U.S. patent application number 10/759716 was filed with the patent office on 2004-07-29 for process for producing arsenic trioxide formulations and methods for treating cancer using arsenic trioxide or melarsoprol.
This patent application is currently assigned to Memorial Sloan-Kettering Cancer Center. Invention is credited to Gabrilove, Janice L., Pandolfi, Pier Paolo, Warrell, Raymond P. JR..
Application Number | 20040146581 10/759716 |
Document ID | / |
Family ID | 22057410 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146581 |
Kind Code |
A1 |
Warrell, Raymond P. JR. ; et
al. |
July 29, 2004 |
Process for producing arsenic trioxide formulations and methods for
treating cancer using arsenic trioxide or melarsoprol
Abstract
The invention relates to the use of arsenic compounds to treat a
variety of leukemia, lymphoma and solid tumors. Further, the
arsenic compounds may be used in combination with other therapeutic
agents, such as a retinoid. The invention also provides a process
for producing arsenic trioxide formulations.
Inventors: |
Warrell, Raymond P. JR.;
(Westfield, NJ) ; Pandolfi, Pier Paolo; (New York,
NY) ; Gabrilove, Janice L.; (New York, NY) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,
KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Assignee: |
Memorial Sloan-Kettering Cancer
Center
New York
NY
|
Family ID: |
22057410 |
Appl. No.: |
10/759716 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10759716 |
Jan 16, 2004 |
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10425785 |
Apr 30, 2003 |
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6723351 |
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10425785 |
Apr 30, 2003 |
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09189965 |
Nov 10, 1998 |
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60064655 |
Nov 10, 1997 |
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Current U.S.
Class: |
424/623 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61K 33/24 20130101; A61K 33/36 20130101; A61K
31/53 20130101; A61K 31/555 20130101; A61P 35/02 20180101; A61K
41/00 20130101; A61K 31/285 20130101; A61K 33/36 20130101; A61K
2300/00 20130101; A61K 41/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/623 |
International
Class: |
A61K 033/36 |
Claims
1. A method for treating acute myelogenous leukemia in a human,
comprising: determining a therapeutically effective dosage of
arsenic trioxide based on the weight of a subject diagnosed with
acute myelogenous leukemia; and administering the therapeutically
effective dosage amount of arsenic trioxide once per day.
2. The method of claim 1, wherein said acute myelogenous leukemia
is acute promyelogenous leukemia.
3. The method of claim 2, wherein said arsenic trioxide is
administered until bone marrow remission, which constitutes a first
administration.
4. The method of claim 3, further comprising a second
administration of 0.15 mg/kg arsenic trioxide once per day for 25
doses.
5. The method of claim 4, wherein said second administration is
administered 3 to 6 weeks after said first administration.
6. The method of claim 4, wherein said second administration is
administered for up to five weeks.
7. The method of claim 6, wherein said second administration is
administered at five doses per week.
8. The method of claim 4, further comprising repeating said second
administration.
9. The method of claim 8, wherein said second administration is
repeated every 3 to 6 weeks.
10. The method of claim 9, wherein said second administration is
repeated until a total of between two and ten cycles of said second
administration are completed.
11. The method of claim 10, further comprising repeating said
second administration until a total of two cycles of said second
administration are completed.
12. The method of claim 10, further comprising repeating said
second administration until a total of ten cycles of said second
administration are completed.
13. The method of claim 2, wherein said arsenic trioxide is
administered for up to sixty days, which constitutes a first
administration.
14. The method of claim 13, further comprising a second
administration of 0.15 mg/kg arsenic trioxide once per day for 25
doses.
15. The method of claim 14, wherein said second administration is
administered 3 to 6 weeks after said first administration.
16. The method of claim 14, wherein said second administration is
administered for up to five weeks.
17. The method of claim 16, wherein said second administration is
administered at five doses per week.
18. The method of claim 14, further comprising repeating said
second administration.
19. The method of claim 18, wherein said second administration is
repeated every 3 to 6 weeks.
20. The method of claim 19, wherein said second administration is
repeated until a total of between two and ten cycles of said second
administration are completed.
21. The method of claim 20, further comprising repeating said
second administration until a total of two cycles of said second
administration are completed.
22. The method of claim 20, further comprising repeating said
second administration until a total of ten cycles of said second
administration are completed.
Description
1. FIELD OF INVENTION
[0001] The present invention relates to methods and compositions
for the treatment of leukemia, lymphoma, and certain other
cancers.
[0002] More specifically, the present invention relates to the
novel uses of arsenic trioxide and an organic arsenic compound for
treating acute leukemia and chronic leukemia.
2. BACKGROUND OF THE INVENTION
[0003] 2.1. Cancer
[0004] Cancer is characterized primarily by an increase in the
number of abnormal cells derived from a given normal tissue,
invasion of adjacent tissues by these abnormal cells, and lymphatic
or blood-borne spread of malignant cells to regional lymph nodes
and to distant sites (metastasis). Clinical data and molecular
biologic studies indicate that cancer is a multistep process that
begins with minor preneoplastic changes, which may under certain
conditions progress to neoplasia.
[0005] Pre-malignant abnormal cell growth as exemplified by
hyperplasia, metaplasia, and dysplasia (for review of such abnormal
growth conditions, see Robbins and Angell, 1976, Basic Pathology,
2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79) precedes the
formation of a neoplastic lesion. A neoplastic lesion may evolve
clonally to grow into a solid tumor, and develop an increasing
capacity for invasion, growth, metastasis, and heterogeneity,
especially under conditions in which the neoplastic cells escape
the host's immune surveillance (Roitt, I., Brostoff, J and Kale,
D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.
17.1-17.12).
[0006] Leukemia refers to malignant neoplasms of the bloodforming
tissues. Transformation to malignancy typically occurs in a single
cell through two or more steps with subsequent proliferation and
clonal expansion. In some leukemias, specific chromosomal
translocations have been identified with consistent leukemic cell
morphology and special clinical features (e.g., translocations of 9
and 22 in chronic myelocytic leukemia, and of 15 and 17 in acute
promyelocytic leukemia). Acute leukemias are predominantly
undifferentiated cell populations and chronic leukemias more mature
cell forms.
[0007] Acute leukemias are divided into lymphoblastic (ALL) and
non-lymphoblastic (ANLL) types. They may be further subdivided by
their morphologic and cytochemical appearance according to the
French-American-British (FAB) classification or according to their
type and degree of differentiation. The use of specific B- and
T-cell and myeloid-antigen monoclonal antibodies are most helpful
for classification. ALL is predominantly a childhood disease which
is established by laboratory findings and bone marrow examination.
ANLL, also known as acute myeloblastic leukemia (AML), occurs at
all ages and is the more common acute leukemia among adults; it is
the form usually associated with irradiation as a causative
agent.
[0008] Chronic leukemias are described as being lymphocytic (CLL)
or myelocytic (CML). CLL is characterized by the appearance of
mature lymphocytes in blood, bone marrow, and lymphoid organs. The
hallmark of CLL is sustained, absolute lymphocytosis
(>5,000/.mu.L) and an increase of lymphocytes in the bone
marrow. Most CLL patients also have clonal expansion of lymphocytes
with B-cell characteristics. CLL is a disease of older persons. In
CML, the characteristic feature is the predominance of granulocytic
cells of all stages of differentiation in blood, bone marrow,
liver, spleen, and other organs. In the symptomatic patient at
diagnosis the total WBC count is usually about 200,000/.mu.L, but
may reach 1,000,000/.mu.L. CML is relatively easy to diagnose
because of the presence of the Philadelphia chromosome.
[0009] The very nature of hematopoietic cancer necessitates using
systemic chemotherapy as the primary treatment modality. Drugs
selected according to sensitivities of specific leukemias are
usually given in combination. Radiation therapy may be used as an
adjunct to treat local accumulations of leukemic cells. Surgery is
rarely indicated as a primary treatment modality, but may be used
in managing some complications. Bone marrow transplantation from an
HLA-matched sibling is sometimes indicated.
[0010] 2.2. Arsenic and its Medical Uses
[0011] Arsenic has been considered to be both a poison and a drug
for a long time in both Western and Chinese medical practices. In
the latter part of the nineteenth century, arsenic was used
frequently in attempts to treat diseases of the blood in the West.
In 1878, it was reported that treatment of a leukemic patient with
Fowler's solution (a solution containing potassium arsenite,
valence +5) reduced markedly the count of white blood cells (Cutler
and Bradford, Am. J. Ned. Sci., January 1878, 81-84). Further
interests in the use of Fowler's solution as a palliative agent to
treat chronic myelogenous leukemia (CML) was described by Forkner
and Scott in 1931 (J. Am. Med. Assoc., 1931, iii, 97), and later
confirmed by Stephens and Lawrence in 1936 (Ann. Intern. Med. 9,
1488-1502). However, while the active chemical ingredient(s) of
Fowler's solution was not determined, its toxicity was well
recognized. Fowler's solution was administered strictly as an oral
composition, and was given to leukemic patients as a solution until
the level of white blood cells was depressed to an acceptable level
or until toxicities (such as skin keratoses and hyperpigmentation)
developed, while the patients enjoyed varying periods of remission.
In the 1960's, Fowler's solution was still used occasionally in
attempts to treat CML, however, most patients with CML were treated
with other chemotherapeutic agents, such as busulfan, and/or
radiation therapy (Monfardini et al., Cancer, 1973,
31:492-501).
[0012] Paradoxically, one of the long recognized effects of
exposure to arsenic, whether the source is environmental or
medicinal, is skin cancer (Hutchinson, 1888, Trans. Path. Soc.
