U.S. patent application number 12/433028 was filed with the patent office on 2009-09-10 for antitumoral treatments.
This patent application is currently assigned to Pharma Mar, S.A.U.. Invention is credited to Debabrata Barnejee, Joseph R. Bertino, Glynn Thomas Faircloth, Saydam Guray, Jose Jimeno.
Application Number | 20090227490 12/433028 |
Document ID | / |
Family ID | 32990868 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090227490 |
Kind Code |
A1 |
Bertino; Joseph R. ; et
al. |
September 10, 2009 |
Antitumoral Treatments
Abstract
Aplidine and aplidine analogues are of use for the treatment of
cancer, in particular in the treatment of leukemias and lymphomas,
especially in combination therapies.
Inventors: |
Bertino; Joseph R.; (New
Brunswick, NJ) ; Barnejee; Debabrata; (New Brunswick,
NJ) ; Guray; Saydam; (New Brunswick, NJ) ;
Jimeno; Jose; (Madrid, ES) ; Faircloth; Glynn
Thomas; (Cambridge, MA) |
Correspondence
Address: |
KING & SPALDING
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036-4003
US
|
Assignee: |
Pharma Mar, S.A.U.
Madrid
ES
|
Family ID: |
32990868 |
Appl. No.: |
12/433028 |
Filed: |
April 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10546750 |
Nov 4, 2005 |
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PCT/US04/07606 |
Mar 12, 2004 |
|
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12433028 |
|
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60454125 |
Mar 12, 2003 |
|
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 19/08 20180101;
A61K 38/15 20130101; A61P 35/00 20180101; A61P 35/04 20180101; A61P
35/02 20180101; A61K 38/15 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/10 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61P 35/00 20060101 A61P035/00; A61P 35/02 20060101
A61P035/02 |
Claims
1. A method of treating leukemia or lymphoma comprising
administering to a patient in need of such treatment a synergistic
amount of aplidine and a drug, wherein said drug is selected from
the group consisting of vinblastine and doxorubicin.
2. The method according to claim 1, wherein said method is a method
of treating leukemia.
3. The method according to claim 2, wherein said drug is
vinblastine.
4. The method according to claim 2, wherein said drug is
doxorubicin.
5. The method according to claim 1, wherein said method is a method
of treating lymphoma.
6. The method according to claim 5, wherein said drug is
vinblastine.
7. The method according to claim 5, wherein said drug is
doxorubicin.
8. The method according to claim 1, wherein said aplidine is
administered prior, with, or after administering said drug selected
from the group consisting of vinblastine and doxorubicin.
9. The method according to claim 8, wherein said aplidine is
administered prior to said drug selected from the group consisting
of vinblastine and doxorubicin.
10. The method according to claim 8, wherein said aplidine is
administered with said drug selected from the group consisting of
vinblastine and doxorubicin.
11. The method according to claim 8, wherein said aplidine is
administered after said drug selected from the group consisting of
vinblastine and doxorubicin.
12. A method of increasing the therapeutic efficacy of a drug
effective in the treatment of leukemia or lymphoma wherein said
drug is selected from the group consisting of vinblastine and
doxorubicin, and wherein said method comprises administering a
synergistic amount of said drug and aplidine to a patient in
thereof.
13. The method according to claim 12, wherein said treatment is
treatment of leukemia.
14. The method according to claim 13, wherein said drug is
vinblastine.
15. The method according to claim 13, wherein said drug is
doxorubicin.
16. The method according to claim 12, wherein said treatment is
treatment of lymphoma.
17. The method according to claim 16, wherein said drug is
vinblastine.
18. The method according to claim 16, wherein said drug is
doxorubicin.
19. A kit for use in the treatment of leukemia or lymphoma which
comprises a dosage form of aplidine, a dosage form of a drug
selected from the group consisting of vinblastine and doxorubicin,
and instructions for the use of aplidine and said drug in
combination for the treatment of cancer.
Description
[0001] This application claims priority as a continuation under 35
U.S.C. .sctn.120 to U.S. patent application Ser. No. 10/546,750,
filed Nov. 4, 2005, which is the entry of the national phase under
35 U.S.C. .sctn.371 of PCT/US2004/007606, filed Mar. 12, 2004,
which claims benefit of priority to U.S. Provisional Application
No. 60/454,125, filed Mar. 12, 2003, the contents of each of which
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to combinations of aplidine or
aplidine analogues with other antitumoral agents, and the use of
these combinations in the treatment of cancer, in particular in the
treatment of leukemias and lymphomas.
BACKGROUND OF THE INVENTION
[0003] Aplidine (Dehydrodidemnin B) is a cyclic depsipeptide that
was isolated from a Mediterranean marine tunicate, Aplidium
albicans, and it is the subject of WO 9109485. It is related to
compounds known as didemnins, and has the following structure:
##STR00001##
[0004] More information on aplidine, aplidine analogues, their
uses, formulations and synthesis can be found in patent
applications WO 98 1352, WO 99 42125, WO 01 76616, WO 01 35974, WO
02 30441 and WO 02 02596. We incorporate by specific reference the
content of each of these PCT texts.
