endpoint supports the idea that squalamine is synergistic with carboplatin in
the inhibition
of these two human lung tumor xenografts. Similarly, it was determined that
squalamine
enhanced the tumor growth delay caused by carboplatin by an enhancement factor
of >2.3
is (Table 7).
Table 7
Human lung tumor type: H460 Calu-6
Tumor Growth Inhibition
(T/C):
Carboplatin 50% 23%
Zo Squalamine 6% 8%
Carboplatin + Squalamine 77% 52%
Tumor Growth Delay:
Carboplatin (C) 2.9 2.5
Carboplatin + Squalamine >6,7 >6.3
D + S)
zs Enhancement Ratio, D + S)/C>2.3 >2.5
EXAMPLE 9
In vivo Evaluation of squalamine activity in combination with carbaplatin plus
paclitaxel
Female Sprague Dawley nulnu mice weighing approximately 20 grams were
implanted subcutaneously by trocar with fragments of MV-522 human lung tumors
so harvested from subcutaneously grown tumors in nude mice hosts. When tumors
reached
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approximately 5 x 5 mm the animals were pair-matched into treatment and
control groups.
Each group contained 10 mice bearing tumors, was followed individually
throughout the
experiment. The administration of drugs (squalamine, paclitaxel plus
carboplatin, or
squalamine with paclitaxel and carboplatin) or vehicle began the day the
animals were pair-
s matched (Day 1 ). Drug doses and schedule were selected based upon prior
measurements
of maximum tolerated doses for paclitaxel plus carboplatin in mice.
Intraperitoneal (i.p.)
dosing of drugs was selected as an acceptable surrogate for intravenous
dosing.
Mice were weighed twice weekly, and tumor measurements were taken by calipers
twice weekly, starting on Day 1. These tumor measurements were converted to mg
tumor
io weight by the well-known formula, LZ x 2 W=tumor weight. The experiments
were
terminated when control tumors reached an estimated mean tumor size of one
gram based
upon in vivo tumor measurements. Upon termination, all mice were weighed,
sacrificed,
and their tumors excised. Excised tumors were weighed prior to their fixation
in 10%
formalin, and the mean excised tumor weight increase from Day I values for
each
is treatment group was calculated for all animals surviving until the
scheduled terminal
sacrifice. In this xenograft model, the [mean excised treated tumor weight
increase divided
by the mean excised control tumor weight increase (T/C)] x 100% was subtracted
from
100% to give the tumor growth inhibition (TGI) value for each group. Mice with
a
reduction in tumor size at the end of the experiment relative to the initial
tumor size were
Zo not included in the calculation of TGI and consequently TGI values may
represent minimal
estimates of tumor response to chemotherapy.
Some drug combinations caused shrinkage of some tumors in the MV-522 tumor
xenograft model. With these combinations, the final weight of a given tumor
(Day 31) was
subtracted from its own calculated weight at the start of treatment on Day 1
for the relevant
zs mice. This difference divided by the initial tumor weight times 100% was
the percent
shrinkage. A mean percent tumor shrinkage was then calculated from the data if
more than
one mouse in a group experienced a reduction in tumor size.
A tumor was considered to have experienced a partial regression if the
reduction in
tumor size was >50% at any time during the study. If the tumor completely
disappeared in
3o a mouse, this was considered a complete response or complete tumor
shrinkage, which was
then confirmed by histologic examination.
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A one-way analysis of variance (ANOVA) with subsequent pairwise comparisons
by Bonferroni's or Tukey's t-test was used for statistical analysis of excised
tumor weight
changes (R.R. Sokal and F.J. Rohlf, ' me , W.H. Freeman and Company, San
Francisco (1981); D.G. Altman, Practical Statistics for Medical Research,
Chapman and
s Hall, New York (I991)). Homogeneity of variances was assessed using Levene's
test and
data with heterogeneous variances were log transformed appropriately before
applying
ANOVA. Data are presented as means t S.E.M. or as percent of vehicle control.
Nonparametric analyses of tumor weights that had non-normal distributions
(determined by
Kolmogorov-Smirnov test) and/or unequal variances were also earned out using a
Kruskal-
io Wallis statistical test, with Mann-Whitney rank sum test for pairwise
comparisons.
Differences between means or ranks as appropriate were considered significant
when
yielding a p-value s 0.05. P-values for squalamine containing regimens are
calculated
relative to the same regimen without squalamine.
Preliminary experiments confirmed that the use of paclitaxel at 10 mg/kg, qd x
5,
is given i.p. in combination with carboplatin at 20 mg/kg, qd x 5, given i.p.
was tolerable to
nude mice and represented the maximal combination dosing that was not toxic.