Lond., 39:352; Neubauer, 1947, Br. J. Cancer, 1:192). There were
even epidemiological data to suggest that the use of Fowler's
solution over long periods could lead to an increased incidence of
cancer at internal sites (Cuzick et al., Br. J. Cancer, 1982,
45:904-911; Kaspar et al., J. Am. Med. Assoc., 1984,
252:3407-3408). The carcinogenicity of arsenic has since been
demonstrated by the fact that it can induce chromosomal aberration,
gene amplification, sister chromatid exchanges and cellular
transformation (See e.g., Lee et al., 1988, Science, 241:79-81; and
Germolec et al., Toxicol. Applied Pharmacol., 1996, 141:308-318).
Because of the known carcinogenic effect of arsenic, its only
therapeutic use in human in Western medicine today is in the
treatment of tropical diseases, such as African trypanosomiasis,
(the organic arsenical, melarsoprol; See Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th edition, chapter 66,
1659-1662, 1997).
[0013] In traditional chinese medicine, arserious acid or arsenic
trioxide paste has been used to treat tooth marrow diseases,
psoriasis, syphilis and rheumatosis (Chen et al., 1995, in Manual
of Clinical Drugs, Shanghai, China, Shanghai Institute of Science
and Technology, p.830). In 1970's, arsenic trioxide had been
applied experimentally to treat acute promyelocytic leukemia (APL)
in China (commented by Mervis, 1996, Science, 273:578). The
clinical efficacy of arsenic trioxide has recently been
re-investigated in 14 of 1.5 patients with refractory APL, where
the use of an intravenous dose at 10 mg/day for 4-9 weeks was
reported to result in complete morphologic remission without
associated bone marrow suppression (Shen et al., 1997, Blood,
89:3354-3360). It was also shown that arsenic trioxide induced
apoptosis (programmed cell death) in vitro in NB4 cells, an APL
cell line, and that apoptosis was apparently associated with
down-regulation of the oncogene bcl-2, and intracellular
redistribution of the chimeric PML/RAR.alpha. protein that are
unique to APL cells (Chen et al., 1996, Blood, 88:1052-1061; Andre
et al., 1996, Exp. Cell Res. 229:253-260). It has been reported
that the biological activity of arsenic is due to the ability of
arsenic to direct the nucleoplasmic fraction of PML to nuclear
bodies for degradation (Zhu et al., 1997, Proc. Natl. Acad. Sci.,
94:3978-3983).
[0014] Although arsenic is well known to be both a poison and a
carcinogenic agent, there have been many reports concerning the use
of arsenic in medical treatment. Further, from the above
discussion, it should be clear that there are many different types
of leukemias, each of which requires a unique treatment protocol
that is modified according to the presence of factors predicting
for a risk of treatment failure. Thus, the development of a broad
spectrum antileu-kemia agent that can be used alone or in
combination with other existing drugs is extremely desirable.
3. SUMMARY OF THE INVENTION
[0015] Despite the conflicting reports in the art concerning
benefits and risks of the administration of arsenic to patients,
applicants have discovered that arsenic trioxide and the organic
arsenical, melarsoprol, have broad applicability in the treatment
of various types of leukemias, lymphomas, and solid tumors.
[0016] The invention described herein encompasses a method of
treating leukemia, lymphoma or solid tumors comprising the
administration of a therapeutically effective and non-lethal amount
of arsenic trioxide or melarsoprol to a human in need of such
therapy. The invention, as mentioned above also encompasses the use
of combination therapy to treat leukemia, especially leukemias
which are refractory to other forms of treatment.
[0017] The invention also encompasses a method for the manufacture
of pharmaceutical compositions comprising arsenic trioxide.
[0018] In accordance with the present invention, arsenic trioxide
or melarsoprol compounds can be used alone or in combination with
other known therapeutic agents (including chemotherapeutics,
radioprotectants and radiotherapeutics) or techniques to either
improve the quality of life of the patient, or to treat leukemia,
lymphoma or solid tumor. The arsenic compounds can be used before,
during or after the administration of one or more known
chemotherapeutic agents, including antitumor agents. In addition,
the arsenic compounds can be used before, during or after radiation
treatment.
[0019] The pharmaceutical compositions of the invention are sterile
solutions suitable for intravenous injection or infusion. In
another embodiment the invention encompasses a composition suitable
for oral delivery; comprising arsenic trioxide or melarsoprol and a
pharmaceutically acceptable excipient or carrier. In another
embodiment, the invention also includes compositions suitable for
topical or transdermal delivery, including but not limited to
iontophoretic methods. Specific therapeutic regimens,
pharmaceutical compositions, and kits are also provided by the
invention.
[0020] Particular compositions of the invention and their uses are
described in the sections and subsections which follow.
4. DETAILED DESCRIPTION OF THE INVENTION
[0021] Methods and compositions for the treatment of leukemia,
lymphoma or solid tumors are described herein. This invention
provides a method of treating acute or chronic leukemia, lymphoma,
or solid tumors in a human which comprises administering to a human
in need of such therapy a therapeutically effective and non-lethal
amount of one or more arsenic compounds, such as arsenic trioxide
or melarsoprol.
[0022] The invention also includes a method of treating leukemia in
a human who has become refractory to other forms of treatment which
comprises administering to a human arsenic trioxide or melarsoprol
in combination with another chemotherapeutic agent, e.g., all-trans
retinoic acid (ATRA).
[0023] The invention also relates to a method for the manufacture
of pharmaceutical compositions comprising arsenic trioxide. It is
preferred that pharmaceutical compositions of the present invention
exhibit reduced toxicity, improved efficacy, improved stability
during storage and use, and that the composition has a
physiologically acceptable pH.
[0024] 4.1. The Arsenic Compounds
[0025] As used herein, "arsenic compound" refers to a
pharmaceutically acceptable form of arsenic trioxide
(As.sub.2O.sub.3) or melarsoprol. Melarsoprol is an organic arsenic
compound which can be synthesized by complexing melarsen oxide with
dimercaprol or commercially purchased (Arsobal.RTM. by Rhone
Poulenc Rorer, Collegeville, Pa.). Since the nonpharmaceutically
formulated raw materials of the invention are well known, they can
be prepared from well-known chemical techniques in the art. (See
for example, Kirk-Othmer, Encyclopedia of Chemical Technology 4th
ed. volume 3 pps. 633-655 John Wiley & Sons).
[0026] As used herein the terms "a therapeutic agent", "therapeutic
regimen", "radioprotectant", "chemotherapeutic" mean conventional
drugs and drug therapies, including vaccines, for treating cancer,
viral infections, and other malignancies, which are known to those
skilled in the art. "Radiotherapeutic"-agents are well known in the
art.
[0027] In accordance with the present invention, arsenic trioxide
or melarsoprol compounds can be used alone or in combination with
other known therapeutic agents (including chemotherapeutics,
radioprotectants and radiotherapeutics) or techniques to either
improve the quality of life of the patient, or to treat leukemia,
lymphoma or solid tumor. For example, the arsenic compounds can be
used before, during or after the administration of one or more
known antitumor agents including but not limited to mustard
compounds, nitrogen mustard, chlorambucil, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil, floxuridine, methotrexate, vincristine,
vinblastine, taxol, etoposide, temiposide, dactinomycin,
daunorubicin, doxorubicin, bleomycin, mitomycin, cisplatin,
carboplatin, estramustine phosphate, hydroxyurea, BCNU,
procarbazine, VM-26, interferons, and all-trans retinoic acid
(ATRA), or other retinoids (See, for example, the Physician Desk
References 1997). In addition, the arsenic compounds can be used
before, during or after radiation treatment.
[0028] In a specific embodiment, the arsenic compound of the
invention and ATRA can be administered as a mixture. In preferred
aspects, the lymphoma, leukemia or solid tumor in the human treated
by the combination is refractory to general methods of treatment,
or is a relapsed case of leukemia.
[0029] Any suitable mode of administration may be used in
accordance with the present invention including but not limited to
parenteral administration such as intravenous, subcutaneous,
intramuscular and intrathecal administration; oral, and intranasal
administration, and inhalation. The mode of administration will
vary according to the type of cancer, and the condition of the
human.
[0030] The pharmaceutical compositions to be used may be in the
form of sterile aqueous or organic solutions; colloidal
suspensions, caplets, tablets and cachets.
[0031] 4.2. Methods of Treatment
[0032] The term "a method for treating leukemia" as used herein
means that the disease and the symptoms associated with the disease
are alleviated, reduced, cured, or placed in a state of remission.
For example, the methods of treatment of the invention can lower
the white blood cell count, or reduce lymphocytosis in a human
under treatment.
[0033] The term "a method for treating lymphoma" as used herein
means that the disease and the symptoms associated with the disease
are alleviated, reduced, cured, or placed in a state of
remission.
[0034] The term "a method for treating solid tumor" as used herein
means that the disease and the symptoms associated with the solid
tumor are alleviated, reduced, cured, or placed in a state of
remission.
[0035] In addition, the term "a method for treating leukemic
infiltration" means that the infiltration of leukemic cells out of
circulation and into other organs and systems and the symptoms
associated with such infiltration are alleviated, reduced, cured,
or placed in a state of remission.
[0036] The term "refractory" when used herein means that the
leukemia is generally resistant to treatment or cure.
[0037] As used herein, "preneoplastic" cell refers to a cell which
is in transition from a normal to a neoplastic form; or cells that
fail to differentiate normally; and morphological evidence,
increasingly supported by molecular biologic studies, indicates
that preneoplasia progresses through multiple steps.
[0038] In one embodiment, the invention provides a method for
treatment of leukemia in a human comprising the administration of a
therapeutically effective and non-lethal amount of arsenic trioxide
or melarsoprol to the human. The invention also provides a
weight-based dosing regimen, not heretofore disclosed, that
maximizes the safety in humans of these otherwise highly toxic
compounds.