[0005] In both animal and human preclinical studies and in clinical
Phase I studies this agent has been shown to have cytotoxic
potential against a broad spectrum of tumor types including
leukemia and lymphoma. See for example: [0006] Faircloth, G. et
al.: "Dehydrodidemnin B (DDB) a new marine derived anticancer agent
with activity against experimental tumour models", 9th NCI-EORTC
Symp New Drugs Cancer Ther (March 12-15, Amsterdam) 1996, Abst 111;
[0007] Faircloth, G. et al.: "Preclinical characterization of
aplidine, a new marine anticancer depsipeptide", Proc Amer Assoc
Cancer Res 1997, 38: Abst 692; [0008] Depenbrock H, Peter R,
Faircloth G T, Manzanares I, Jimeno J, Hanauske A R.: "In vitro
activity of Aplidine, a new marine-derived anti-cancer compound, on
freshly explanted clonogenic human tumour cells and haematopoietic
precursor cells" Br. J. Cancer, 1998; 78: 739-744; [0009] Faircloth
G, Grant W, Nam S, Jimeno J, Manzanares I, Rinehart K.:
"Schedule-dependency of Aplidine, a marine depsipeptide with
antitumor activity", Proc. Am. Assoc. Cancer Res. 1999; 40: 394;
Broggini M, Marchini S, D'Incalci M, Taraboletti G, Giavazzi R,
Faircloth G, Jimeno J.: "Aplidine blocks VEGF secretion and
VEGF/VEGF-R1 autocrine loop in a human leukemic cell line", Clin
Cancer Res 2000; 6 (suppl): 4509; [0010] Erba E, Bassano L, Di
Liberti G, Muradore I, Chiorino G, Ubezio P, Vignati S, Codegoni A,
Desiderio M A, Faircloth G, Jimeno J and D'Incalci M.: "Cell cycle
phase perturbations and apoptosis in tumour cells induced by
aplidine", Br J Cancer 2002; 86: 1510-1517; [0011] Paz-Ares L,
Anthony A, Pronk L, Twelves C, Alonso S, Cortes-Funes H, Celli N,
Gomez C, Lopez-Lazaro L, Guzman C, Jimeno J, Kaye S.: "Phase I
clinical and pharmacokinetic study of aplidine, a new marine
didemnin, administered as 24-hour infusion weekly" Clin. Cancer
Res. 2000; 6 (suppl): 4509; [0012] Raymond E, Ady-Vago N, Baudin E,
Ribrag V, Faivre S, Lecot F, Wright T, Lopez Lazaro L, Guzman C,
Jimeno Ducreux M, Le Chevalier T, Armand J P.: "A phase I and
pharmacokinetic study of aplidine given as a 24-hour continuous
infusion every other week in patients with solid tumor and
lymphoma", Clin. Cancer Res. 2000; 6 (suppl): 4510; [0013] Maroun
J, Belanger K, Seymour L, Soulieres D, Charpentier D, Goel R,
Stewart D, Tomiak E, Jimeno J, Matthews S.: "Phase I study of
aplidine in a 5 day bolus q 3 weeks in patients with solid tumors
and lymphomas", Clin. Cancer Res. 2000; 6 (suppl): 4509; [0014]
Izquierdo M A, Bowman A, Martinez M, Cicchella B, Jimeno J, Guzman
C, Germa J, Smyth J.: "Phase I trial of Aplidine given as a 1 hour
intravenous weekly infusion in patients with advanced solid tumors
and lymphoma", Clin. Cancer Res. 2000; 6 (suppl): 4509.
[0015] Mechanistic studies indicate that aplidine can block VEGF
secretion in ALL-MOLT4 cells and in vitro cytotoxic activity at low
concentrations (5 nM) has been observed in AML and ALL samples from
pediatric patients with de novo or relapsed ALL and AML. Aplidine
appears to induce both a G1 and a G2 arrest in drug treated
leukemia cells in vitro. Apart from down regulation of the VEGF
receptor, little else is known about the mode(s) of action of
aplidine.
[0016] In phase I clinical studies with aplidine, L-carnitine was
given as a 24 hour pretreatment or co-administered to prevent
myelotoxicity, see for example WO 02 30441. Co-administration of
L-carnitine was proven to be able to improve the recovery of the
drug induced muscular toxicity and has allowed for dose escalation
of aplidine.
[0017] Thus in clinical Phase I studies aplidine was not myelotoxic
at maximum tolerated doses, except for mild lymphopenia. These
characteristics make aplidine a potentially useful agent for the
treatment of leukemia. Adding aplidine to the current chemotherapy
for leukemia could improve efficacy without the necessity of dose
reductions of drugs with proven antileukemic activity, because of
increased myelotoxicity. This seems especially relevant for the
treatment of relapsed ALL and newly diagnosed and relapsed AML,
since these are diseases with a relatively poor prognosis, which
are currently being treated with myelotoxic drug combinations.