The
combination of paclitaxel and carboplatin at these doses and with this dosing
regimen in
the in vivo MV-522 human lung tumor xenograft model produced a mean excised
tumor
size of 365.9 ~ 66.0 mg at Day 3 I or a TGI of 60.3% (p<0.01 ), with no
evident tumor
zo shrinkage for any animals in this treatment group. By contrast, triple
combination
chemotherapy with squalamine, paclitaxel and carboplatin produced a mean
excised tumor
growth of-0.1 ~ 19.3 mg (p<_0.001) or a net tumor stasis for the triple
therapy combination
group of animals, despite no chemotherapy during the last three weeks prior to
terminal
sacrifice. The TGI index for the squalamine/paclitaxel/carboplatin arm of this
study was
Zs 96.1 %. The benefit of chemotherapy on tumor size became apparent by Day 7
and
differences in mean tumor sizes between the two paclitaxel and carboplatin-
containing
arms of this study increased thereafter. Tumors in the squalamine-treated
animals scored at
terminal sacrifice were significantly smaller than those seen in animals which
received only
paclitaxel and carboplatin. No mice receiving only paclitaxel and carboplatin
were noted to
so undergo tumor shrinkage as assessed at the end of the experiment, but four
mice in the
triple combination chemotherapy cohort displayed tumor shrinkage over the
course of the
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experiment, with a mean reduction in tumor size for these animals of 54.8%.
There were
no complete tumor regressions in any treatment group receiving
paclitaxel/carboplatin
and/or squalamine, and only in the triple combination chemotherapy group were
there
partial regressions (6 of 7 surviving animals). The transient reduction in
tumor sizes was
s maximal on Day 14 of chemotherapy, as was seen with squalamine plus
cisplatin.
The initial selection of squalamine dose and dosing regimen for combination
chemotherapy was based on previous experiences with the maximum tolerated
daily dose
of squalamine and squalamine pharmacokinetics in rodents. Possible maintenance
of
squalamine activity with paclitaxel plus carboplatin at lower squalamine doses
was
io separately investigated. A dose ranging experiment with the MV-522 lung
tumor xenagraft
model was undertaken with paclitaxel and carboplatin using squalamine at daily
doses of
0.2-20 mg/kg on Days 1-5 and 8-9. This experiment was terminated on Day 32 and
final
excised tumor weights and percent TGI values were determined, with mean values
calculated on the basis of these observations (Table 7). At terminal
sacrifice, mean excised
is control tumor growth was 674.3 ~ 75.8 mg, while the excised tumor growth in
animals
treated with paclitaxel and carboplatin alone was 223.4 t 82.5 mg. This
corresponded to a
TGI for paclitaxel plus carboplatin of 66.9%, which is similar to the 60.3%
TGI seen in the
first experiment for this double drug treatment (see above). The mean excised
tumor
growth values for squalamine-treated groups were 132.0 ~ 46.4 mg (TGI = 74.2%)
at a
Zo daily squalamine dose of 0.2 mg/kg (p>0.05) and less than 77.0 mg (TGI >
78.8%) at 2.0,
10.0 and 20.0 mg/kg/day squalamine (Table 7). The maximal enhancement seen
with
multiple daily doses of squalamine occurred with 2.U mg/kg/day dosing; at this
dose, mean
excised tumor growth was 22.9 t 25.0 mg, corresponding to a TGI of 92.1 % (p =
0.017).
Statistically significant results were only observed at doses of 2.0 (p=0.017)
and 10.0
zs (p=0.035) mg/kg/day squalamine and were not observed at 20 mg/kg/day
(p>0.05)
squalamine. However, a meta-analysis across this experiment and the previous
one does
show 20 mg/kg/day squalamine significantly enhances the effect of paclitaxel
plus
carboplatin (p<0,p01). The advantage of adding squalamine to paclitaxel and
carboplatin
treatment of the MV-522 tumor was apparent at all doses by Day 5 and persisted
3o throughout the course of the experiment.
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No mice receiving paclitaxel and carboplatin alone had tumor shrinkages, but
there
were at lease two mice in each squalamine-treated group whose tumors were
reduced in
size over the course of the experiment, with a maximum of 5 mice out of 10
displaying
tumor shrinkage at Day 32 in each of the groups receiving 2 or 10 mg/kg/day
squalamine.
s No mice were observed with complete tumor shrinkages in any treatment group,
but
significant partial tumor regressions were seen in all treatment groups
(including
paclitaxel/carboplatin alone) which were most evident at Day 1 S following
initiation of
chemotherapy. In the two squalamine treated groups with the greatest tumor
response (2
and 10 mg/kg/day squalamine), 19 of 20 mice had a reduction in tumor burden of
at least
io 50% at Day 15.