[0039] Arsenic trioxide (As.sub.2O.sub.3) inhibits growth and
induce apoptosis in NB4 acute promyelocytic leukemic cells. Acute
promyelocytic leukemia (APL) is associated with the t(15;17)
translocation, which generates a PML/RAR.alpha. fusion protein
between PML, a growth suppressor localized on nuclear
matrix-associated bodies, and RAR.alpha., a nuclear receptor for
retinoic acid (RA). PML/RAR.alpha. was proposed to block myeloid
differentiation through inhibition of nuclear receptor response, as
does a dominant negative RAR.alpha. mutant. In addition, in APL
cells, PML/RAR.alpha. displaces PML and other nuclear body (NB)
antigens onto nuclear microspeckles, likely resulting in the loss
of PML and/or NB functions. It has been suggested that high
concentrations of arsenic trioxide promote apoptosis, whereas low
concentrations induce partial differentiation in NB4 cells as well
as cells derived from APL patients. It was postulated that
As.sub.2O.sub.3 works through its ability to specifically cause
PML-RAR.alpha. in APL cells to be relocalized to nuclear bodies for
degradation (Zhu et al., 1997, Proc. Natl. Acad. Sci. USA,
94:3978-3983). However, these findings tend to limit the use of
arsenic trioxide to a subset of leukemias. See Konig et al., 1997,
Blood, 90:562-570.
[0040] Unexpectedly, the inventors have discovered that both
As.sub.2O.sub.3 and melarsoprol are able to inhibit cell growth,
and induce apoptosis in various myeloid leukemia cell lines in a
PML and PML-RAR.alpha. independent manner. Thus, the inventors have
discovered that, contrary to the earlier findings, arsenic trioxide
and melarsoprol are both effective against a broad range of
leukemias regardless of the underlying molecular mechanism that
causes the neoplasia. Working examples of the effect of arsenic
compounds on a number of leukemic cell lines are provided in
Sections 5.1 and 5.2.
[0041] Accordingly, the arsenic compounds of the present invention
can be used against a variety of leukemias, including but not
limited to;
[0042] Acute lymphoblastic leukemia (ALL)
[0043] Acute lymphoblastic B-cell leukemia
[0044] Acute lymphoblastic T-cell leukemia
[0045] Acute myeloblastic leukemia (AML)
[0046] Acute promyelocytic leukemia (APL)
[0047] Acute monoblastic leukemia
[0048] Acute erythroleukemic leukemia
[0049] Acute megakaryoblastic leukemia
[0050] Acute myelomonocytic leukemia
[0051] Acute undifferentiated leukemia
[0052] Chronic myelocytic leukemia (CML)
[0053] Chronic lymphocytic leukemia (CLL)
[0054] The skilled artisan will recognize that other leukemias may
be treated in accordance with the present invention.
[0055] In another embodiment, the invention provides a method for
treatment of lymphoma in a human comprising the administration of a
therapeutically effective and non-lethal amount of arsenic trioxide
or melarsoprol to the human. Lymphoma that can be treated by the
methods of the invention include but are not limited to high grade
lymphoma, intermediate grade lymphoma, low grade lymphoma, and the
various subclassifications.
[0056] In yet another embodiment, the invention provides a method
for treatment of solid tumors, including metastasises, in humans
comprising the administration of a therapeutically effective and
non-lethal amount of arsenic trioxide or melarsoprol to the human.
Solid tumors that can be treated by the methods of the invention
include but are not limited to: cancer of the digestive tract,
oesophagus, liver, stomach, and colon; skin; brain; bone; breast;
lung; and soft tissues, including but not limited to various
sarcomas, and preferably prostate cancer.
[0057] In various embodiments, the leukemic or tumor cells are
infiltrating other organs and systems in a human, for example, the
central nervous system. The methods of the invention are also
applicable to reduce the number of preneoplastic cells in a human
in which there is an abnormal increase in the number of
preneoplastic cells.
[0058] In a specific embodiment, the invention provides a method of
treatment of acute promyelolytic leukemia (APL) in a human
comprising the administration of a therapeutically effective and
non-lethal amount of melarsoprol to the human. The inventors
discovered, as described in Section 5.2, that concentrations of
melarsoprol that are cytotoxic in vitro can readily be achieved in
vivo.
[0059] In one specific embodiment, the invention provides a method
of treatment of chronic myelogenous leukemia (CML) in a human
comprising the administration of a therapeutically effective and
non-lethal amount of arsenic trioxide to the human. The inventors
discovered, as described in Section 5.3, that arsenic trioxide can
also induce apoptosis in a CML cell line. The therapeutic benefits
of the pharmaceutical compositions of the invention comprising
arsenic trioxide is far superior to that of potassium arsenite,
commonly formulated as Fowler's solution.
[0060] In yet another specific embodiment, the invention provides a
method of treatment of acute promyelocytic leukemia (APL) in a
human, in which the APL is associated with a translocation of the
RAR.alpha. locus on chromosome 17 to chromosome 11, comprising the
administration of a therapeutically effective amount of arsenic
trioxide or melarsoprol to the human. In the majority of APL cases,
RAR.alpha. on chromosome 17 translocates and fuses with the PML
gene located on chromosome 15, i.e., t(15;17). In a few cases
RAR.alpha. translocates to chromosome 11 where it fuses to the PLZF
gene. Patients harboring the t(15;17) are uniquely sensitive to
treatment with all-trans retinoic acid (ATRA), yielding complete
remission rates of 75% to 95%. APL associated with the t(11;17)
(PLZF-RAR.alpha.) shows a distinctly worse prognosis with poor
response to chemotherapy and little or no response to treatment
with ATRA, thus defining a new APL syndrome. The present invention
provides that arsenic trioxide or melarsoprol can be used to treat
such cases of APL. Transgenic animal models of APL associated with
t(15;17) and t(11;17) for testing the therapeutic benefits and
dosages of arsenic compounds of the invention are described in
Section 5.4 hereinbelow.
[0061] Humans having leukemia are sometimes refractory to
conventional methods of treatment by reason of having undergone
anti-leukemic therapy (e.g., chemotherapy). Thus, the invention
provides a method of treatment of leukemia in a human who is not
responding to conventional therapy comprising the administration of
a therapeutically effective and non-lethal amount of a combination
of arsenic compound and another chemotherapeutic agent, such as but
not limited to, all-trans retinoic acid (ATRA) or other retinoids,
to the human. The arsenic compound can either be arsenic trioxide
or melarsoprol or a pharmaceutically acceptable salt thereof. The
invention also encompasses the treatment of retinoid-resistant
patients with an arsenic compound.
[0062] In specific embodiments, the arsenic compound of the
invention and the chemotherapeutic agent can be administered either
as a mixture or sequentially. When administered sequentially, the
arsenic compound may be administered before or after the
chemotherapeutic agent, so long as the first administered agent is
still providing antileukemic activity in the human when the second
agent is administered. Any of the modes of administration described
herein may be used to deliver the combination. In preferred
aspects, the leukemia in the human treated by the combination is
refractory to general methods of treatment, or is a relapsed case
of leukemia.
[0063] 4.3. Process for the Manufacture of Sterile Arsenic Trioxide
Solution
[0064] The arsenic compounds of the invention may be formulated
into sterile pharmaceutical preparations for administration to
humans for treatment of leukemias, lymphomas and solid tumors.
Compositions comprising a compound of the invention formulated in a
compatible pharmaceutical carrier may be prepared, packaged,
labelled for treatment of and used for the treatment of the
indicated leukemia, lymphoma, or solid tumor.
[0065] In one aspect, the invention provides a method for the
manufacture of a pharmaceutical composition comprising a
therapeutic effective and non-lethal amount of arsenic trioxide
(As.sub.2O.sub.3). Arsenic trioxide (raw material) is a solid
inorganic compound that is commercially available in a very pure
form. However, it is difficult to dissolve As.sub.2O.sub.3 in
aqueous solution. Further, the inventors are unaware of any
published teachings on how to formulate As.sub.2O.sub.3 as a
pharmaceutical composition suitable for injection directly into a
human. Arsenic is present in solution in the +5 valence state
(pentavalent) or the +3 valence state (trivalent). For example,
potassium arsenite (KAS02; which is present in Fowler's solution)
and salts of arsenious acid contain pentavalent arsenic. It is
known that one form of arsenic is more toxic than the other.
(Goodman & Gilman's The Pharmacological Basis of Therapeutics,
9th edition, chapter 66, 1660, 1997). A fresh solution of arsenic
trioxide containing arsenic in the trivalent state will be
gradually oxidized to pentavalent state if exposed to air for a
prolonged period, and as a result of the accumulation of
pentavalent arsenic, the relative toxicity of a solution of
As.sub.2O.sub.3 will change over time. (Id.) Furthermore, it is
observed that the total amount of arsenic in solution decreases
over time. This loss of material is caused by the progressive
conversion of arsenic in the solution into arsine (AsH.sub.3) which
is a gaseous compound at room temperature. This is particularly
problematic in pharmaceutical applications if the concentration of
an active ingredient in the injected material cannot be controlled.
It is also undesirable to allow arsine to escape from the solution
into the atmosphere because arsine is also toxic.
[0066] The inventors have experimented and successfully developed a
method for formulating arsenic trioxide which overcomes the
above-described problems of solubility and stability. The method
comprises solubilizing solid high purity As.sub.2O.sub.3 in an
aqueous solution at high pH, such as pH greater than 12. For
example, a 5 M solution of sodium hydroxide may be used. To aid
solubilization and obtain a clear and homogenous solution,
mechanical stirring and/or gentle heating may be applied. A
solution of As.sub.2O.sub.3 can also be obtained by dissolving the
solid compound overnight. Typically, a solution of 1 M
As.sub.2O.sub.3 is obtained by this method. However, this solution
is too basic to be useful as a pharmaceutical composition.