SUMMARY OF THE INVENTION
[0018] We have for the first time established that aplidine and
aplidine analogues potentiate other anticancer agents and therefore
can be successfully used in combination therapy for the treatment
of cancer. This invention is directed to pharmaceutical
compositions, pharmaceutical dosage forms, kits and methods for the
treatment of cancer using these combination therapies.
[0019] In accordance with one aspect of this invention, we provide
effective combination therapies based on aplidine and aplidine
analogues, using other drugs which are effective in the treatment
of cancer. Preferably the other drug is effective in the treatment
of leukemia and/or lymphoma. Most preferably the other drug is
selected from the group consisting of methotrexate, cytosine
arabinoside, mitoxantrone, vinblastine, methylprednisolone and
doxorubicin.
[0020] In another embodiment the invention encompasses a method of
treating primary and/or metastatic cancer comprising administering
to a patient in need of such treatment a therapeutically effective
amount of aplidine or an aplidine analogue, or a pharmaceutically
acceptable prodrug, salt, solvate or hydrate thereof, and a
therapeutically effective amount of another drug which is effective
in the treatment of cancer or a pharmaceutically acceptable
prodrug, salt, solvate or hydrate thereof, administered prior,
during, or after administering aplidine or aplidine analogue.
[0021] Preferably the other drug is effective in the treatment of
leukemia and/or lymphoma. Most preferably the other drug is
selected from the group consisting of methotrexate, cytosine
arabinoside, mitoxantrone, vinblastine, methylprednisolone and
doxorubicin. The other drugs may form part of the same composition,
or be provided as a separate composition for administration at the
same time or at a different time.
[0022] The cancer to be treated is preferably a leukemia or a
lymphoma, most preferably ALL, AML, CML, MML or CLL.
[0023] In another aspect the invention encompasses a method of
increasing the therapeutic efficacy of a drug effective in the
treatment of cancer, preferably a drug effective in the treatment
of leukemia and/or lymphoma, most preferably a drug selected from
the group consisting of methotrexate, cytosine arabinoside,
mitoxantrone, vinblastine, methylprednisolone and doxorubicin, or a
pharmaceutically acceptable prodrug, salt, solvate or hydrate
thereof, which comprises administering to a patient in need thereof
an amount of aplidine or an aplidine analogue, or a
pharmaceutically acceptable prodrug, salt, solvate or hydrate
thereof. Aplidine or the aplidine analogue is administered prior,
during, or after administering the other drug.
[0024] Aplidine or an aplidine analogue is able to increase the
therapeutic efficacy of some cancer drugs. In one aspect, the
result is synergism, rather than additive. Such synergistic
combinations represent a preferred aspect of the present invention.
Synergism may be indicated by use of the Chou-Talalay method, or
other methods. In other instances, antagonism may be found.
[0025] In a further aspect the invention encompasses a
pharmaceutical composition comprising aplidine or an aplidine
analogue, or a pharmaceutically acceptable prodrug, salt, solvate
or hydrate thereof, and another drug effective in the treatment of
cancer. Preferably the other drug is effective in the treatment of
leukemia and/or lymphoma. Most preferably the other drug is
selected from the group consisting of methotrexate, cytosine
arabinoside, mitoxantrone, vinblastine, methylprednisolone and
doxorubicin.
[0026] The invention also encompasses a kit for use in the
treatment or prevention of cancer which comprises a dosage form of
aplidine or an aplidine analogue, or a pharmaceutically acceptable
prodrug, salt, solvate or hydrate thereof, a dosage form of another
drug effective in the treatment of cancer, or a pharmaceutically
acceptable prodrug, salt, solvate or hydrate thereof, and
instructions for the use of each actor in combination for the
treatment or prevention of cancer. Preferably the other drug is
effective in the treatment of leukemia and/or lymphoma. Most
preferably the other drug is selected from the group consisting of
methotrexate, cytosine arabinoside, mitoxantrone, vinblastine,
methylprednisolone and doxorubicin.
[0027] In a further aspect, the invention is directed to the use of
aplidine for the treatment of chronic lymphocytic leukemia.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1. Aplidine inhibits growth of CLL cells in culture
[0029] FIG. 2. Aplidine is a potent inhibitor of preB-ALL cells in
culture
[0030] FIG. 3. The cytotoxic dose-response curve of CCRF-CEM (FIG.