The persistence of squalamine's effect on tumor growth in combination with
paclitaxel and carboplatin more than three weeks after the last dosing with
squalamine led
to an investigation of the required period of time over which a squalamine
dose was given
that is important. A separate arm of the experiment described in the previous
paragraph
is studied tumor growth for animal groups given squalamine b.i.d. as a single
daily dose of
0.2-40 mg/kg with paclitaxel and carboplatin. There was a squalamine dose-
related
enhancement of tumor growth inhibition of paclitaxel and carboplatin for a
single daily
dose of squalamine. The best responses were seen at single daily squalamine
doses of 10
and 20 mg/kg. At these doses, the mean excised tumor growths were 113.1 mg at
10
zo mg/kg and 96.6 mg at 20 mg/kg squalamine by Day 32, values which comparably
favorably to mean excised tumor values of 674.3 mg for control tumors and
223.4 mg for
paclitaxel and carboplatin-treated tumors. However, no squalamine dose
achieved
statistical significance when compared to paclitaxel and carboplatin alone.
Partial tumor
regressions were seen in 2-4 squalamine-treated mice during the experiment and
durable
is partial tumor shrinkages were seen in 2-3 animals in each squalamine-
treated group when
they were evaluated at the end of the experiment, but no reduction in tumor
size was seen
with any mice treated with paclitaxel and carboplatin alone. There also was
one complete
regression observed in a mouse which received 20 mg/kg squalamine on day S
along with
paclitaxel and carboplatin. The mean percent tumor shrinkages in the
squalamine-treated
3o animals with tumor regressions was in the range of 21.0-50.4%.
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EXAMPLE 10
PCT/US99/20645
Squalamine activity in combinatian with radiation therapy
Syngeneic mammary carcinoma MCA-4 tumor cells (5 x 105) were injected
intramuscularly into the right legs of C3Hf/Kam male mice (4 months of age).
When the
s tumors reached 6 mm in diameter, the mice were randomly assigned to receive:
(a) no
treatment, (b) squalamine alone (20 mg/kg/day b.i.d. for 11-14 days), (c) 15
Gy of gamma
irradiation, or (d) squalamine plus 15 Gy of gamma rays. Tumor growth was
followed until
tumors reached at least 12 mm in diameter. The effect of squalamine on tumor
radioresponse
was determined by absolute and normalized tumor growth delays (AGD and NGD).
AGD is
io defined as the time in days for tumors treated with radiation (or
squalamine) to grow from 8
to 12 mm minus the time in days for untreated tumors to grow from 8-12 mm. NGD
is defined
as the time in days for tumors treated with both squalamine and radiation to
grow from 8 to
12 mm minus the time in days for tumors treated with squaIamine only to grow
from 8 to I2
mm. An enhancement factor (EF) was determined as the ratio of NGD to AGD. The
EF for
is squalamine with 15 Gy gamma rays calculated from the observed times for
tumor growth
(Table 8) is >1.5.
Table 8
EXAMPLE 11
Squalamine activity in combination with cisplatin for ovarian cancer
is Human ovarian tumor cells were injected subcutaneously (5 x 10' cells per
animal)
in the mid-back region of female Swiss nude mice. The human ovarian tumors
were either
the parental 2008 cell line (cf. R.J. Pietras et al., nc ne _9, 1829-1838
(1994)) or a
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transfected variant overexpressing the Her-2/neu receptor. The overexpression
of the Her-
2/neu receptor is considered to make the resulting xenograft tumors more
angiogenic and
less responsive to chemotherapy. After one week, animals were randomized to
groups
receiving no treatment (controls), a single dose of cisplatin (4 mg/kg), daily
treatment with
s squalamine (2 mg/kg, days 1-10) or cisplatin plus squalamine. The growth of
tumors was
then scored on day 28 following initiation of chemotherapy. The results were
very similar
with the parental 2008 tumor and the transfected variant overexpressing the
Her-2/neu
receptor. Exemplary results with the parental 2008 cell line are shown in
Table 9. These
results can be quantified by calculating a tumor growth inhibition index for
each treatment
io group. In this xenograft model, the [day 28 mean treated tumor weight
increase divided by
the day 28 mean control tumor weight increase (T/C)] x 100% was subtracted
from 100%
to give the tumor growth inhibition (TGI) value for each group. Cisplatin had
minimal
effect on 2008 tumor cell growth in this model, but squalamine significantly
reduced day
28 mean tumor size compared to controls. The combination of cisplatin plus
squalamine
is was highly effective at slowing tumor growth.
Table 9
Treatment ConditionDay 28 Mean Tumor Tumor Growth
Size(mm3) Inhibition
(TGI) Index (%)
None (controls} 6197
Cisplatin 5108 18%
ao Squalamine 2254 41
Cisplatin plus squalamine356 94%