[0067] To adjust the pH of the As.sub.2O.sub.3 solution, the
solution is first diluted in water, for example, to a concentration
of about 1 mg/mL, pH 12. The As.sub.2O.sub.3 solution is then
back-titrated with acid, such as, hydrochloric acid (1 M to 5 M
HCl), with constant stirring until the pH is about 8.0 to 8.5.
Highly concentrated HCl is not suitable as it causes precipitation
to occur in the As.sub.2O.sub.3 solution. The partially
neutralized. As.sub.203 solution may then be sterilized for
example, by filtration (e.g., through a 0.22 .mu.m filter), and
stored in sterile vials.
[0068] To make a pharmaceutical composition that can be directly
injected into a subject, the composition must be sterile, standard
techniques known to the skilled artisan for sterilization can be
used. See, e.g., Remington's Pharmaceutical Science. The pH of the
partially neutralized As.sub.2O.sub.3 solution may be further
adjusted to near physiological pH by dilution (10-100 fold) with a
pharmaceutical carrier, such as a 5% dextrose solution. For
example, 10 mL of a partially neutralized As.sub.2O.sub.3 solution
can be added to 500 mL of a 5% dextrose solution to yield a final
pH of about 6.5 to 7.5. The method of the invention reduces the
oxidation of arsenic in solution. Pharmaceutical compositions
containing arsenic trioxide manufactured by the method of the
invention show improved stability and long shelf life.
[0069] 4.4. Pharmaceutical Composition and Modes of
Administration
[0070] According to the invention, the arsenic compounds and their
physiologically acceptable solvates may be formulated for oral or
parenteral administration.
[0071] For oral administration, the pharmaceutical preparation may
be in liquid form, for example, solutions, syrups or suspensions,
or may be presented as a drug product for reconstitution with water
or other suitable vehicle before use. Such liquid preparations may
be prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily esters, or fractionated vegetable oils); and
preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic
acid). The pharmaceutical compositions may take the form of, for
example, tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art.
[0072] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0073] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or is
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampules or in multidose containers, with
an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0074] The invention also provides kits for carrying out the
therapeutic regimens of the invention. Such kits comprise in one or
more containers therapeutically effective amounts of the arsenic
compounds in pharmaceutically acceptable form. The arsenic compound
in a vial of a kit of the invention may be in the form of a
pharmaceutically acceptable solution, e.g., in combination with
sterile saline, dextrose solution, or buffered solution, or other
pharmaceutically acceptable sterile fluid. Alternatively, the
complex may be lyophilized or desiccated; in this instance, the kit
optionally further comprises in a container a pharmaceutically
acceptable solution (e.g., saline, dextrose solution, etc.),
preferably sterile, to reconstitute the complex to form a solution
for injection purposes.
[0075] In another embodiment, a kit of the invention further
comprises a needle or syringe, preferably packaged in sterile form,
for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of arsenic
compounds by a clinician or by the patient.
[0076] The magnitude of a therapeutic dose of an arsenic compound
in the acute or chronic management of leukemia will vary with the
severity of the condition to be treated and the route of
administration. The dose, and perhaps dose frequency, will also
vary according to the age, body weight, condition and response of
the individual patient. In general, the daily dose ranges of
arsenic trioxide for the conditions described herein are generally
from about 0.05 to about 5 mg per kg body weight administered in
divided doses administered parenterally or orally or topically. A
preferred total daily dose is from about 2.5 to about 40 mg of
arsenic trioxide. Preferably the arsenic trioxide formulation of
the invention is given daily for a maximum of 60 days, or until
remission, followed by two to ten additional cycles, each lasting
about 25 days in duration. For example, depending on the body
weight of a patient with acute promyelocytic leukemia, a daily dose
of arsenic trioxide greater than or less than 10 mg can be
administered. Alternatively, following the weight-based dosing
regimen, a therapeutic effect can be obtained with a daily dose of
arsenic trioxide less than 10 mg.
[0077] For treatment of solid tumor, a preferred dosing regimen
involves intravenous infusion of about 0.1 to about 5 mg per kg
body weight per day for 5 days. This five-day treatment protocol is
repeated once per month until the tumor growth tumor is inhibited
or when the tumor shows signs of regression.
[0078] As for melarsoprol, the total daily dose ranges for the
conditions described herein are generally from about 0.1 to about 5
mg/kg body weight administered in divided doses administered
parenterally or orally or topically. A preferred total daily dose
is from about 0.5 to about 4 mg melarsoprol per kg body weight.
[0079] The effect of the therapy with arsenic trioxide or
melarsoprol on development and progression of cancer can be
monitored by any methods known in the art, including but not
limited to determining: levels of tumor specific antigens and
putative biomarkers, e.g., carcinoembryonic antigens (CEA),
alpha-fetoprotein; and changes in morphology and/or size using
computed tomographic scan and/or sonogram.
[0080] Desirable blood levels may be maintained by a continuous
infusion of an arsenic compound as ascertained by plasma levels. It
should be noted that the attending physician would know how to and
when to terminate, interrupt or adjust therapy to lower dosage due
to toxicity, or bone marrow, liver or kidney dysfunctions.
Conversely, the attending physician would also know how to and when
to adjust treatment to higher levels if the clinical response is
not adequate (precluding toxic side effects).
[0081] Again, any suitable route of administration may be employed
for providing the patient with an effective dosage of an arsenic
compound. For example, oral, transdermal, iontophoretic, parenteral
(subcutaneous, intramuscular, intrathecal and the like) may be
employed. Dosage forms include tablets, troches, cachet,
dispersions, suspensions, solutions, capsules, patches, and the
like. (See, Remington's Pharmaceutical Sciences.)
[0082] The pharmaceutical compositions of the present invention
comprise an arsenic compound as the active ingredient,
pharmaceutically acceptable salt thereof, and may also contain a
pharmaceutically acceptable carrier, and optionally, other
therapeutic ingredients, for example all trans retinoic acid. The
term "pharmaceutically acceptable salts" refers to salts prepared
from pharmaceutically acceptable non-toxic acids and bases,
including inorganic and organic acids and bases.
[0083] The pharmaceutical compositions include compositions
suitable for oral, mucosal routes, transdermal, iontophoretic,
parenteral (including subcutaneous, intramuscular, intrathecal and
intravenous), although the most suitable route in any given case
will depend on the nature and severity of the condition being
treated.
[0084] In the case where an intravenous injection or infusion
composition is employed, a suitable dosage range for use is, e.g.,
from about one to about 40 mg arsenic trioxide total daily; about
0.001 to about 10 mg arsenic trioxide per kg body weight total
daily, or about 0.1 to about 10 mg melarsoprol per kg body weight
total daily.
[0085] In addition, the arsenic carrier could be delivered via
charged and uncharged matrices used as drug delivery devices such
as cellulose acetate membranes, also through targeted delivery
systems such as fusogenic liposomes attached to antibodies or
specific antigens.
[0086] In practical use, an arsenic compound can be combined as the
active ingredient in intimate admixture with a pharmaceutical
carrier according to conventional pharmaceutical compounding
techniques. The carrier may take a wide variety of forms depending
on the form of preparation desired for administration, e.g., oral
or parenteral (including tablets, capsules, powders, intravenous
injections or infusions). In preparing the compositions for oral
dosage form any of the usual pharmaceutical media may be employed,
e.g., water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents, and the like; in the case of oral
liquid preparations, e.g., suspensions, solutions, elixirs,
liposomes and aerosols; starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents, and the like in the case of oral solid
preparations e.g., powders, capsules, and tablets. In preparing the
compositions for parenteral dosage form, such as intravenous
injection or infusion, similar pharmaceutical media may be
employed, e.g., water, glycols, oils, buffers, sugar, preservatives
and the like know to those skilled in the art. Examples of such
parenteral compositions include, but are not limited to Dextrose 5%
w/v, normal saline or other solutions. The total dose of the
arsenic compound may be administered in a vial of intravenous
fluid, e.g., ranging from about 2 ml to about 2000 ml. The volume
of dilution fluid will vary according to the total dose
administered. For example, arsenic trioxide supplied as a 10 ml
aqueous solution at 1 mg/ml concentration is diluted in 10 to 500
ml of 5% dextrose solution, and used for intravenous infusion over
a period of time ranging from about ten minutes to about four
hours.
[0087] An exemplary course of treatment of a patient with leukemia,
lymphoma, or solid cancer can involve daily administration by
intravenous infusion of arsenic trioxide in an aqueous solution at
a daily dose of about 0.01 to 1 mg arsenic trioxide per kg of body
weight of the patient. Preferably, about 0.15 mg arsenic trioxide
per kg body weight per day is used. The course of treatment may
continue until bone marrow remission is observed or when side
effects are becoming serious. The course of treatment may be
repeated for up to ten times over approximately 10 months with a
break of about 3 to 6 weeks in between courses. The post-remission
course of treatment involves infusion of arsenic trioxide at a
daily dose of about 0.15 mg per kg of body weight of the patient on
a daily or weekdays-only basis for a cumulative total of 25
days.
5. EXAMPLES
[0088] Described below are examples of uses of the arsenic
compounds of the invention in treatment of various types of
leukemia. Through these and other experiments the arsenic trioxide
formulation of the invention were found to be well-tolerated in
humans. For example, three APL patients were given 0.10 mg of the
arsenic trioxide formulation of the invention once daily (flat
dose) intravenous dose.