3A), SKI-DLCL (FIG. 3B) and K562 (3C) cells following aplidine
treatment for 96 hours
[0031] FIG. 4. Chou-Talalay analysis of combination of aplidine and
AraC in CCRF-CEM cells
[0032] FIG. 5. Chou-Talalay analysis of combination of aplidine and
AraC in SKI-DLCL cells
[0033] FIG. 6. Chou-Talalay analysis of combination of aplidine and
mitoxantrone in CCRF-CEM cells
[0034] FIG. 7. Chou-Talalay analysis of combination of aplidine and
mitoxantrone in SKI-DLCL cells
[0035] FIG. 8. Chou-Talalay analysis of combination of aplidine and
methotrexate in CCRF-CEM cells
[0036] FIG. 9. Chou-Talalay analysis of combination of aplidine and
doxorubicin in CCRF-CEM cells
[0037] FIG. 10. Chou-Talalay analysis of combination of aplidine
and vinblastine in CCRF-CEM cells
[0038] FIG. 11. Chou-Talalay analysis of combination of aplidine
and doxorubicin in SKI-DLCL cells
[0039] FIG. 12. Chou-Talalay analysis of combination of aplidine
and vinblastine in SKI-DLCL cells
[0040] FIG. 13. Chou-Talalay analysis of combination of aplidine
and methylprednisolone in SKI-DLCL cells
[0041] FIG. 14. Combination of IC.sub.20 of aplidine lowered the
IC.sub.50 of AraC in CCRF-CEM (FIG. 14A) and SKI-DLCL (FIG. 14B)
cells after incubation for 96 hours
[0042] FIG. 15. The effect of aplidine on in vivo tumor size as a
single agent and in combination with AraC
DETAILED DESCRIPTION OF THE INVENTION
[0043] By cancer it is meant to include tumors, neoplasias, and any
other malignant tissue or cells. The present invention is directed
to the use of aplidine or an aplidine analogue in combination for
the treatments of cancer in general, but more preferably for the
treatment of different leukemias and lymphomas.
[0044] In order to study the possible potentiation of other
anticancer agents with aplidine we have initiated a systematic
study of drug combinations for possible use in leukemias and
lymphomas. Aplidine was found to be an effective in vitro cytotoxic
agent against primary cells from a patient with preB-ALL (DM4) as
well as against fresh cells obtained from six chronic lymphocytic
leukemia (CLL) patients. The IC.sub.50 value was 10 nM for 3 day
exposure with the DM4 line and after a 11 day exposure with the
primary CLL samples.
[0045] Drug combination studies were carried out on established
cell lines rather than primary cells. We studied three cell lines
viz. K562, CCRF-CEM and SKI-DLCL representing acute myeloid
leukemia, lymphoblastic lymphoma and diffuse B cell large cell
lymphoma respectively. The data in the examples show that Aplidine
potentiates the effect of methotrexate, cytosine arabinoside,
mitoxantrone, vinblastine, methylprednisolone as well as
doxorubicin in K562, CCRF-CEM and SKI-DLCL cells by lowering the
IC.sub.50s for the drugs.
[0046] Thus we have found that aplidine is a potent cytotoxic agent
against cells of several hematologic malignancies. Significantly,
we have established for the first time that aplidine inhibits
growth of CLL cells in culture. We also found that aplidine
enhances the cytotoxicity of agents used in the treatment of
leukemias, such as methotrexate (MTX), cytosine arabonoside (AraC),
mitoxantrone (Mitox), vinblastine (Vinb), methylprednisolone
(Metpred) and doxorubicin (DOX).
[0047] Leukemia is classified by how quickly it progresses. Acute
leukemia is fast-growing and can overrun the body within a few
weeks or months. By contrast, chronic leukemia is slow-growing and
progressively worsens over years.
[0048] The blood-forming (hematopoietic) cells of acute leukemia
remain in an immature state, so they reproduce and accumulate very
rapidly. Therefore, acute leukemia needs to be treated immediately,
otherwise the disease may be fatal within a few months.
Fortunately, some subtypes of acute leukemia respond to available
therapies and they are curable. Children often develop acute forms
of leukemia, which are managed differently from leukemia in
adults.
[0049] In chronic leukemia, the blood-forming cells eventually
mature, or differentiate, but they are not "normal". They remain in
the bloodstream much longer than normal white blood cells, and they
are unable to combat infection well.
[0050] Leukemia also is classified according to the type of white
blood cell that is multiplying--that is, lymphocytes (immune system
cells), granulocytes (bacteria-destroying cells), or monocytes
(macrophage-forming cells). If the abnormal white blood cells are
primarily granulocytes or monocytes, the leukemia is categorized as
myelogenous, or myeloid, leukemia. On the other hand, if the
abnormal blood cells arise from bone marrow lymphocytes, the cancer
is called lymphocytic leukemia.
[0051] Other cancers, known as lymphomas, develop from lymphocytes
within the lymph nodes, spleen, and other organs. Such cancers do
not originate in the bone marrow and have a biological behavior
that is different from lymphocytic leukemia.
[0052] There are over a dozen different types of leukemia, but four
types occur most frequently. These classifications are based upon
whether the leukemia is acute versus chronic and myelogenous versus
lymphocytic, that is:
Acute myelogenous leukemia (AML): also known as acute
nonlymphocytic leukemia (ANLL)--is the most common form of adult
leukemia. Most patients are of retirement age (average age at
diagnosis=65 years), and more men are affected than women.