[0089] 5.1. Arsenic Trioxide and Melarsoprol Induce Apoptosis in
Myeloid Leukemia Cell Lines
[0090] The activity of As.sub.2O.sub.3 and melarsoprol against
myeloid leukemia cell lines, including the APL cell line NB4-306 (a
retinoic acid-resistant cell line derived from NB4 that no longer
expresses the intact PML-RAR.alpha. fusion protein), HL60, KG-1,
and the myelomonocytic cell line U937 was investigated. To examine
the role of PML in mediating arsenical activity, the inventors also
tested these agents using murine embryonic fibroblasts (MEFs) and
bone marrow (BM) progenitors in which the PML gene had been
inactivated by homologous recombination. Unexpectedly, it is found
that both compounds inhibited cell growth and induced apoptosis in
all cell lines tested. Melarsoprol was more potent than
As.sub.2O.sub.3 at equimolar concentrations ranging from 10.sup.-7
to 10.sup.-5 mol/L. As.sub.2O.sub.3 relocalized PML and
PML-RAR.alpha. onto nuclear bodies, which was followed by PML
degradation in NB4 as well as in HL60 and U937 cell lines. Although
melarsoprol was more potent in inhibiting growth and inducing
apoptosis, it did not affect PML and/or PML-RAR.alpha. nuclear
localization. Moreover, both As.sub.203 and melarsoprol comparably
inhibited growth and induced apoptosis of PML+/+ and PML-/- MEF,
and inhibited colony-forming unit erythroid (CFU-E) and CFU
granulocyte-monocyte formation in BM cultures of PML+/+ and PML-/-
progenitors. A detailed description of the methods, materials, and
results of these experiments is provided in Wang et al., Blood,
1998, 92:1497-1504.
[0091] Results from the experiments show that the cytotoxic effect
of both arsenicals in these cell lines is not mediated by
mechanisms that are dependent on PML or PML-RAR.alpha. expression.
In most lines, melarsoprol was somewhat more potent compared with
As.sub.2O.sub.3 in inhibiting growth and inducing apoptosis, and
the effects of both drugs were dose dependent. As previously
reported, it is confirmed that As.sub.2O.sub.3 relocalized PML
protein onto nuclear bodies and induced PML and PML-RAR.alpha.
degradation in NB4 cells while triggering spoptosis. However,
similar effects were also observed in HL60 and U937 cells which do
not harbor the PML-RAR.alpha. fusion gene. Moreover, melarsoprol
induced apoptosis in all the cell lines tested without altering PML
and/or PML-RAR.alpha..
[0092] The differentiating action of As.sub.2O.sub.3 and
melarsoprol, appeared negligible in vitro, and did not appear to
depend on the expression and/or modulation of PML and/or
PML-RAR.alpha. either. In fact, the small effect observed by the
inventors in long-term cultures. (up to 2 weeks), was comparable in
all the cell lines tested with both compounds.
[0093] It is also found that bcl-2 downregulation, which has been
previously linked to the antileukemic effects of As.sub.203 in APL,
was also not dependent on expression of PML-RAR.alpha. protein,
because it occurred in the NB4 subclone 306 in which the intact
protein is not detectable. Finally, to test whether PML expression
was essential to the antileukemic effects of arsenicals, both
agents were tested in mouse embryonic fibroblasts and BM cells from
animals wherein wild-type PML had been eliminated by homologous
recombination. In these cells wholly lacking PML expression, both
As.sub.2O.sub.3 and melarsoprol were equally effective in
inhibiting growth and inducing apoptosis, and both had similar
effects on normal CFU-E and CFU-GM colony formation. Moreover, no
differences between wild-type and PML-/- cells were observed.
Without being limited by any theory, together, these data strongly
support theory that the antileukemic effects of these arsenicals
occurs independently of both PML and PML-RAR.alpha. expression.
These results are in keeping with the medicinal history of
arsenicals for diseases that are not characterized by alterations
in PML protein such as, for instance, chronic myelocytic
leukemia.
[0094] The results indicate that both As.sub.2O.sub.3 and
melarsoprol are broadly active as antileukemic agents in both
myeloid and lymphoid diseases. In conclusion, the data indicate
that cytotoxic activity is not mediated by the PML protein and
therefore is not limited to diseases that are associated with
alterations in PML expression. Thus, the arsenic compounds of the
invention have a potentially broader therapeutic role that is not
confined to APL.
[0095] 5.2. Clinical Study of Melarsoprol in Patients with Advanced
Leukemia
[0096] Melarsoprol, an organic arsenical synthesized by complexing
melarsen oxide with dimercaprol, has primarily been used for the
treatment of African trypanosomiasis. The effects of melarsoprol
upon induction of apoptosis in cell lines representative of chronic
B-cell lymphoproliferative disorders have been investigated, and
the results are described below.
[0097] Melarsoprol (supplied as Arsobal [36 mg/mL] by Rhone Poulenc
Rorer, Collegeville, Pa.) was diluted in propylene glycol at a
stock concentration of 10.sup.-4 mol/L and stored at room
temperature. As.sub.2O.sub.3 (Sigma, St. Louis, Mo.) was dissolved
in 1.65 mol/L sodium hydroxide (NaOH) at a stock solution of 10-3
mol/L. Serial dilution (10.sup.-6 to 10.sup.-9 mol/L) were made in
RPMI 1640 media. An Epstein-Barr virus (EBV)-transformed
B-prolymphocytic cell line (JVM-2), an EBV-transformed B-cell
chronic lymphocytic leukemia (B-CLL) cell line (183CLL), and one
non-EBV-transformed B-CLL cell line (WSU-CLL) were used as targets.
Dose-response experiments with melarsoprol (10.sup.-7 to 10.sup.-9
mol/L) were performed over 96 hours.
[0098] Unexpectedly, the inventors found that melarsoprol caused a
dose- and time-dependent inhibition of survival and growth in all
three cell lines. In contrast, As.sub.2O.sub.3 at similar
concentrations had no effect on either viability or growth. After
24 hours, all three cell lines treated with melarsoprol (10.sup.-7
mol/L) exhibited morphologic characteristics of apoptosis. A
prominent concentration-dependent downregulation of bcl-2 mRNA
after 24 hours of exposure to melarsoprol in WSU-CLL 183CLL, and
JVM-2 cells was observed. Decrease of bcl-2 protein expression was
also observed in all three cell lines, whereas As.sub.2O.sub.3 had
no effect on this parameter.
[0099] Given that the in vitro data above have shown unexpectedly
broad antileukemic activity for melarsoprol against both myeloid
and lymphoid cells, and generally at lower concentrations than
As.sub.2O.sub.3, a study was initiated to evaluate the
pharmacokinetics, safety, and potential efficacy of melarsoprol in
human patients with relapsed or refractory leukemia.
[0100] Eligible patients were treated with a brief IV injection
daily for 3 days, repeated weekly for 3 weeks, with an additional 3
wk course in responding pts. The initial dose was 1 mg/kg on Day 1,
2 mg/kg on Day 2, and 3.6 mg/kg on Day 3 and all days thereafter.
Parallel in vitro studies included culture sensitivity of fresh
leukemic cells to both melarsoprol and As.sub.2O.sub.3, along with
serial flow cytometric studies of surface antigen expression,
apoptosis, and bcl-2 expression. Three patients with AML and one
with CML have entered the study.
[0101] Using a method based on high performance liquid
chromatography that is sensitive to approximately 10 mg/ml,
preliminary pharmacokinetic data show that peak plasma drug
concentrations were obtained immediately after injection with a
Cmax that ranged from 1.2 ng/ml on day 1 to 2.4 ng/ml on day 3.
While the initial distribution phase was rapid, a prolonged Thy has
suggested release from a deep compartment. Plasma areas under the
concentration.times.time curves (AUC's) were proportional to the
administered dose, ranging from 0.48 ng.multidot.hr/ml on Day 1 to
1.48 ng-hr/ml on Day 3. Detectable concentrations of the drug-were
found in plasma one week after initial dosing. The drug has been
relatively well-tolerated. Adverse effects have included transient
pain at the injection site and mild nausea. No signs of "reactive
encephalopathy" (occasionally observed during treatment of CNS
trypanosomiasis) have been observed.
[0102] Results from these studies suggest that melarsoprol may have
broader activity than inorganic As.sub.2O.sub.3, and that
concentrations which are cytotoxic to leukemic cells in vitro, and
thus therapeutic, are readily achieved in vivo. 5.3. Arsenic
Trioxide Induces Apoptosis In K562 Chronic Myelogenous Leukemia
(CML) Cells
[0103] A Philadelphia chromosome positive CML cell line K562 is
used to determine if arsenic trioxide (As.sub.2O.sub.3) promotes
apqptosis in CML. Suspension cultures of cells in log phase were
exposed to As.sub.2O.sub.3 at concentrations of 1.times.10.sup.-5
M, 5.times.10.sup.-6 M, and 1.times.10.sup.-6 M. Aliquots of cells
were analyzed at various time points over the course of 72 hours to
assess viability and apoptosis. Viability was measured using trypan
blue exclusion; at the same time, apoptosis was detected by
morphology, flow cytometry, and DNA gel electrophoresis.
[0104] Arsenic trioxide at a concentration of 1.times.10.sup.-6 M
had no effect on K562 cell growth or viability. The greatest effect
on cell growth and survival was seen with 1.times.10.sup.-5 M
As.sub.203 K562 cell growth and viability data after 72 hours of
exposure to As.sub.2O.sub.3 are recorded in Table 1:
1 TABLE 1 % Cell Growth Impairment % Viability p value control 0
92.1 .+-. 0.9 5 .times. 10.sup.-6 M As.sub.2O.sub.3 63.0 78.8 .+-.