Fortunately, because of recent advances in treatment, AML can be
kept in remission (lessening of the disease) in approximately 60%
to 70% of adults who undergo appropriate therapy. Initial response
rates are approximately 65-75% but the overall cure rates are more
on the order of 40-50%. Chronic myelogenous leukemia (CML) is known
as a myeloproliferative disorder--that is, it is a disease in which
bone marrow cells proliferate (multiply) outside of the bone marrow
tissue. CML is easy to diagnose, since it has a genetic
peculiarity, or marker, that is readily identifiable under a
microscope. About 95% of CML patients have a genetic translocation
between chromosomes 9 and 22 in their leukemic cells. The
Philadelphia chromosome causes uncontrolled reproduction and
proliferation of all types of white blood cells and platelets
(blood clotting factors). CML is not yet curable by standard
methods of chemotherapy or immunotherapy. Acute lymphocytic
leukemia (ALL)--also known as acute lymphoblastic leukemia--is a
malignant disease caused by the abnormal growth and development of
early nongranular white blood cells, or lymphocytes. The leukemia
originates in the blast cells of the bone marrow (B-cells), thymus
(T-cells), and lymph nodes. ALL occurs predominantly in children,
peaking at 4 years of age. Chronic lymphocytic leukemia (CLL) is
the most common leukemia in North America and in Europe. It is a
disease of older adults and is very rare among people who are
younger than 50 years of age. Men with CLL outnumber women by a
2-to-1 average. CLL is thought to result from the gradual
accumulation of mature, long-lived lymphocytes. Therefore, this
cancer is caused not so much by overgrowth as it is by the extreme
longevity and build-up of malignant cells. Although the rate of
accumulation varies among individuals, the extensive tumor burden
eventually causes complications in all CLL patients.
[0053] The compositions of the present invention may comprise both
components (drugs) in a single pharmaceutically acceptable
formulation. Alternatively, the components may be formulated
separately and administered in combination with one another.
Various pharmaceutically acceptable formulations well known to
those of skill in the art can be used in the present invention.
Selection of an appropriate formulation for use in the present
invention can be performed routinely by those skilled in the art
based upon the mode of administration and the solubility
characteristics of the components of the composition.
[0054] Examples of pharmaceutical compositions containing Aplidine
or an aplidine analogue include liquid (solutions, suspensions or
emulsions) with suitable composition for intravenous
administration, and they may contain the pure compound or in
combination with any carrier or other pharmacologically active
compounds. Solubilised aplidine shows substantial degradation under
heat and light stress testing conditions, and a lyophilised dosage
form was developed, see WO99/42125 incorporated herein by
reference.
[0055] Administration of aplidine or compositions of the present
invention is based on a Dosing Protocol preferably by intravenous
infusion. We prefer that infusion times of up to 72 hours are used,
more preferably 1 to 24 hours, with about 1, about 3 or about 24
hours most preferred. Short infusion times which allow treatment to
be carried out without an overnight stay in hospital are especially
desirable. However, infusion may be around 24 hours or even longer
if required. Infusion may be carried out at suitable intervals with
varying patterns, illustratively once a week, twice a week, or more
frequently per week, repeated each week optionally with gaps of
typically one week.
[0056] The correct dosage of the compounds of the combination will
vary according to the particular formulation, the mode of
application, and the particular situs, host and tumour being
treated. Other factors like age, body weight, sex, diet, time of
administration, rate of excretion, condition of the host, drug
combinations, reaction sensitivities and severity of the disease
shall be taken into account. Administration can be carried out
continuously or periodically within the maximum tolerated dose.
Further guidance for the administration of aplidine is given in WO
0135974 which is incorporated herein by reference in its
entirety.
[0057] For the present invention, analogues of aplidine can be used
in place of APL, aplidine itself. Typically such compounds are as
defined in WO 0202596. Examples of compounds for the present
invention include the preferred compounds given in WO 0202596, and
in particular we import into this patent specification the
discussion of preferred compounds and related aspects given in WO
0202596. More preferably, the analogues are structurally close to
aplidine, and usually differ from aplidine in respect of one amino
acid or the terminal sidechain. The different amino acid can be in
the cyclic part of the molecule or in the sidechain. Many examples
of such compounds are given in WO 0202596, and they are candidates
for use in the present invention.
EXAMPLES
Example 1
[0058] Aplidine was tested against various primary cells from
patients with hematologic malignancies. The cells used were:
[0059] fresh cells obtained from six chronic lymphocytic leukemia
patients
[0060] primary cell from a patient with preB-ALL (DM4)
[0061] Patient samples were obtained with prior consent and CLL
cells were isolated by density gradient centrifugation over
histopaque. The media used was RPMI supplemented with 10%
autologous serum and L-glutamine. The cultures were incubated with
10 nM aplidine and cell viability was measured days 3, 7, 11 and 18
and compared with viability of untreated cells and STI 571 (0.5
mM).
[0062] The results of these studies are shown in FIGS. 1-2.
Example 2
[0063] In order to study the possible potentiation of other
anticancer agents we undertook a study of drug combinations for
possible use in leukemias and lymphomas.
[0064] Drug combination studies were carried out on established
cell lines rather than primary cells. We studied three cell lines,
viz. K562 as a model for acute myeloid leukemia, CEM representing
acute lymphocytic leukemia and SKI-DLCL representing diffuse large
cell lymphoma. Combination studies with IC.sub.20 and IC.sub.50
dose of aplidine with a dose range of methotrexate, cytosine
arabinoside and doxorubicin were tested to determine if aplidine
could potentiate the effect of these drugs.
[0065] The results are shown in table 1:
TABLE-US-00001 Additional Drug IC.sub.50 Dox IC.sub.50 MTX
IC.sub.50 Ara-C No Aplidine 18 nM 5 nM 30 nM IC.sub.20 Aplidine
(0.5 nM) 1 nM 500 pM 6 nM p < 0.01, p < 0.05, p < 0.05
[0066] Clearly, these data show that aplidine potentiates the
effect of doxorubicin, methotrexate and cytosine arabinoside by
lowering very significantly the IC.sub.50s for the drugs.
Example 3
In Vitro Studies to Determine the Effect of Aplidine as a Single
Agent on CCRF-CEM, SKI-DLCL and K562 Cell Lines
[0067] CCRF-CEMS, SKI-DLCL and K562 cells are maintained in RPMI
1640 supplemented with 10% FCS. To determine the cytotoxic effect
of aplidine on all cell lines and to obtain the IC.sub.50 of
aplidine in these cell lines, cells were plated into 96 well plates
and incubated for 96 hours in humidified and 5% CO2 containing
incubator. Cell viability is measured by XTT assay in an automated
plate reader. We found aplidine to be cytotoxic to all cell lines
with an IC.sub.50 dose of 0.5-1.0 nM (FIG. 3).
Example 4
Studies on In Vitro Effect of Aplidine+Drug Combination with Fixed
Doses of IC.sub.50:IC.sub.50 on all Cell Lines
[0068] Methotrexate, cytosine arabinoside C (ara-C), mitoxantrone,
methylprednisolone, vinblastine and doxorubicin were tested in
combination with aplidine.
[0069] Chou-Talalay analysis was used to analyze the drug
combinations. When Combination Index (CI) obtained by this analysis
is less than 1, the drugs are synergistic; when CI is 1, the drugs
are additive; and, if CI is greater than 1, the drugs are
antagonistic.
[0070] All the cytotoxicity studies were performed by using XTT or
MTS. We first determined the IC.sub.50 dose of these drugs in
SKI-DLCL, CCRF-CEM and K562 cell lines. We investigated drug
combinations using IC.sub.50(Aplidine):IC.sub.50(DrugX) fixed
ratio.
[0071] In table 2 is shown the combination of aplidine and Ara-C
with the dose of (IC.sub.50:IC.sub.50) in CCRF-CEM cells.
TABLE-US-00002 TABLE 2 Viability (% Ratio Dose of APL Dose of AraC
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 52.7
IC.sub.50(AraC) 0 10 nM 56.4 x16 8 nM 160 nM 4.7 x8 4 nM 80 nM 7.9
x4 2 nM 40 nM 7.6 x2 1 nM 20 nM 7.8 IC.sub.50:IC.sub.50 0.5 nM 10
nM 10.6 x 1/2 0.25 nM 5 nM 16.2 x 1/4 1.125 NM 2.5 nM 36.7 x 1/8
0.0625 nM 1.25 nM 70.8
[0072] The results of Chou-Talalay analysis of combination of
aplidine and Ara-C in CCRF-CEM cells can be seen in FIG. 4. The CT
for this combination in CCRF-CEM cells is 0.469.
[0073] In table 3 is shown the combination of aplidine and Ara-C
with the dose of (IC.sub.50:IC.sub.50) in SKI-DLCL cells.
TABLE-US-00003 TABLE 3 Viability (% Ratio Dose of APL Dose of AraC
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(AraC) 0 30 nM 50 x16 8 nM 480 nM 12 x8 4 nM 240 nM 10.7
x4 2 nM 120 nM 14.1 x2 1 nM 60 nM 17.4 IC.sub.50:IC.sub.50 0.5 nM
30 nM 23.1 x 1/2 0.25 nM 15 nM 25.4 x 1/4 1.125 nM 7.5 nM 25.5 x
1/8 0.0625 nM 3.75 nM 50.8
[0074] The results of Chou-Talalay analysis of combination of
aplidine and Ara-C in SKI-DLCL cells can be seen in FIG. 5. The CT
for this combination in SKI-DLCL cells is 0.306.
[0075] In table 4 is shown the combination of aplidine and Ara-C
with the dose of (IC.sub.50:IC.sub.50) in K562 cells.