0.5 0.0001 1 .times. 10.sup.-5 M As.sub.2O.sub.3 75.3 61.9 .+-. 2.9
0.0223
[0105] Evidence that this arsenic-induced decrease in viability
represented apoptosis was analyzed. Morphologic features of
apoptosis including membrane blebbing and nuclear condensation were
evident in stained cytospins of K562 cells incubated with 10.sup.-5
M As.sub.2O.sub.3 for 72 hours. This correlated with evidence of
DNA internucleosomal damage as visualized by gel electrophoresis of
DNA extracted from K562 cells exposed to 10.sup.-5, M
As.sub.2O.sub.3. Quantitative assessment of apoptosis, as measured
by the TUNEL method demonstrated that 75.6%.+-.8.6 (1.times.10-5 M
As.sub.2O.sub.3) cells exhibited apoptosis as compared with
6.3%.+-.3.0 (control) cells at 72 hours. Treatment of K562 cells
with 10.sup.-5 M As.sub.2O.sub.3 resulted in an upregulation of p21
mRNA, as detected by Northern analysis, suggesting an arrest of the
cells in the G1 phase of the cell cycle. This data indicates
arsenic trioxide as a therapeutic agent for CML.
[0106] 5.4. Therapeutic Trials with Retinoic Acid and Arsenic
[0107] Trioxide (As.sub.2O.sub.3) In PML-RAR.alpha. AND
PLZF-RAR.alpha. Transgenic Mice
[0108] Acute promyelocytic leukemia (APL) is associated with
chromosomal translocations which invariably involve the
translocation of the Retinoic Acid Receptor a (RAR.alpha.) locus on
chromosome 17 to other loci in the genome, such as in the majority
of APL cases, the PML gene located on chromosome 15, and in a few
cases the PLZF gene on chromosome 11. Patients harboring the
t(15;17) are sensitive to treatment with A11-Trans Retinoic Acid
(ATRA), yielding complete remission rates of 75% to 95%. APL
associated with the t(11;17) (PLZF-RAR.alpha.) shows a poor
response to ATRA.
[0109] To test the efficacy of As.sub.2O.sub.3 in the treatment of
APL, models of the disease were created in transgenic mice.
Transgenic mice were generated by standard techniques in which the
expression of the PML-RAR.alpha. or PLZF-RAR.alpha. fusion proteins
is placed under the control of a myeloid-promyelocytic specific
human Cathepsin-G (hCG) minigene. Both hCG-PML/RAR.alpha. and
hCG-PLZF-RAR.alpha. transgenic mice develop myeloid leukemia with
features of APL similar to those in humans.
[0110] Therapeutic trials on these leukemic mice with the following
regimens were started: 1) ATRA: 1.5 .mu.g per gram of body weight
per day administered orally; and 2) ATRA: 6 .mu.g per gram of body
weight per day administered intraperitoneally. Mice were bled once
a week to evaluate the response.
[0111] PML/RAR.alpha. leukemias responded well to ATRA with high
remission rates (80% with regimen 1). Surprisingly, in vitro, ATRA
induced differentiation, and inhibited growth of leukemic cells as
well as leukemic colony formation in bone marrow and spleen
progenitors assays in both PML-RAR.alpha. and PLZF-RAR.alpha.
leukemias. Furthermore, in ex vivo experiments, leukemic cells from
PLZF-RAR.alpha. mice lost their tumorigenic capacity when
transplanted in recipient nude mice upon pre-incubation with ATRA,
while untreated cells were tumorigenic. However, in vivo,
PLZF-RAR.alpha. leukemias responded poorly to ATRA (28% with
regimen 1), while higher doses of ATRA appeared more effective (50%
with regimen 2). In conclusion, leukemias in PLZF-RAR.alpha.
transgenic mice are sensitive to ATRA treatment, but might require
therapeutic regimens with high doses of ATRA. These findings have
direct implications in the treatment of APL patients with
t(11;17).
[0112] In both PML-RAR.alpha. and PLZF-RAR.alpha. leukemias, ATRA
prolonged survival, but leukemias relapsed shortly after remission
was achieved, and were refractory to further ATRA-treatment. The
two transgenic mouse models is also used to test the efficacy and
dosage of As.sub.2O.sub.3, and ATRA+As.sub.2O.sub.3 in combination
for treatment of APL patients resistant to ATRA, and in APL
associated with the t(11;17). A regimen of As.sub.2O.sub.3 at 6
.mu.g per day or a combination of As.sub.2O.sub.3 at 6 .mu.g and
ATRA at 1.5 or 6 .mu.g per gram of body weight per day is
administered intraperitoneally. Mice are bled weekly to evaluate
the remission of the APL.
[0113] 5.5. Manufacture and Stability of Pharmaceutical
Formulation
[0114] Solid ultrapure arsenic trioxide (As.sub.2O.sub.3) was
solubilized in a solution of 5 M sodium hydroxide (NaOH). The
suspension was stirred at ambient temperature for 5 minutes which
yielded a clear, homogenous solution. The As.sub.2O.sub.3 solution
(2 mL, 1.0 M) was added to 393.6 mL of H.sub.2O in a 500 ml
Erlenmeyer flask, which yielded an As.sub.2O.sub.3 concentration of
1 mg/mL at pH=12. A 5.0 M HCl solution was prepared by dilution of
HCl (49.26 mL, 37% wt/wt, 10/15 M) with H.sub.2O. (50.74-mL) in a
250 mL Erlenmeyer flask. The HCl solution was later transferred via
syringe to a 1000 mL empty evacuated container. The As.sub.2O.sub.3
solution was back titrated with HCl (0.725 mL, 5.0 M) to pH 8.0.
Approximately 10 mL of the backtitrated As.sub.2O.sub.3 solution
was filtered through a Millex-GS 0.22 .mu.m filter unit and was
added to each of approximately 30 sterile evacuated sterile vials.
To make the pharmaceutical composition which would be injected
intravenously into patients, 10 mL of this solution was withdrawn
from two of the vials and was added to a 500 mL 5%-dextrose
solution which yielded a final pH of 6.5.
[0115] The high purity of the bulk starting material was confirmed
(see Table 1) by atomic absorptiometry. Duplicate samples of four
intermediate or final-step solutions were assayed for total arsenic
content. Assay bulk powder confirmed the extremely high purity of
the starting material. Data for arsenic content of the intermediate
and finished product solutions are presented in Table 2 below.
[0116] The data below show that the solutions are stable in that
there does not appear to be any indication of weight loss of
arsenic over time.
2TABLE 2 Arsenic content (ppm) of intermediate formulation and
finished product solution of arsenic trioxide. Sample Code A-01*
A-02 A-03 A-04 A-05 Aliquot A 140,600 600 707 629 680 Aliquot B
139,000 564 703 688 687 Assay 1.1% 6% 0.57% 8.7% Variance *Identity
of sample codes: A-01: Intermediate product solution after initial
solubilization in NaOH. A-02: Intermediate product solution prior
to HCl titration. A-03: Intermediate product prior to Millex
filtration. A-04: Finished product from sterile 10 ml fill vial
immediately after manufacturing. A-05: Finished product from capped
vials two months after manufacturing.
6. EXAMPLES: CLINICAL TRIALS IN APL PATIENTS
[0117] Arsenic trioxide was evaluated in patients with APL to
determine whether this agent induced either cytodifferentiation or
apoptosis. Twelve patients who had relapsed from extensive prior
therapy were treated with arsenic trioxide at doses ranging from
0.06 to 0.2 mg/kg per day until a bone marrow remission was
achieved. Bone marrow mononuclear cells were serially monitored by
flow cytometry for immunophenotype, fluorescence in situ
hybridization (FISH), reverse transcription polymerase chain
reaction (RT-PCR) assay for PML/RAR-.alpha. expression, and Western
blot expression of the apoptosis-associated proteins, caspases 1, 2
and 3. The results showed that low-doses of arsenic trioxide are
highly effective for inducing complete remission in relapsed
patients with APL. Clinical response is associated with incomplete
cytodifferentiation and induction of apoptosis with caspase
activation in leukemic cells.
[0118] 6.1. Methods
[0119] Clinical protocol: Eligibility requirements included a
diagnosis of APL confirmed by cytogenetics or fluorescence in situ
hybridization (FISH) analysis for a t(15;17) translocation, or by
reverse transcriptase polymerase reaction (RT-PCR) assay for
PML/RAR-.alpha.. Patients must have relapsed from standard therapy
that had included all-trans retinoic acid plus a combination of
cytotoxic drugs. Signed informed consent was required, and the
protocol was reviewed and approved by this center's institutional
review board
[0120] Arsenic trioxide treatment: Arsenic trioxide was supplied as
an aqueous solution in 10 ml vials containing 1 mg/ml of drug. The
drug was further diluted in 500 ml of 5% dextrose solution and
infused intravenously over 2 to 4 hours once per day. While the
initial cohort of patients received either 10 or 15 mg/day as a
flat dose, the referral of two children prompted the invention of a
weight-based regimen (0.15 mg/kg/day) that was heretofore unknown.
The drug was given daily until bone marrow remission was observed.
Patients who achieved complete remission were eligible for
treatment with additional courses of therapy 3 to 6 weeks after the
preceding course. Subsequent courses were generally given at a dose
of 0.15 mg/kg/day for a cumulative total of 25 days, administered
either daily or on a weekdays-only schedule, for a maximum total of
6 courses over approximately 10 months.