TABLE-US-00004 Viability (% Ratio Dose of APL Dose of AraC of
control) Control 0 0 100 IC.sub.50(APL) 1 nM 0 50 IC.sub.50(AraC) 0
30 nM 50 x16 16 nM 480 nM 11.8 x8 8 nM 240 nM 15.2 x4 4 nM 120 nM
15.5 x2 2 nM 60 nM 17 IC.sub.50:IC.sub.50 1 nM 30 nM 22.1 x 1/2 0.5
nM 15 nM 25.6 x 1/4 0.25 nM 7.5 nM 31.1 x 1/8 0.125 nM 3.75 nM
44.2
[0076] The CI for this combination in K562 cells is 0.502.
[0077] In table 5 is shown the combination of aplidine and
mitoxantrone with the dose of (IC.sub.50:IC.sub.50) in CCRF-CEM
cells.
TABLE-US-00005 Viability (% Ratio Dose of APL Dose of Mitoxantrone
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(Mitox) 0 30 nM 56 x16 8 nM 480 nM 9.9 x8 4 nM 240 nM 11.6
x4 2 nM 120 nM 11.9 x2 1 nM 60 nM 13.8 IC.sub.50:IC.sub.50 0.5 nM
30 nM 20.6 x 1/2 0.25 nM 15 nM 39.7 x 1/4 1.125 nM 7.5 nM 60.7 x
1/8 0.0625 nM 3.75 nM 76.5
[0078] The results of Chou-Talalay analysis of combination of
aplidine and mitoxantrone in CCRF-CEM cells can be seen in FIG. 6.
The CI for this combination in CCRF-CEM cells is 0.911.
[0079] In table 6 is shown the combination of aplidine and
mitoxantrone with the dose of (IC.sub.50:IC.sub.50) in SKI-DLCL
cells.
TABLE-US-00006 Viability (% Dose of APL Dose of Mitoxantrone of
control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(Mitox) 0 5 nM 50 x16 8 nM 80 nM 17 x8 4 nM 40 nM 29 x4 2
nM 20 nM 22.6 x2 1 nM 10 nM 19.9 IC.sub.50:IC.sub.50 0.5 nM 5 nM
32.2 x 1/2 0.25 nM 2.5 nM 53.1 x 1/4 1.125 nM 1.25 nM 58.6 x 1/8
0.0625 nM 0.625 nM 70.1
[0080] The results of Chou-Talalay analysis of combination of
aplidine and mitoxantrone in SKI-DLCL cells can be seen in FIG. 7.
The CI for this combination in SKI-DLCL cells is 0.646.
[0081] In table 7 is shown the combination of aplidine and
mitoxantrone with the dose of (IC.sub.50:IC.sub.50) in K562
cells.
TABLE-US-00007 Viability (% Dose of APL Dose of Mitoxantrone if
control) Control 0 0 100 IC.sub.50(APL)1 1 nM 0 50 IC.sub.50(Mitox)
0 7.5 nM 50.7 x16 16 nM 120 nM 9.9 x8 8 nM 60 nM 11.6 x4 4 nM 30 nM
11.9 x2 2 nM 15 nM 13.8 IC.sub.50:IC.sub.50 1 nM 7.5 nM 20.6 x 1/2
0.5 nM 3.75 nM 39.7 x 1/4 0.25 nM 1.8 nM 60.7 x 1/8 0.125 nM 0.9 nM
76.5
[0082] The CI for this combination in K562 cells is 0.487.
[0083] In table 8 is shown the combination of aplidine and
methotrexate with the dose of (IC.sub.50:IC.sub.50) in CCRF-CEM
cells.
TABLE-US-00008 Viability (% Ratio Dose of APL Dose of Methotrexate
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(MTX) 0 10 nM 50 x16 8 nM 160 nM 5 x8 4 nM 80 nM 13 x4 2
nM 40 nM 11 x2 1 nM 20 nM 12 IC.sub.50:IC.sub.50 0.5 nM 10 nM 20 x
1/2 0.25 nM 5 nM 30 x 1/4 1.125 nM 2.5 nM 88 x 1/8 0.0625 nM 1.25
nM 100
[0084] The results of Chou-Talalay analysis of combination of
aplidine and methotrexate in CCRF-CEM cells can be seen in FIG. 8.
The CI for this combination in CCRF-CEM cells is 0.950.
[0085] The results of Chou-Talalay analysis of combination of
aplidine and Doxorubicin in CCRF-CEM cells can be seen in FIG. 9.
The CI for this combination in CCRF-CEM cells is 1.952.
[0086] The results of Chou-Talalay analysis of combination of
aplidine and vinblastine in CCRF-CEM cells can be seen in FIG. 10.
The CI for this combination in CCRF-CEM cells is 2.046.
[0087] In table 9 is shown the combination of aplidine and
doxorubicin with the dose of (IC.sub.50:IC.sub.50) in SKI-DLCL
cells.
TABLE-US-00009 Viability (% Ratio Dose of APL Dose of Doxorubicin
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(Doxo) 0 5 nM 50 x16 8 nM 80 nM 9.4 x8 4 nM 40 nM 8.6 x4 2
nM 20 nM 8 x2 1 nM 10 nM 9.7 IC.sub.50:IC.sub.50 0.5 nM 5 nM 21 x
1/2 0.25 nM 2.5 nM 40 x 1/4 1.125 nM 1.25 nM 45 x 1/8 0.0625 nM
0.625 nM 49
[0088] The results of Chou-Talalay analysis of combination of
aplidine and doxorubicin in SKI-DLCL cells can be seen in FIG. 11.
The CI for this combination in SKI-DLCL cells is 0.478.
[0089] In table 10 is shown the combination of aplidine and
vinblastine with the dose of (IC.sub.50:IC.sub.50) in SKI-DLCL
cells.
TABLE-US-00010 Viability (% Ratio Dose of APL Dose of Vinblastine
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(Vinb) 0 4 nM 50 x16 8 nM 64 nM 15 x8 4 nM 32 nM 17 x4 2
nM 16 nM 17 x2 1 nM 8 nM 21 IC.sub.50:IC.sub.50 0.5 nM 4 nM 29 x
1/2 0.25 nM 2 nM 25 x 1/4 1.125 nM 1 nM 28 x 1/8 0.0625 nM 0.5 nM
38
[0090] The results of Chou-Talalay analysis of combination of
aplidine and vinblastine in SKI-DLCL cells can be seen in FIG. 12.
The CI for this combination in SKI-DLCL cells is 0.760.
[0091] In table 11 is shown the combination of aplidine and
methylprednisolone with the dose of (IC.sub.50:IC.sub.50) in
SKI-DLCL cells.
TABLE-US-00011 Viability (% Dose of APL Dose of methylprednisolone
of control) Control 0 0 100 IC.sub.50(APL) 0.5 nM 0 50
IC.sub.50(Metpred) 0 160 nM 51 x16 8 nM 2560 nM 10.8 x8 4 nM 1280
nM 17.3 x4 2 nM 640 nM 16.7 x2 1 nM 320 nM 17.4 IC.sub.50:IC.sub.50
0.5 nM 160 nM 24.7 x 1/2 0.25 nM 80 nM 32.4 x 1/4 1.125 nM 40 nM
39.1 x 1/8 0.0625 nM 20 nM 50
[0092] The results of Chou-Talalay analysis of combination of
aplidine and methylprednisolone in SKI-DLCL cells can be seen in
FIG. 13. The CI for this combination in SKI-DLCL cells is
0.646.
Example 5
[0093] We have also investigated the cytotoxic effect of
combination of IC.sub.20 (APL) with a variable dose of AraC on
CCRF-CEM and SKI-DLCL cell lines. Aplidine in both cell lines
potentiated the effect of AraC, the IC.sub.50 dose of AraC was
reduced from 30 nM to 1.6 nM in SKI-DLCL cell line, and from 10 nM
to 0.8 nM in CCRF-CEM cell line respectively (FIG. 14). Data was
obtained after cell incubation for 96 hours and using XTT assay.
The results represent means of three different experiments.
Example 6
In Vivo Studies
[0094] We have performed in vivo experiments to study the effect of
aplidine alone and in combination with other drugs for lymphoid
malignancies.
Determination of Maximum Tolerated Dose (MTD) in C.B.-17 Scid/Scid
(Scid Mice)
[0095] We have used an in vivo model of human lymphoma in SCID mice
for this purpose. Specifically, we have used CCRF-CEMS cells and
CB.17 scid/scid mice. We have experience with this model and have
evaluated drug treatments using this xenograft (Lacerda J. F. et
al. Blood 85 (10): 2675-2679 (1995)). We found that a total dose of
1 mg/kg/week given in five daily doses is the aplidine maximum dose
that can be tolerated by mice.
Determination of In Vivo Antitumor Effect of Aplidine as a Single
Agent and in Combination with AraC in SCID Mice Xenograft Model
[0096] SCID mice were inoculated subcutaneously in the right flank
with 10.sup.7 CEM-T leukemic cells. They were observed twice weekly
for tumor formation at the site of inoculation. After establishment
of palpable tumor, aplidine was injected as single agent and in
combination with several doses of AraC to determine the antitumor
effect. Mice were randomized to receive aplidine alone at doses of
0.75 mg/kg and 1 mg/kg, AraC alone at 50 mg/kg, or combination of
Aplidine and AraC for all dose combinations. The AraC dose chosen
for this combination is the dose at which the tumor growth was
inhibited but no tumor regression occurred. All drugs were
administered intra-peritoneally, and tumor size was compared to a
control group of mice not receiving any treatment and for
combination groups, compared to tumor sizes with single agent
treatment.
[0097] The most effective combination was found to be AraC-50
mg/kg+aplidine-0.75 mg/kg (FIG. 15).
[0098] These findings in respect of aplidine can be extended to
aplidine analogues, derivatives and related compounds. For example,
the present invention provides a combination of a compound such as
those of WO 02 02596 with an anticancer drug, preferably an
anti-leukemia drug or anti-lymphoma drug, notably methotrexate,
cytosine arabinoside, mitoxantrone, vinblastine, methylprednisolone
or doxorubicin.
* * * * *