[0121] Monitoring during study: Patients with coagulopathy were
transfused with platelets and fresh-frozen plasma to maintain the
platelet count and fibrinogen at target levels .gtoreq.50,000
cells/cu mm and .gtoreq.100 mg/dL, respectively. Blood counts,
coagulation studies, serum chemistry profiles, urinalyses, and
electrocardiograms were serially obtained. A bone marrow aspiration
and/or biopsy was performed at baseline and periodically thereafter
until remission was documented.
[0122] Conventional response criteria were observed, which included
recovery of bone marrow to <5% blasts, peripheral blood
leukocytes.gtoreq.3,000 cells/cu mm, and platelets.gtoreq.100,000
cells/cu mm.
[0123] Cellular immunophenotype studies: Heparinized bone marrow or
blood samples were collected and mononuclear cells were isolated by
Ficoll-Hypaque centrifugation. Surface membrane antigens were
detected by direct immunofluorescence staining using fluorescein
isethiocynate (FITC) or phycoerythrin conjugated monoclonal
antibodies: CD16 (Leu 11a), CD11b, CD33 (Leu M9), HLA-DR, CD45, and
CD14, purchased from either Becton-Dickinson (Mountainview, Calif.)
or Immunotech Immunology (Marseille, France). Dual-color staining
was performed by incubating cells simultaneously with two
monoclonal antibodies, including CD33-PE/CD11b-FITC and
CD33-PE/CD16-FITC. Negative controls using irrelevant monoclonal
immunoglobulins of the same isotype were analyzed concurrently.
Flow cytometric analyses were performed on an EPICS Profile II flow
cytometer (Coulter Electronics) equipped with a 488 nm argon laser.
Forward and side-scatter cell parameters were measured and combined
with CD45/CD14 staining to identify populations of interest and to
exclude monocytes from the analysis gate. The Multiparameter Data
Acquisition and Display System (MDADS, Coulter Electronics) was
used to acquire and analyze data.
[0124] Fluorescence in situ hybridization (FISH): Selected
specimens that had undergone immunofluorescence staining for CD33
and CD11b were sorted for cells that coexpressed both antigens
using a FACStar Plus cell-sorter (Becton-Dickinson) Separated cells
were incubated in culture media at 37.degree. C. for one hour,
treated with hypotonic solution 0.075M KCl for 5 minutes, fixed in
3:1 methanol:acetic acid fixative, and airdried. Interphase FISH
was performed using a specific PML/RAR-.alpha. translocation
dual-color probe (Vysis; Downer's Grove, Ill.). Briefly, DNA from
interphase cells was denatured by immersing slides in a solution of
50% formamide/2.times.SSC at 73.degree. C. for 5 minutes; the
slides were then dehydrated in alcohol and air dried. A mixture of
probe in hybridization mixture was applied, covered with a cover
slip, and sealed with rubber cement. Hybridization was carried out
at 37.degree. C. in a moist chamber for approximately 12 to 16
hours. Following hybridization, unbound probe was removed by
washing the slides at 45.degree. C. in 50% formamide/2.times.SSC
solution three times for 10 minutes each, followed by a wash in
2.times.SSC/0.1 NP-40 solution at 45.degree. C. for 5 minutes.
Slides were then air dried and counter-stained with
4',6-diamidino-2-phenylindole and covered with a glass coverslip.
Analysis of interphase cells for fluorescent signals was performed
with a Photometrics Sensys camera fitted to a Zeiss axioscope. A
minimum of 300 cells was studied for each sample.
[0125] Western blot analysis: Cells were lysed in a buffer
containing 50 mM Tris-HCl, 0.5 mM ethylene glycol
[bis]-[aminioacyl] tetra acetic acid, 170 mM NaCl, 1 mM
dithiothreitol, 0.2% NP-40, 0.01 U/mL aprotinin, 10 .mu.g/mL
leupeptin, 10 .mu.g/mL pepstatin, and 1 .mu.M phenylmethylsulfonyl
fluoride (all from Sigma). The lysates were then sonicated using a
ultrasonic homogenizer (4710 series, Cole Parmer Instruments,
Chicago, Ill.) and centrifuged at 7,500 g (Sorvall Instruments,
Newtown, Conn.). Protein content of the lysates was determined
using a BioRad Protein Assay Kit (Bio-Rad Laboratories, Hercules,
Calif.) at 595 nm with a BSA standard. A sample buffer containing
10% glycerol, 0.4% SDS, 0.3% bromphenol blue, 0.2% pyronin Y, in
1.times. stacking buffer (Tris base 0.5 M, 0.8% SDS), 20%
2-mercaptoethanol, was added to the cell lysates, which were
heat-denatured at 95.degree. C. for 3 min. Subsequently, 15
.mu.g/lane of protein was loaded on a SDS-polyacrylamide gel
containing 12.5% polyacrylamide and was size-fractionated by
electrophoresis. Proteins were electroblotted onto Tras-Blot.RTM.
transfer medium (Bio-Rad) and stained with Ponceau-S as an internal
loading control. Rabbit anti-human monoclonal antibodies, including
caspase 1, caspase 2 (both from Santa Cruz Biotechnology, Santa
Cruz, Calif.), and caspase 3 (PharMingen, San Diego, Calif.) were
added, and bound antibodies were detected using the ECL.TM.
chemiluminescence detection system (Amersham, Arlington Heights,
Ill.). Protein bands were quantified by computer densitometry.
[0126] RT-PCR analysis for PML/RAR-.alpha.: RT-PCR was performed
using methods previously described (Miller et al., 1992, Proc.
Natl. Acad. Sci. 89:2694-8; Miller et al., 1993, Blood,
82:1689-94).
[0127] 6.2. Results
[0128] Patients: Twelve patients with relapsed or refractory APL
were treated. All patients had received extensive prior therapy
with retinoids and cytotoxic drugs (Table 3). Two patients had
relapsed from allogeneic bone marrow transplantation, one of whom
had also failed donor T-cell reinfusion. One patient was being
maintained on hemodialysis for chronic renal failure.
[0129] Clinical Efficacy: Eleven of the 12 patients achieved a
complete remission after arsenic trioxide treatment. The patient
who entered on hemodialysis sustained an intracranial hemorrhage on
day 1 and died on day 5. The median duration of therapy in
responding patients was 33 days (range, 12 to 39 days), the median
daily dose was 0.16 mg/kg (range, 0.06 to 0.2 mg/kg), and the
median cumulative dose during induction was 360 mg (range, 160 to
515 mg) (Table 3). Complete remission by all criteria was attained
at a median time of 47 days (range, 24 to 83 days) after initiation
of therapy. Remission by bone marrow criteri--the determining
factor for discontinuing therapy--was achieved first, usually
followed in sequence by recovery of peripheral blood leukocytes and
platelets. Over the range of doses used in this study, no
differences in efficacy or time to response were obvious. After 2
courses of therapy, 8 of 11 patients tested had converted their
RT-PCR assays for PML/RAR-.alpha. from positive to negative.
[0130] All 11 patients in complete remission completed at least 1
post-remission treatment course with arsenic trioxide. Four, two,
and one patient each have completed a total of three, four and five
treatment courses, respectively. The median duration of remission
is 5+months (range, 1 to 9+months). However, 3 of the 11 patients
relapsed during the second treatment course; none of these patients
had converted their RT-PCR assays, and each appeared to have
rapidly acquired drug resistance. Two of these individuals have
since expired from progressive leukemia.
[0131] Adverse Events: The clinical condition of patients in this
study was highly variable, which reflected their extensive prior
therapy. The protocol did not require hospitalization; three
patients completed induction therapy entirely as outpatients, and
one other individual was hospitalized solely for placement of a
venous catheter. However, 8 patients were hospitalized for
complications of leukemia, 5 of whom required transfer to an
intensive care unit, endotracheal intubation and assisted
ventilation for complications that included pulmonary hemorrhage,
renal failure, sepsis, graft vs. host disease non-specific
pulmonary infiltrates, or hypotension. One patient required
insertion of a permanent pacemaker after second-degree heart block
developed in the setting of severe metabolic acidosis,
hyperkalemia, hypotension, and renal insufficiency. However, the
heart block reversed despite rechallenge with further arsenic
trioxide therapy. The drug was temporarily withheld due to serious
intercurrent medical complications in 5 patients for a median of 2
days (range, 1 to 5 days). Two patients developed symptoms similar
to that of the "retinoic acid syndrome"; both were presumptively
treated with dexamethasone and improved. Only 2 patients required
no platelet transfusions whatsoever; the median number of platelet
units transfused was 61 (range, 0 to 586 units).
[0132] The median total peripheral blood leukocyte count at entry
was 4,700 cells/cu mm (range, 500 to 144,000 cells/cu mm). Six
patients developed leukocytosis (i.e., .gtoreq.20,000 cells/cu mm)
that ranged from 20,800 to 144,200 cells/cu mm. No: additional
therapy was administered to these patients, and the leukocytosis
resolved in all cases without further intervention.
[0133] Common adverse reactions included lightheadedness during the
infusion, fatigue, musculoskeletal pain, and mild hyperglycemia.
Three patients developed dysesthias presumably due to peripheral
neuropathy. However, 2 of these patients had been immobilized for
prolonged periods during assisted ventilation, and the other
patient had an antecedent neuropathic history.
[0134] Immunophenotype studies: APL is characterized by cells that
express CD33, an antigen typically associated with primitive
myeloid cells. Arsenic trioxide therapy induced a progressive
decrease in the proportion of cells that solely expressed CD33,
along with an increase in the proportion of cells that expressed
CD11b, an antigen associated with mature myeloid elements. While
these changes would be anticipated from any agent that induced
remission in APL, arsenic trioxide also induced expression of cells
that simultaneously expressed both antigens. In most cases, these
dual-expressing cells dominated the myeloid cell population, and
they persisted for extended periods after complete remission was
achieved by clinical criteria.
[0135] Fluorescence in situ hybridization analysis: Bone marrow
mononuclear cells taken from a patient both early and later in
complete remission were sorted by flow cytometry for coexpression
of CD33 and CD11b. Using fluorescence in situ hybridization (FISH)
analysis, three hundred cells were examined early in remission.
Similar to control APL cells, the majority of these cells yielded a
hybrid signal, indicating a translocation between PML and
RAR-.alpha. genes and their origin from the neoplastic clone.
However, when cells from the same patient were again sorted using
these same parameters later in remission, only the normal pattern
of fluorescence signals was detected, indicating their derivation
from normal hematopoietic progenitors.
[0136] Western blot analysis: Protein extracts from bone marrow
mononuclear cells were serially examined by Western blot analysis.
The analysis showed that the precursor forms of caspase 2 and
caspase 3 were upregulated in vivo in response to arsenic trioxide
treatment. Moreover, this treatment also induced expression of
cleaved fragments of caspase 1, indicating activation of the
enzyme. There is also some indication that expression of the
cleaved form of caspase 3 is increased. The antibody used in these
experiments does not react with the cleaved form of caspase 2.
[0137] 6.3. Discussion
[0138] In this study, with few exceptions, patients admitted to the
trial had sustained multiple relapses and were resistant to
conventional chemotherapy, retinoids, or bone marrow
transplantation. At entry, patients in this study suffered from
numerous leukemia-related complications, including respiratory
failure, disseminated Varicella zoster infection, cavitary
aspergillosis, chronic renal failure, and graft-vs.-host disease.
Moreover, 5 of the 12 patients required admission to an intensive
care unit for assisted ventilation and supportive care, but these
complications were not directly related to arsenic trioxide
therapy.
[0139] Virtually all patients with a confirmed diagnosis of APL
attained remission without the early mortality associated with
retinoid therapy. Although less commonly observed compared with
all-trans retinoic acid treatment, arsenic trioxide induced
striking leukocytosis in several patients. Upon withholding other
cytotoxic drugs, the leukocytosis disappeared as patients attained
remission. Despite 3 early relapses, 8 of 11 patients tested
converted RT-PCR assays for PML/RAR-.alpha. (a molecular marker of
residual disease) to negative, a phenomenon that is unusual after
all-trans retinoic acid treatment alone. Finally, arsenic trioxide
is active in APL over at least a three-fold dose range from 0.06 to
0.20 mg/kg.
[0140] All-trans retinoic acid induces "terminal" differentiation
of APL cells, but the cytodifferentiating effects of arsenic
trioxide appear to be incomplete. Arsenic induces a population of
cells that simultaneously express surface antigens characteristic
of both mature and immature cells (i.e. CD11b and CD33,
respectively). Early during induction, these cells retain the
t(15;17) translocation that characterizes APL. Unexpectedly, these
cells persisted in the bone marrow despite the achievement of a
clinically complete remission; however, later in remission, the
coexpressing cells--while still readily detectable--were no longer
positive by in situ hybridization. The morphologic appearance of
leukemic cells during therapy is also far less distinctive than
that observed during therapy with all-trans retinoic acid. In fact,
leukemic cells from many patients displayed few morphologic changes
for 10 or more days, after which the proportion of leukemic cells
progressively decreased.
[0141] Following "non-terminal" differentiation, arsenic trioxide
appeared to induce apoptosis, coincident with increased expression
and conversion of cysteine proteases (termed caspases) from
inactive precursors to activated enzymes. The caspase pathway has
only recently been characterized as an important pathway of
programmed cell death. Initially recognized due to homology between
the C. elegans protein ced-3 and mammalian interleukin-1.beta.
converting enzyme (ICE), the family of caspases now encompasses at
least 10 different proteins that cleave a number of polypeptides.
In leukemic cell lines, caspase activation is inducible with a
number of cytotoxic agents, including all-trans retinoic acid.
Since these enzymes induce widespread proteolysis, it is
conceivable that PML/RAR-.alpha. is a caspase substrate.
[0142] A final similarity shared by arsenic trioxide and all-trans
retinoic acid is the rapid development of clinical resistance in
some individuals. Leukemic cells taken from two patients who
relapsed retained in vitro sensitivity over concentrations ranging
from 10.sup.-4 M to 10.sup.-7M. Relative arsenic resistance due to
decreased intracellular transport has been described in association
with down-regulation of membrane transporters encoded by the ars
operon in bacterial cells.
[0143] Resistance in mammalian cells is less well-characterized,
but alterations in membrane transport or efflux are probably
important factors.
[0144] In summary, arsenic trioxide induces complete remission in
patients with APL who have relapsed from extensive prior therapy.
This drug causes partial but incomplete cytodifferentiation of
leukemic cells, followed by caspase activation and induction of
apoptosis.
3TABLE 3 Clinical characteristics and induction therapy results of
patients with acute promyelocytic leukemia treated with arsenic
trioxide. Treatment Time To Platelets Leukocytes Age No. of
duration Daily Dose Cumulative Remission .gtoreq.100,000/
.gtoreq.3,000/ (yrs) Relapses (days) (mg/kg) Dose (mg) (days) cu mm
cu mm 36 1* 36 .16 360 54 36 54 45 3*.sup.a 39 .12 390 83 39 83 31
3.sup.a,b 37 .18 370 41 39 41 25 2 16 .06 160 24 16 16 62 2*.sup.d
30 .11 300 41 41 31 75 1 12 .20 180 30 30 30 40 1* 33 .16 495 47 47
43 13 2*.sup.a,b 27 .18 270 50 41 52 9 1* 33 .17 165 28 28 28 70
1.sup.c 28 .16 420 77 77 49 28 2* 36 .15 515 54 47 54 25 3 5 .15 75
.dagger. .dagger. .dagger.
[0145] All patients had previously received one or more courses of
all-trans retinoic acid, plus an anthracycline antibiotic plus
cytosine arabinoside. * Denotes individuals with proven retinoid
resistance (i.e. lack of response during reinduction or relapse
while on retinoid maintenance); .dagger. Denotes patient who died
early. Other treatment: .sup.a mitoxantrone/etoposide; .sup.b
allogeneic bone marrow transplantation; .sup.c
methotrexate/vincristine/6-mercaptopurine; .sup.d 9-Cis retinoic
acid plus M195 (anti-CD33 monoclonal antibody).
7. EXAMPLES: CLINICAL USE IN LYMPHOMA
[0146] Based upon the initial discovery of the antitumor effects of
arsenic trioxide in vitro against B-cell-lymphocyte lines, the
inventors treated one patient with intermediategrade large cell
lymphoma who had relapsed from multiple forms of conventional
therapy, including autologous bone marrow transplantation. Despite
rapid progression of his disease prior td starting the arsenic
trioxide therapy, treatment with arsenic trioxide effected a major
(>50%) shrinkage in the size of his cancerous lymph nodes and
spleen, which was also associated with a major improvement of his
quality of life.
8. EXAMPLES: CLINICAL USE IN NON-HEMATOPOIETIC CANCER
[0147] Arsenic trioxide was also used to treat colon cancer. In a
preliminary test, one patient with colon cancer who received one
treatment with arsenic trioxide showed a major reduction in his
serum CEA (carcinoembryonic antigen) level. The patient received
daily intravenous infusion of 0.1-5 mg arsenic trioxide per kg body
weigh per day for five days. A change in the level of CEA from
19,901 ng/ml to 15,266 ng/ml, a 23% reduction, was observed. It is
well known that the a reduced level of serum CEA is associated with
ant-itumor response.
[0148] Clinical data confirms that arsenic trixoide can also be
used to treat other non-hematopoietic cancer, such as colon
cancer.
9. EXAMPLES: PHARMACOKINETICS STUDIES
[0149] Several dose-ranging studies were conducted to examine the
pharmacokinetics (PK) and biological effects of As.sub.2O.sub.3 in
patients with APL and in patients with other hematologic diseases.
In patients with APL, marrow mononuclear cells were serially
monitored by flow cytometry for immunophenotype, fluorescence in
situ hybridization (FISH), and Western blot expression of the
apoptosis-associated proteins, caspases 1, 2 and 3. Cells that
coexpressed CD11b and CD33, and which by FISH analysis carried the
t(15;17) translocation, progressively increased during treatment
and persisted early in complete remission. As.sub.2O.sub.3 also
induced in vivo expression of the proenzymes of caspase 2 and
caspase 3, and activation of both caspase 1 and caspase 3. PK
analysis of blood and urine for elemental arsenic (As) content
showed that As was distributed in both plasma and red blood cell
fractions of whole blood. Parallel elimination curves suggested
that these 2 compartments were freely exchangeable, and decayed
from peak values with initial half lives of about 60 mins. The mean
AUC on day 1 was about 400 ng-hr/ml. Approximately 20% of the
administered dose was recovered in urine within the first 24
hrs.
[0150] We then initiated a dose-ranging study in patients with
diseases other than APL using a daily intravenous dosing schedule
for a cumulative total of 25 days per treatment course every 3-5
weeks at dose levels of 0.1 and 0.15 mg per kg body weight per day.
To date, 10 patients have been accrued, including patients with CLL
(2 patients), AML (3 patients), lymphoma (4 patients), and CML (1
patient). Five patients were removed from the study early due to
rapid progression, and 5 completed the planned 25-day course. Over
this dose range, the drug has proved well-tolerated; adverse
effects have included skin rash, lightheadedness during the
infusion, fatigue, and QTc prolongation on EKG. Results from this
ongoing study show that clinical use of As.sub.2O.sub.3 induces
partial differentiation and apoptosis in APL, but that the
therapeutic effects of this agent are not confined to this
disorder.
[0151] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0152] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *