U.S. patent application number 13/851053 was filed with the patent office on 2014-01-02 for gemcitabine combination therapy.
This patent application is currently assigned to CELGENE CORPORATION. The applicant listed for this patent is Mitchell KEEGAN, William MCCULLOCH. Invention is credited to Mitchell KEEGAN, William MCCULLOCH.
Application Number | 20140005122 13/851053 |
Document ID | / |
Family ID | 38832254 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140005122 |
Kind Code |
A1 |
MCCULLOCH; William ; et
al. |
January 2, 2014 |
GEMCITABINE COMBINATION THERAPY
Abstract
The present invention provides compositions and methods for the
treatment of cell proliferative disorders using at least one DAC
inhibitor and gemcitabine.
Inventors: |
MCCULLOCH; William;
(Raleigh, NC) ; KEEGAN; Mitchell; (Marlborough,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MCCULLOCH; William
KEEGAN; Mitchell |
Raleigh
Marlborough |
NC
MA |
US
US |
|
|
Assignee: |
CELGENE CORPORATION
Summit
NJ
|
Family ID: |
38832254 |
Appl. No.: |
13/851053 |
Filed: |
March 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13482940 |
May 29, 2012 |
|
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13851053 |
|
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12298265 |
Feb 5, 2010 |
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PCT/US07/09295 |
Apr 13, 2007 |
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13482940 |
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60794599 |
Apr 24, 2006 |
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Current U.S.
Class: |
514/19.9 ;
514/49 |
Current CPC
Class: |
A61K 38/15 20130101;
A61P 35/00 20180101; A61K 31/7068 20130101; A61K 45/06 20130101;
A61K 2300/00 20130101; A61K 31/7068 20130101; A61K 38/12
20130101 |
Class at
Publication: |
514/19.9 ;
514/49 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61K 45/06 20060101 A61K045/06; A61K 31/7068 20060101
A61K031/7068 |
Claims
1. A method comprising steps of: administering to a subject
suffering from or susceptible to a cell proliferative disorder,
combination therapy of a DAC inhibitor and gemcitabine.
2. The method of claim 1 wherein the cell proliferative disorder
involves a tumor.
3. The method of claim 2, wherein the tumor is a Ras-expressing
tumor.
4. The method of claim 2, wherein the tumor is a pancreatic
tumor.
5. The method of claim 1, wherein the DAC inhibitor is
romidepsin.
6. The method of claim 1 further comprising administering
electrolyte supplementation.
7. The method of claim 1, wherein the cell proliferative disorder
is cutaneous T-cell lymphoma.
8. The method of claim 1, wherein the cell proliferative disorder
is peripheral T-cell lymphoma.
9. The method of claim 1, wherein the cell proliferative disorder
is a hematological malignancy.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 13/482,940, filed May 29, 2012, abandoned,
which is a continuation of U.S. application Ser. No. 12/298,265,
filed Feb. 5, 2010, abandoned, which is a U.S. national phase
application under 35 U.S.C. .sctn.371 of international PCT
application number PCT/US2007/009295, filed Apr. 13, 2007, which
claims priority under 35 U.S.C. .sctn.119(e) to U.S. provisional
application, U.S. Ser. No. 60/794,599, filed Apr. 24, 2006. Each of
the above applications are incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] Dysregulation or loss of control of cell division can result
in the development of any of a variety of cell proliferative
disorders, many of which are debilitating or deadly. Although much
has been learned about mechanisms involved in cell proliferation,
and therefore about common biological principles underlying a
variety of different disorders, there remains a need for the
development of new and/or improved therapies for the treatment of
such conditions.
[0003] There is a particular need for the development of improved
therapies for the treatment of tumors that express the Ras
oncogene. Ras-expressing tumors are often more resistant to
standard therapies. Furthermore, many of the most deadly cancers
involver Ras-expressing tumors. For example, 90-95% of pancreatic
tumors are Ras-expressing. Similarly, 40-45% of colorectal tumors,
40% of bladder tumors, 15-20% of non small cell lung carcinomas
express Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS),
which are not themselves cancer but are bone marrow disorders
characterized by abnormal cell maturation that typically progress
to cancer (AML), also express Ras. There is a profound need for the
development of therapies for these and other Ras-expressing
diseases and disorders.
SUMMARY OF THE INVENTION
[0004] The present invention encompasses the finding that
combinations of DAC inhibitors with gemcitabine are have particular
utility in the treatment of proliferative diseases. Among other
things, the invention establishes the particular utility of DAC
inhibitor/gemcitabine combination therapy in treatment of tumors
expressing the Ras oncogene. In certain particular embodiments,
combination therapy with romidepsin and gemcitabine is provided,
for example for use in the treatment of proliferative disorders
generally and/or for use in the treatment of tumors expressing the
Ras oncogene.
[0005] The present invention provides methods of treating a
proliferative disorder by administering a combination of one or
more DAC inhibitors and gemcitabine.
[0006] The present invention further provides methods of treating
tumors that express the Ras oncogene by administering a DAC
inhibitor together with gemcitabine. In some embodiments, such
methods involve determining that a tumor expresses the Ras
oncogene, and then, administering combination therapy with a DAC
inhibitor and gemcitabine.
[0007] Determination that a tumor expresses the Ras oncogene can
involve testing for expression of the Ras oncogene and/or can
involve determining that the tumor is of a type that typically
expresses the Ras oncogene.
[0008] The present invention provides combination regimens, and
unit dosages of pharmaceutical compositions useful in such
regimens. The present invention further provides kits for
combination therapy of DAC inhibitors and gemcitabine.
DEFINITIONS
[0009] Alicyclic: The term "alicyclic," as used herein, denotes a
monovalent group derived from a monocyclic or bicyclic saturated
carbocyclic ring compound by the removal of a single hydrogen atom.
Examples include, but not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and
bicyclo[2.2.2]octyl. Such alicyclic groups may be further
substituted.
[0010] Aliphatic: An "aliphatic group" is non-aromatic moiety that
may contain any combination of carbon atoms, hydrogen atoms,
halogen atoms, oxygen, nitrogen or other atoms, and optionally
contain one or more units of unsaturation, e.g., double and/or
triple bonds. An aliphatic group may be straight chained, branched
or cyclic and preferably contains between about 1 and about 24
carbon atoms, more typically between about 1 and about 12 carbon
atoms. In addition to aliphatic hydrocarbon groups, aliphatic
groups include, for example, polyalkoxyalkyls, such as polyalkylene
glycols, polyamines, and polyimines, for example. Such aliphatic
groups may be further substituted.
[0011] Aryl: The term "aryl," as used herein, refers to a mono- or
polycyclic carbocyclic ring system having one or two aromatic rings
including, but not limited to phenyl, naphthyl, tetrahydronaphthyl,
indanyl, idenyl and the like. In accordance with the invention, any
of the aryls, substituted aryls, heteroaryls and substituted
heteroaryls described herein, can be any aromatic group. Aromatic
groups can be substituted or unsubstituted.
[0012] Cell Proliferative Disorder, Disease, or Condition: The term
"cell proliferative disease or condition" is meant to refer to any
condition characterized by aberrant cell growth, preferably
abnormally increased cellular proliferation.
[0013] Combination Therapy: According to the present invention, a
DAC inhibitor may desirably be administered in combination with
gemcitabine. Such therapy will commonly involve administration of
multiple individual doses of a DAC inhibitor and/or of gemcitabine,
spaced out over time. Doses of a DAC inhibitor and gemcitabine may
be administered in the same amounts and/or according to the same
schedule or alternatively may be administered in different amounts
and/or according to different schedules.
[0014] DAC Inhibitor: In general, any agent that specifically
inhibits a deacetylase is considered to be a DAC inhibitor. Any
agent that specifically inhibits a histone deacetylase is
considered to be an HDAC inhibitor. Those of ordinary skill in the
art will appreciate that, unless otherwise set forth herein or
known in the art, DAC inhibitors may be administered in any form
such as, for example, salts, esters, prodrugs, metabolites, etc.
Furthermore. DAC inhibitors that contain chiral centers may be
administered as single stereoisomers or as mixtures, including
racemic mixtures, so long as the single stereoisomer or mixture has
DAC inhibitor activity.
[0015] DAC Inhibitor Therapy: As used herein, the phrase "DAC
inhibitor therapy" refers to the regimen by which a DAC inhibitor
is administered to an individual. Commonly, DAC inhibitor therapy
will involve administration of multiple individual doses of a DAC
inhibitor, spaced out over time. Such individual doses may be of
different amounts or of the same amount. Furthermore, those of
ordinary skill in the art will readily appreciate that different
dosing regimens (e.g., number of doses, amount(s) of doses, spacing
of doses) are typically employed with different DAC inhibitors.
[0016] Electrolyte: In general, the term "electrolyte", as used
herein, refers to physiologically relevant free ions.
Representative such free ions include, but are not limited to
sodium (Na.sup.+), potassium (K.sup.+), calcium (Ca.sup.2+),
magnesium (Mg.sup.2), chloride (CI.sup.-), phosphate (PO4.sup.3-),
and bicarbonate (HCO.sub.3.sup.-).
[0017] Electrolyte Supplementation: The term "electrolyte
supplementation", as used herein, refers to administration to a
subject of a composition comprising one or more electrolytes in
order to increase serum electrolyte levels in the subject. For
purposes of the present invention, when electrolyte supplementation
is administered "prior to, during, or after" combination therapy,
it may be administered prior to initiation of combination therapy
inhibitor therapy (i.e., prior to administration of any dose) or
prior to, concurrently with, or after any particular dose or
doses.
[0018] Halogen: The term "halogen", as used herein, refers to an
atom selected from tluorine, chlorine, bromine, and iodine.
[0019] Heteroaryl: The term "heteroaryl", as used herein, refers to
a mono- or polycyclic (e.g. bi-, or tri-cyclic or more) aromatic
radical or ring having from five to ten ring atoms of which one or
more ring atom is selected from, for example, S, O and N; zero, one
or two ring atoms are additional heteroatoms independently selected
from, for example, S, O and N; and the remaining ring atoms are
carbon, wherein any N or S contained within the ring may be
optionally oxidized. Heteroaryl includes, but is not limited to,
pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,
thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,
benzooxazolyl, quinoxalinyl, and the like.
[0020] Heterocyclic: The term "heterocyclic", as used herein,
refers to a non-aromatic 5-, 6- or 7-membered ring or a bi- or
tri-cyclic group fused system, where (i) each ring contains between
one and three heteroatoms independently selected from oxygen,
sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double
bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the
nitrogen and sulfur heteroatoms may optionally be oxidized, (iv)
the nitrogen heteroatom may optionally be quaternized, (v) any of
the above rings may be fused to a benzene ring, and (vi) the
remaining ring atoms are carbon atoms which may be optionally
oxo-substituted. Representative heterocycloalkyl groups include,
but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,
pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,
piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,
thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and
tetrahydrofuryl. Such heterocyclic groups may be further
substituted.
[0021] Initiation: As used herein, the term "initiation" when
applied to therapy can refer to a first administration of an active
agent (e.g. a DAC inhibitor or gemcitabine) inhibitor to a patient
who has not previously received a DAC inhibitor. Alternatively or
additionally, the term "initiation" can refer to administration of
a particular dose of a DAC inhibitor and/or of gemcitabine during
therapy of a patient.
[0022] Pharmaceutically acceptable carrier or excipient: As used
herein, the term "pharmaceutically acceptable carrier or excipient"
means a non-toxic, inert solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0023] Pharmaceutically acceptable ester: As used herein, the term
"pharmaceutically acceptable ester" refers to esters which
hydrolyze in vivo and include those that break down readily in the
human body to leave the parent compound or a salt thereof. Suitable
ester groups include, for example, those derived from
pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic
acids, in which each alkyl or alkenyl moiety advantageously has not
more than 6 carbon atoms. Examples of particular esters include,
but are not limited to, formates, acetates, propionates, butyrates,
acrylates and ethylsuccinates.
[0024] Pharmaceutically acceptable prodrug: The term
"pharmaceutically acceptable prodrugs" as used herein refers to
those prodrugs of the compounds of the present invention which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals with undue
toxicity, irritation, allergic response, and the like, commensurate
with a reasonable benefit/risk ratio, and effective for their
intended use, as well as the zwitterionic forms, where possible, of
the compounds of the present invention. "Prodrug", as used herein
means a compound which is convertible in vivo by metabolic means
(e.g. by hydrolysis) to a compound of the invention. Various forms
of prodrugs are known in the art, for example, as discussed in
Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et
al. (ed.). Methods in Enzymology, vol. 4, Academic Press (1985);
Krogsgaard-Larsen, et al., (ed), "Design and Application of
Prodrugs, Textbook of Drug Design and Development". Chapter 5,
113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews,
8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et
seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug
Delivery Systems. American Chemical Society (1975); and Bernard
Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug
Metabolism Chemistry, Biochemistry And Enzymology," John Wiley and
Sons, Ltd. (2002).
[0025] Pharmaceutically acceptable salt: As used herein, the term
"pharmaceutically acceptable salt" refers to those salts which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response and the like, and are
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example. S. M.
Berge, et al. describes pharmaceutically acceptable salts in detail
in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be
prepared in situ during the final isolation and purification of the
compounds of the invention, or separately by reacting the free base
function with a suitable organic acid. Examples of pharmaceutically
acceptable include, but are not limited to, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
maleic acid, tartaric acid, citric acid, succinic acid or malonic
acid or by using other methods used in the art such as ion
exchange. Other pharmaceutically acceptable salts include, but are
not limited to adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, arid the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,
sulfonate and aryl sulfonate.
[0026] Stable: The term "stable", as used herein, refers to
compounds which possess stability sufficient to allow manufacture
and which maintains the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein (e.g.,
therapeutic or prophylactic administration to a subject). In
general, combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds.
[0027] Substituted: The terms "substituted aryl", "substituted
heteroaryl", or "substituted aliphatic," as used herein, refer to
aryl, heteroaryl, aliphatic groups as previously defined,
substituted by independent replacement of one, two, or three or
more of the hydrogen atoms thereon with substituents including, but
not limited to, --F, --CI, --Br, --I, --OH, protected hydroxyl,
--NO.sub.2, --CN, --C.sub.1-C.sub.12-alkyl optionally substituted
with, for example, halogen, C.sub.2-C.sub.12-alkenyl optionally
substituted with, for example, halogen, --C.sub.2-C.sub.12-alkynyl
optionally substituted with, for example, halogen, --NH.sub.2,
protected amino, --NH--C.sub.1-C.sub.12-alkyl,
--NH--C.sub.2-C.sub.12-alkenyl, --NH--C.sub.2-C.sub.12-alkenyl,
--NH--C.sub.3-C.sub.12-cycloalkyl, --NH-aryl, --NH-heteroaryl,
--NH-heterocycloalkyl, -dialkylamino, -diarylamino,
-diheteroarylamino, --O--C.sub.1-C.sub.12-alkyl,
--O--C.sub.2-C.sub.12-alkenyl, --O--C.sub.2-C.sub.12-alkenyl,
--O--C.sub.3-C.sub.12-cycloalkyl, --O-aryl, --O-heteroaryl.
--O-heterocycloalkyl, --C(O)--C,-C.sub.12-alkyl,
--C(O)--C.sub.2-C.sub.12-alkenyl, --C(O)--C.sub.2-C.sub.12-alkenyl,
--C(O)--C.sub.3-C.sub.12-cycloalkyl, --C(O)-aryl,
--C(O)-heteroaryl, --C(O)-heterocycloalkyl, --CONH.sub.2,
--CONH--C.sub.1-C.sub.12-alkyl, --CONH--C.sub.2-C.sub.12-alkenyl,
--CONH--C.sub.2-C.sub.12-alkenyl, --CONH--C.sub.3-C,2-cycloalkyl,
--CONH-aryl, --CONH-heteroaryl, --CONH-heterocycloalkyl,
--OCO.sub.2--C.sub.1-C.sub.12-alkyl,
--OCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--OCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--OCO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --OCO.sub.2-aryl,
--OCO.sub.2-heteroaryl, --OCO.sub.2-heterocycloalkyl,
--OCONH.sub.2, --OCONH--C.sub.1-C.sub.12-alkyl,
--OCONH--C.sub.2-C.sub.12-alkenyl,
--OCONH--C.sub.2-C.sub.12-alkenyl, --OCONH--C.sub.3-C,2-cycloalkyl,
--OCONH-aryl, --OCONH-heteroaryl, --OCONH-heterocycloalkyl,
--NHC(O)--C.sub.1C.sub.12-alkyl,
--NHC(O)--C.sub.2-C.sub.12-alkenyl,
--NHC(O)--C.sub.2-C.sub.12-alkenyl,
--NHC(O)--C.sub.3-C.sub.12-cycloalkyl, --NHC(O)-aryl,
--NHC(O)-heteroaryl, --NHC(O)-heterocycloalkyl,
--NHCO.sub.2--C.sub.1-C.sub.12-alkyl,
--NHCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NH.sub.1--CO.sub.2-C.sub.2-C.sub.12-alkenyl,
--NHCO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --NHCO.sub.2-aryl,
--NHCO.sub.2-heteroaryl, --NHCO.sub.2-heterocycloalkyl,
--NHC(O)NH.sub.2, --NHC(O)NH--C.sub.1C.sub.12-alkyl,
--NHC(O)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(O)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(O)NH--C.sub.3-C.sub.12-cycloalkyl. --NHC(O)NH-aryl,
--NHC(O)NH-heteroaryl, --NHC(O)NH-heterocycloalkyl, NHC(S)NH.sub.2,
--NHC(S)NH--C.sub.1-C.sub.12-alkyl,
--NHC(S)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(S)NH--C.sub.2-C,2-alkenyl,
--NHC(S)NH--C.sub.3-C.sub.12-cycloalkyl, --NHC(S)NH-aryl,
--NHC(S)NH-heteroaryl, --NHC(S)NH-heterocycloalkyl,
--NHC(NH)NH.sub.2, --NHC(NH)NH--C,-C.sub.12-alkyl,
--NHC(NH)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)NH--C.sub.3-C.sub.12-cycloalkyl, --NHC(NH)NH-aryl,
--NHC(NH)NH-heteroaryl, --NHC(NH)NH-heterocycloalkyl,
--NHC(NH)--C.sub.1-C)2-alkyl, --NHC(NH)--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)--C.sub.3-C.sub.12-cycloalkyl, --NHC(NH)-aryl,
--NHC(NH)-heteroaryl, --NHC(NH)-heterocycloalkyl,
--C(NH)NH--C.sub.1-C.sub.12-alkyl,
--C(NH)NH--C.sub.2-C.sub.12-alkenyl,
--C(NH)NH--C.sub.2-C.sub.12-alkenyl,
--C(NH)NH--C.sub.3-C.sub.12-cycloalkyl, --C(NH)NH-aryl,
--C(NH)NH-heteroaryl, --C(NH)NH-heterocycloalkyl,
--S(O)--C.sub.1-C.sub.12-alkyl, --S(O)--C.sub.2-C.sub.12-alkenyl,
--S(O)--C.sub.2-C.sub.12-alkenyl,
--S(O)--C.sub.3-C.sub.12-cycloalkyl, --S(O)-aryl,
--S(O)-heteroaryl, --S(O)-heterocycloalkyl-SO.sub.2NH.sub.2,
--SO.sub.2NH--C.sub.1-C.sub.12-alkyl,
--SO.sub.2NH--C.sub.2-C.sub.12-alkenyl,
--SO.sub.2NH--C.sub.2-C.sub.12-alkenyl,
--SO.sub.2NH--C.sub.3-C.sub.12-cycloalkyl, --SO.sub.2NH-aryl,
--SO.sub.2NH-heteroaryl, --SO.sub.2NH-heterocycloalkyl,
--NHSO.sub.2--C.sub.1-C.sub.12-alkyl,
--NHSO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NHSO.sub.2--C.sub.2-C,2-alkenyl,
--NHSO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --NHSO.sub.2-aryl,
--NHSO.sub.2-heteroaryl, --NHSO.sub.2-heterocycloalkyl,
--CH.sub.2NH.sub.2, --CH.sub.2SO.sub.2CH.sub.3, -aryl, -arylalkyl,
-heteroaryl, -heteroarylalkyl, -heterocycloalkyl,
--C.sub.3-C.sub.12-cycloalkyl, polyalkoxyalkyl, polyalkoxy,
-methoxymethoxy, -methoxyethoxy, --SH, --S--C.sub.1-C.sub.12-alkyl,
--S--C.sub.2-C.sub.12-alkenyl, --S--C.sub.2-C.sub.1-alkenyl,
--S--C.sub.3-C.sub.12-cycloalkyl, --S-aryl, --S-heteroaryl,
--S-heterocycloalkyl, or methylthiomethyl. It is understood that
the aryls, heteroaryls, alkyls, and the like can be further
substituted.
[0028] Susceptible to: The term "susceptible to", as used herein
refers to an individual having higher risk (typically based on
genetic predisposition, environmental factors, personal history, or
combinations thereof) of developing a particular disease or
disorder, or symptoms thereof, than is observed in the general
population.
[0029] Therapeutically effective amount: The term "therapeutically
effective amount" of an active agent or combination of agents is
intended to refer to an amount of agent(s) which confers a
therapeutic effect on the treated subject, at a reasonable
benefit/risk ratio applicable to any medical treatment. The
therapeutic effect may be objective (i.e., measurable by some test
or marker) or subjective (i.e., subject gives an indication of or
feels an effect). An effective amount of a particular agent may
range from about 0.1 mg/Kg to about 500 mg/Kg, preferably from
about 1 to about 50 mg/Kg. Effective doses may also vary depending
on route of administration, as well as the possibility of co-usage
with other agents. It will be understood, however, that the total
daily usage of any particular active agent utilized in accordance
with the present invention will be decided by the attending
physician within the scope of sound medical judgment. The specific
therapeutically effective dose level for any particular patient
will depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or contemporaneously with the
specific compound employed; and like factors well known in the
medical arts.
[0030] Therapeutic agent. As used herein, the phrase "therapeutic
agent" refers to any agent that, when administered to a subject,
has a therapeutic effect and/or elicits a desired biological and/or
pharmacological effect.
[0031] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a
biologically active agent that partially or completely alleviates,
ameliorates, relives, inhibits, delays onset of, reduces severity
of and/or reduces incidence of one or more symptoms or features of
a particular disease, disorder, and/or condition. Such treatment
may be of a subject who does not exhibit signs of the relevant
disease, disorder and/or condition and/or of a subject who exhibits
only early signs of the disease, disorder, and/or condition.
Alternatively or additionally, such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0032] As indicated, the present invention demonstrates that
combinations of DAC inhibitors and gemcitabine are particularly
useful in the treatment of proliferative disorders.
Cell Proliferative Disorders, Diseases, or Conditions
[0033] In some embodiments, the invention provides methods for
treating cell proliferative disorders, diseases or conditions. In
general, cell proliferative disorders, diseases or conditions
encompass a variety of conditions characterized by aberrant cell
growth, preferably abnormally increased cellular proliferation. For
example, cell proliferative disorders, diseases, or conditions
include, but are not limited to, cancer, immune-mediated responses
and diseases (e.g., transplant rejection, graft vs host disease,
immune reaction to gene therapy, autoimmune diseases,
pathogen-induced immune dysregulation, etc.), certain circulatory
diseases, and certain neurodegenerative diseases.
[0034] In certain embodiments, the invention relates to methods of
treating cancer. In general, cancer is a group of diseases which
are characterized by uncontrolled growth and spread of abnormal
cells. Examples of such diseases are carcinomas, sarcomas,
leukemias, lymphomas and the like.
[0035] For example, cancers include, but are not limited to
leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL),
peripheral T-cell lymphomas, lymphomas associated with human T-cell
lymphotropic virus (HTLV) such as adult T-cell leukemia/lymphoma
(ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute
nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic
myelogenous leukemia, acute myelogenous leukemia. Hodgkin's
disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic
syndrome, mesothelioma, common solid tumors of adults such as head
and neck cancers (e.g., oral, laryngeal and esophageal),
genitourinary cancers (e.g., prostate, bladder, renal, uterine,
ovarian, testicular, rectal and colon), lung cancer, breast cancer,
pancreatic cancer, melanoma and other skin cancers, stomach cancer,
brain tumors, liver cancer and thyroid cancer, and/or childhood
solid tumors such as brain tumors, neuroblastoma, retinoblastoma,
Wilms' tumor, bone tumors, and soft-tissue sarcomas.
[0036] In some embodiments, the invention relates to treatment of
leukemias. For example, in some embodiments, the invention relates
to treatment of chronic lymphocytic leukemia, chronic myelogenous
leukemia, acute lymphocytic leukemia acute myelogenous leukemia,
and/or adult T cell leukemia/lymphoma. In certain embodiments, the
invention relates to the treatment of AML. In certain embodiments,
the invention relates to the treatment of ALL. In certain
embodiments, the invention relates to the treatment of CML. In
certain embodiments, the invention relates to the treatment of
CLL.
[0037] In some embodiments, the invention relates to treatment of
lymphomas. For example, in some embodiments, the invention relates
to treatment of Hodgkin's or non-Hodgkin's (e.g., T-cell lymphomas
such as peripheral T-cell lymphomas, cutaneous T-cell lymphomas,
etc.) lymphoma.
[0038] In some embodiments, the invention relates to the treatment
of myelomas and/or myelodysplastic syndromes. In some embodiments,
the invention relates to treatment of solid tumors. In some such
embodiments the invention relates to treatment of solid tumors such
as lung, breast, colon, liver, pancreas, renal, prostate, ovarian,
and/or brain. In some embodiments, the invention relates to
treatment of pancreatic cancer. In some embodiments, the invention
relates to treatment of renal cancer. In some embodiments, the
invention relates to treatment of prostate cancer. In some
embodiments, the invention relates to treatment of sarcomas. In
some embodiments, the invention relates to treatment of soft tissue
sarcomas. In some embodiments, the invention relates to methods of
treating one or more immune-mediated responses and diseases.
[0039] For example, in some embodiments, the invention relates to
treatment of rejection following transplantation of synthetic or
organic grafting materials, cells, organs or tissue to replace all
or part of the function of tissues, such as heart, kidney, liver,
bone marrow, skin, cornea, vessels, lung, pancreas, intestine,
limb, muscle, nerve tissue, duodenum, small-bowel,
pancreatic-islet-cell, including xeno-transplants, etc.; treatment
of graft-versus-host disease, autoimmune diseases, such as
rheumatoid arthritis, systemic lupus erythematosus, thyroiditis,
Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis,
type I diabetes uveitis, juvenile-onset or recent-onset diabetes
mellitus, uveitis, Graves' disease, psoriasis, atopic dermatitis,
Crohn's disease, ulcerative colitis, vasculitis, auto-antibody
mediated diseases, aplastic anemia, Evan's syndrome, autoimmune
hemolytic anemia, and the like; and further to treatment of
infectious diseases causing aberrant immune response and/or
activation, such as traumatic or pathogen induced immune
dysregulation, including for example, that which are caused by
hepatitis B and C infections, HIV. Staphylococcus aureus infection,
viral encephalitis, sepsis, parasitic diseases wherein damage is
induced by an inflammatory response (e.g., leprosy). In some
embodiments, the invention relates to treatment of graft vs host
disease (especially with allogenic cells), rheumatoid arthritis,
systemic lupus erythematosus, psoriasis, atopic dermatitis, Crohn's
disease, ulcerative colitis and/or multiple sclerosis.
[0040] Alternatively or additionally, in some embodiments, the
invention relates to treatment of an immune response associated
with a gene therapy treatment, such as the introduction of foreign
genes into autologous cells and expression of the encoded product.
In some embodiments, the invention relates to treatment of
circulatory diseases, such as arteriosclerosis, atherosclerosis,
vasculitis, polyarteritis nodosa and/or myocarditis.
[0041] In some embodiments, the invention relates to treatment of
any of a variety of neurodegenerative diseases, a non-exhaustive
list of which includes: [0042] I. Disorders characterized by
progressive dementia in the absence of other prominent neurologic
signs, such as Alzheimer's disease; Senile dementia of the
Alzheimer type; and Pick's disease (lobar atrophy); [0043] II.
Syndromes combining progressive dementia with other prominent
neurologic, abnormalities such as A) syndromes appearing mainly in
adults (e.g., Huntington's disease. Multiple system atrophy
combining dementia with ataxia and/or manifestations of Parkinson's
disease, Progressive supranuclear palsy
(Steel-Richardson-Olszewski), diffuse Lewy body disease, and
corticodentatonigral degeneration); and B) syndromes appearing
mainly in children or young adults (e.g., Hallervorden-Spatz
disease and progressive familial myoclonic epilepsy); [0044] III.
Syndromes of gradually developing abnormalities of posture and
movement such as paralysis agitans (Parkinson's disease),
striatonigral degeneration, progressive supranuclear palsy, torsion
dystonia (torsion spasm; dystonia musculorum deformans), spasmodic
torticollis and other dyskinesis, familial tremor, and Gilles de la
Tourette syndrome; [0045] IV. Syndromes of progressive ataxia such
as cerebellar degenerations (e.g., cerebellar cortical degeneration
and olivopontocerebellar atrophy (OPCA)); and spinocerebellar
degeneration (Friedreich's ataxia and related disorders); [0046] V.
Syndromes of central autonomic nervous system failure (Shy-Drager
syndrome); [0047] VI. Syndromes of muscular weakness and wasting
without sensory changes (motorneuron disease such as amyotrophic
lateral sclerosis, spinal muscular atrophy (e.g. infantile spinal
muscular atrophy (Werdnig-Hoffman), juvenile spinal muscular
atrophy (Wohlfart-Kugelberg-Welander) and other forms of familial
spinal muscular atrophy), primary lateral sclerosis, and hereditary
spastic paraplegia; [0048] VII. Syndromes combining muscular
weakness and wasting with sensory changes (progressive neural
muscular atrophy; chronic familial polyneuropathies) such as
peroneal muscular atrophy (Charcot-Marie-Tooth), hypertrophic
interstitial polyneuropathy (Dejerine-Sottas), and miscellaneous
forms of chronic progressive neuropathy; [0049] VIII. Syndromes of
progressive visual loss such as pigmentary degeneration of the
retina (retinitis pigmentosa), and hereditary optic atrophy
(Leber's disease).
[0050] In some embodiments, the neurodegenerative disease is
Alzheimer's disease, Parkinson's disease, and/or Huntington's
disease.
[0051] In some embodiments, the invention relates to treatment of
disorders, diseases or conditions associated with chromatin
remodeling.
[0052] In some embodiments, the invention relates to treatment of
tumors expressing the Ras oncogene, as discussed more fully in
commonly owned co-pending United States Patent Publication
Application Number US 2009/0305956 published Dec. 10, 2009, and
entitled "TREATMENT OF RAS-EXPRESSING TUMORS", a complete copy of
which is attached hereto as Exhibit A. As indicated above,
Ras-expressing tumors are often more resistant to standard
therapies. Ras-expressing tumors are often more resistant to
standard therapies. Furthermore, many of the most deadly cancers
involve Ras-expressing tumors. For example, 90-95% of pancreatic
tumors are Ras-expressing. Similarly, 40-45% of colorectal tumors,
40% of bladder tumors, 15-20% of non small cell lung carcinomas
express Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS),
which are not themselves cancer but are bone marrow disorders
characterized by abnormal cell maturation that typically progress
to cancer, also express Ras. There is a profound need for the
development of therapies for these and other Ras-expressing
diseases and disorders.
DAC Inhibitors
[0053] Deacetylase inhibitors, as that term is used herein are
compounds which are capable of inhibiting the deacetylation of
proteins in vivo, in vitro or both. In many embodiments, the
invention relates to HDAC inhibitors, which inhibit the
deacetylation of histones. However, those of ordinary skill in the
art will appreciate that HDAC inhibitors often have a variety of
biological activities, at least some of which may well be
independent of histone deacetylase inhibition.
[0054] As indicated, DAC inhibitors inhibit the activity of at
least one deacetylase. Where the DAC inhibitor is an HDAC
inhibitor, an increase in acetylated histones occurs and
accumulation of acetylated histones is a suitable biological marker
for assessing the activity of HDAC inhibitors. Therefore,
procedures which can assay for the accumulation of acetylated
histones can be used to determine the HDAC inhibitory activity of
agents of interest. Analogous assays can determine DAC inhibitory
activity
[0055] It is understood that agents which can inhibit deacetylase
activity (e.g., histone deacetylase activity) typically can also
bind to other substrates and as often can inhibit or otherwise
regulate other biologically active molecules such as enzymes.
[0056] Suitable DAC or HDAC inhibitors according to the present
invention include, for example, 1) hydroxamic acid derivatives; 2)
Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4)
benzamides; 5) electrophilic ketones; and/or any other class of
compounds capable of inhibiting histone deacetylase. Examples of
such DAC inhibitors include, but are not limited to: [0057] A)
HYDROXAMIC ACID DERIVATIVES such as Suberoylanilide Hydroxamic Acid
(SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95:3003, 1998);
M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Richon et al.,
supra); pyroxamide; CBHA; Trichostatin analogues such as
Trichostatin A (TSA) and Trichostatin C (Koghe et al. Biochem.
Pharmacol. 56:1359, 1998); Salicylihydroxamic Acid (SBHA) (Andrews
et al., International J. Parasitology 30:761, 2000); Azelaic
Bishydroxamic Acid (ABHA) (Andrews et al., supra);
Azelaic-1-Hydroxamate-9-Anilide (AAHA) (Qiu et al., Mol. Biol. Cell
1:2069, 2000); 6-(3-Chlorophenylureido) carpoic Hydroxamic Acid
(3C1-UCHA), Oxamflatin [(2E)-5-[3-[(phenylsulfonyl-)amino
phenyl]-pent-2-en-4-ynohydroxamic acid (Kim et al. Oncogene, 18:
2461, 1999); A-161906. Scriptaid (Su et al. 2000 Cancer Research,
60:3137, 2000); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews
et al., supra); and MW2996 (Andrews et al., supra). [0058] B)
CYCLIC TETRAPEPTIDES such as Trapoxin A (TPX)-Cyclic Tetrapeptide
(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amin-o-8-oxo-9,10-
-epoxy decanoyl)) (Kijima et al., J. Biol. Chem. 268:22429, 1993);
FR901228 (FK 228, FR901228. Depsipeptide. Romidepsin) (Nakajima et
al., Ex. Cell Res. 241:12, 1998); FR225497 Cyclic Tetrapeptide
(Mori et al., PCT Application WO 00/08048. Feb. 17, 2000); Apicidin
Cyclic Tetrapeptide [cyclo
(NO-methyl-L-trvptophanyl-L-isoleucinyl-D-pipe-colinyl-L-2-amino-8-
oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA
93:13143, 1996); Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin
IIa, and Apicidin IIb (P. Dulski et al., PCT Application WO
97/11366); CHAP, HC-Toxin Cyclic Tetrapeptide (Bosch et al., Plant
Cell 7:1941, 1995); WF27082 Cyclic Tetrapeptide (PCT Application WO
98/48825); and Chiamydocin (Bosch et al., supra). [0059] C) SHORT
CHAIN FATTY ACID (SCFA) DERIVATIVES such as: Sodium Butyrate
(Cousens et al., J. Biol. Chem. 254:1716, 1979); Isovalerate
(McBain et al., Biochem. Pharm. 53:1357, 1997); Valerate (McBain et
al., supra); 4 Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer
Research, 15:879, 1995); Phenylbutyrate (PB) (Wang et al., Cancer
Research, 59:2766, 1999); Propionate (McBain et al., supra);
Butyramide (Lea and Tulsyan, supra); Isobutyramide (Lea and
Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra);
3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et
al., Cancer Research, 60:749, 2000); Valproic acid and Valproate.
[0060] D) BENZAMIDE DERIVATIVES such as CI-994; MS-275
[N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamid-
e] (Saito et al., Proc. Natl. Acad. Sci. USA 96:4592, 1999;
3'-amino derivative of MS-27-275 (Saito et al. supra); MGCD0103
(MethylGene), or related compounds. [0061] E) ELECTROPHILIC KETONE
DERIVATIVES such as trifluoromethyl ketones (Frey et al, Bioorganic
& Med. Chem. Lett., 12: 3443, 2002; U.S. Pat. No. 6,511,990)
and 9.alpha.-keto amides such as N-methyl-.alpha.-ketoamides.
[0062] F) OTHER DAC Inhibitors such as Depudecin (Kwon et al.,
Proceedings of the National Academy of Sciences USA, 95:3356,
1998), and other compounds.
[0063] Suitable DAC inhibitors for use in accordance with the
present invention particularly include, for example, CRA-024781
(Celera Genomics), PXD-101 (CuraGene), LAQ-824 (Novartis AG),
LBH-589 (Novartis AG). MGCD0103 (MethylGene), MS-275 (Schering AG),
romidepsin (Gloucester Pharmaceuticals), and/or SAHA (Alton
Pharma/Merck).
[0064] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (I):
##STR00001##
wherein [0065] m is 1, 2, 3 or 4; [0066] n is 0, 1, 2 or 3; [0067]
p and q are independently 1 or 2; [0068] X is O, NH, or NR.sub.8;
[0069] R.sub.1, R.sub.2, and R.sub.3 are independently hydrogen;
unsubstituted or substituted, branched or unbranched, cyclic or
acyclic aliphatic; unsubstituted or substituted, branched or
unbranched, cyclic or acyclic heteroaliphatic; unsubstituted or
substituted aryl; or unsubstituted or substituted heteroaryl;
[0070] R.sub.4, R.sub.5, R.sub.5, R.sub.7 and R.sub.5 are
independently hydrogen; or substituted or unsubstituted, branched
or unbranched, cyclic or acyclic aliphatic; and pharmaceutically
acceptable forms thereof. In certain embodiments, m is 1. In
certain embodiments, n is 1. In certain embodiments, p is 1. In
certain embodiments, q is 1. In certain embodiments, X is O. In
certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are
unsubstituted, or substituted, branched or unbranched, acyclic
aliphatic. In certain embodiments, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are all hydrogen.
[0071] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (II):
##STR00002##
wherein: [0072] m is 1.2, 3 or 4; [0073] n is 0, 1.2 or 3; [0074] q
is 2 or 3; [0075] X is O, NH, or NR.sub.8; [0076] Y is OR.sub.8, or
SR.sub.8; [0077] R.sub.2 and R.sub.3 are independently hydrogen;
unsubstituted or substituted, branched or unbranched, cyclic or
acyclic aliphatic; unsubstituted or substituted, branched or
unbranched, cyclic or acylic heteroaliphatic; unsubstituted or
substituted aryl; or unsubstituted or substituted heteroaryl;
[0078] R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
independently selected from hydrogen; or substituted or
unsubstituted, branched or unbranched, cyclic or acyclic aliphatic;
and pharmaceutically acceptable forms thereof. In certain
embodiments, m is 1. In certain embodiments, n is 1. In certain
embodiments, q is 2. In certain embodiments, X is O. In other
embodiments, X is NH. In certain embodiments. R.sub.2 and R.sub.3
are unsubstituted or substituted, branched or unbranched, acyclic
aliphatic. In certain embodiments, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are all hydrogen.
[0079] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (III):
##STR00003##
wherein
[0080] A is a moiety that is cleaved under physiological conditions
to yield a thiol group and includes, for example, an aliphatic or
aromatic acyl moiety (to form a thioester bond); an aliphatic or
aromatic thioxy (to form a disulfide bond); or the like; and
pharmaceutically acceptable forms thereof. Such aliphatic or
aromatic groups can include a substituted or unsubstituted,
branched or unbranched, cyclic or acyclic aliphatic group; a
substituted or unsubstituted aromatic group; a substituted or
unsubstituted heteroaromatic group; or a substituted or
unsubstituted heterocyclic group. A can be, for example,
--COR.sub.1, --SC(.dbd.O)--O--R.sub.1, or --SR.sub.2, R.sub.1 is
independently hydrogen; substituted or unsubstituted amino;
substituted or unsubstituted, branched or unbranched, cyclic or
acyclic aliphatic; substituted or unsubstituted aromatic group;
substituted or unsubstituted heteroaromatic group; or a substituted
or unsubstituted heterocyclic group. In certain embodiment, R.sub.1
is hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl,
isobutyl, benzyl, or bromobenzyl, R.sub.2 is a substituted or
unsubstituted, branched or unbranched, cyclic or acyclic aliphatic
group; a substituted or unsubstituted aromatic group; a substituted
or unsubstituted heteroaromatic group; or a substituted or
unsubstituted heterocyclic group. In certain embodiments, R.sub.2
is methyl, ethyl, 2-hydroxyethyl, isobutyl, fatty acids, a
substituted or unsubstituted benzyl, a substituted or unsubstituted
aryl, cysteine, homocysteine, or glutathione.
[0081] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (IV) or
(IV'):
##STR00004##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same or
different and represent an amino acid side chain moiety, each
R.sub.6 is the same or different and represents hydrogen or
C.sub.1-C.sub.4 alkyl, and Pr.sup.1 and Pr.sup.2 are the same or
different and represent hydrogen or thiol-protecting group. In
certain embodiments, the amino acid side chain moieties are those
derived from natural amino acids. In other embodiments, the amino
acid side chain moieties are those derived from unnatural amino
acids. In certain embodiments, each amino acid side chain is a
moiety selected from --H, --C.sub.1-C.sub.6 alkyl,
--C.sub.2-C.sub.6 alkenyl, -L-O--C(O)--R', -L-C(O)--O--R'', -L-A,
-L-NR''R'', -L-Het-C(O)--Het-R'', and -L-Het-R'', wherein L is a
C.sub.1-C.sub.6 alkylene group, A is phenyl or a 5- or 6-membered
heteroaryl group, each R' is the same or different and represents
C.sub.1-C.sub.4 alkyl, each R'' is the same or different and
represent H or C.sub.1-C.sub.6 alkyl, each -Het- is the same or
different and is a heteroatom spacer selected from --O--,
--N(R''')-, and --S--, and each R''' is the same of different and
represents H or C.sub.1-C.sub.4 alkyl. In certain embodiments,
R.sub.6 is --H. In certain embodiments, Pr.sup.1 and Pr.sup.2 are
the same or different and are selected from hydrogen and a
protecting group selected from a benzyl group which is optionally
substituted by C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 acyloxy,
hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl,
diphenylmethyl, phenyl, t-butyl, adamanthyl, C.sub.1-C.sub.6
acyloxymethyl, C.sub.1-C.sub.6 alkoxymethyl, tetrahydropyranyl,
benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl,
benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its
derivatives, benzoyl and its derivatives, carbamoyl,
phenylcarbamoyl, and C.sub.1-C.sub.6 alkylcarbamoyl. In certain
embodiments, Pr.sup.1 and Pr.sup.2 are hydrogen. Various romidepsin
derivatives of formula (IV) and (IV') are disclosed in published
PCT application WO 2006/129105, published Dec. 7, 2006; which is
incorporated herein by reference.
[0082] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (V):
##STR00005##
wherein [0083] B is a substituted or unsubstituted, saturated or
unsaturated aliphatic group, a substituted or unsubstituted,
saturated or unsaturated alicyclic group, a substituted or
unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, or a substituted or unsubstituted
heterocyclic group; R.sub.20 is hydroxylamino, hydroxyl, amino,
alkylamino, dialkylamino, or alkyloxy group; R.sub.21 and R.sub.22
are independently selected from hydrogen, hydroxyl, a substituted
or unsubstituted, saturated or unsaturated aliphatic group, a
substituted or unsubstituted, saturated or unsaturated alicyclic
group, a substituted or unsubstituted aromatic group, a substituted
or unsubstituted heteroaromatic group, or a substituted or
unsubstituted heterocyclic group. In a particular embodiment of
Formula IV, R.sub.20 is a hydroxylamino, hydroxyl, amino,
methylamino, dimethylamino or methyloxy group and B is a
C.sub.6-alkyl. In yet another embodiment of Formula IV. R.sub.21 is
a hydrogen atom, R.sub.22 is a substituted or unsubstituted phenyl
and B is a C.sub.6-alkyl. In further embodiments of Formula IV,
R.sub.21 is hydrogen and R.sub.22 is an .alpha.-, .beta.-, or
.gamma.-pyridine.
[0084] Other examples of DAC or HDAC inhibitors can be found in,
for example, U.S. Pat. Nos. 5,369,108, issued on Nov. 29, 1994,
5,700,811, issued on Dec. 23, 1997, 5,773,474, issued on Jun. 30,
1998, 5,932,616 issued on Aug. 3, 1999 and 6,511,990, issued Jan.
28, 2003 all to Breslow et al.; U.S. Pat. Nos. 5,055,608, issued on
Oct. 8, 1991, 5,175,191, issued on Dec. 29, 1992 and 5,608,108,
issued on Mar. 4, 1997 all to Marks et al.; U.S. Provisional
Application No. 60/459,826, filed Apr. 1, 2003 in the name of
Breslow et al.; as well as, Yoshida. M., et al., Bioassays 17,
423-430 (1995); Saito, A., et al., PNAS USA 96, 4592-4597, (1999);
Furamai R, et al., PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et
al., Cancer Res. 61(11), 4459-4466 (2001); Su, G. H., et al.,
Cancer Res. 60, 3137-3142 (2000); Lee, B. I, et al., Cancer Res.
61(3), 931-934; Suzuki, T., et al., J. Med. Chem. 42(15), 3001-3003
(1999); published PCT Application WO 01/18171 published on Mar. 15,
2001 Sloan-Kettering Institute for Cancer Research and The Trustees
of Columbia University; published PCT Application WO02/246144 to
Hoffmann-La Roche; published PCT Application WO02/22577 to
Novartis; published PCT Application WO02/30879 to Prolifix;
published PCT Applications WO 01/38322 (published May 31, 2001), WO
01/70675 (published on Sep. 27, 2001) and WO 00/71703 (published on
Nov. 30, 2000) all to Methylgene, Inc.; published PCT Application
WO 00/21979 published on Oct. 8, 1999 to Fujisawa Pharmaceutical
Co., Ltd.; published PCT Application WO 98/40080 published on Mar.
11, 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current
patent status of histone deacetylase inhibitors Expert Opin. Ther.
Patents (2002) 12(9): 1375-1384 and references cited therein).
[0085] Specific non-limiting examples of DAC or HDAC inhibitors are
provided in the Table below. It should be noted that the present
invention encompasses any compounds which both are structurally
similar to the compounds represented below and are capable of
inhibiting histone deacetylases.
TABLE-US-00001 Title MS-275 ##STR00006## DEPSIPEPTIDE ##STR00007##
Cl-994 ##STR00008## Apicidin ##STR00009## A-161906 ##STR00010##
Scriptaid ##STR00011## PXD-101 ##STR00012## CHAP ##STR00013##
LAQ-824 ##STR00014## Butyric Acid ##STR00015## Depudecin
##STR00016## Oxamflatin ##STR00017## Trichostatin C
##STR00018##
[0086] DAC or HDAC inhibitors for use in accordance with the
present invention may be prepared by any available means including,
for example, synthesis, semi-synthesis, or isolation from a natural
source.
[0087] DAC or HDAC inhibitors for use in accordance with the
present invention may be isolated or purified. For example,
synthesized compounds can be separated from a reaction mixture, and
natural products can be separated from their natural source, by
methods such as column chromatography, high pressure liquid
chromatography, and/or recrystallization.
[0088] A variety of synthetic methodologies for preparing DAC or
HDAC inhibitors are known in the art. As can be appreciated by the
skilled artisan, further methods of
[0089] A variety of synthetic methodologies for preparing DAC or
HDAC inhibitors are known in the art. As can be appreciated by the
skilled artisan, further methods of synthesizing the compounds of
the formulae herein will be evident to those of ordinary skill in
the art. Additionally, the various synthetic steps may be performed
in an alternate sequence or order to give the desired compounds.
Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein are known in the art and include,
for example, those such as described in R. Larock, Comprehensive
Organic Transformations, VCH Publishers (1989); T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed. John
Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995), and subsequent editions thereof.
[0090] DAC or HDAC inhibitors for use in accordance with the
present invention may be modified as compared with presently known
DAC or HDAC inhibitors, for example, by appending appropriate
functionalities to enhance selective biological properties. Such
modifications are known in the art and may include those which
increase biological penetration into a given biological system
(e.g., blood, lymphatic system, central nervous system), increase
oral availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0091] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in
accordance with the present invention may contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric forms that may be defined,
in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)-
or (L)- for amino acids. The present invention encompasses all such
possible isomers, as well as their racemic and optically pure forms
to the extent that they have DAC inhibitory activity.
[0092] In general, optical isomers may be prepared from their
respective optically active precursors by the procedures described
above, or by resolving the racemic mixtures. The resolution can be
carried out in the presence of a resolving agent, by chromatography
or by repeated crystallization or by some combination of these
techniques which are known to those skilled in the art. Further
details regarding resolutions can be found in Jacques, et al.
Enantiomers, Racemates, and Resolutions (John Wiley & Sons,
1981).
[0093] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in
accordance with the present invention may contain olefinic double
bonds, other unsaturation, or other centers of geometric asymmetry.
The present invention encompasses both E and Z geometric isomers or
cis- and trans-isomers to the extent that they have DAC inhibitory
activity. The present invention likewise encompasses all tautomeric
forms that have DAC inhibitory activity. In general, where a
chemical structure is presented, the configuration of any
carbon-carbon double bond appearing herein is selected for
convenience only and is not intended to designate a particular
configuration unless the text so states or it is otherwise clear
from context; thus a carbon-carbon double bond or carbon-heteroatom
double bond depicted arbitrarily herein as trans may be cis, trans,
or a mixture of the two in any proportion.
[0094] DAC inhibitors (e.g., HDAC inhibitors) are particularly
useful in the treatment of neoplasms in vivo. However, they may
also be used in vitro for research or clinical purposes (e.g.,
determining the susceptibility of a patient's disease to a
particular DAC inhibitor). In certain embodiments, the neoplasm is
a benign neoplasm. In other embodiments, the neoplasm is a
malignant neoplasm. Any cancer may be treated using a DAC inhibitor
alone or in combination with another pharmaceutical agent.
[0095] In certain embodiments, the malignancy is a hematological
malignancy. Manifestations can include circulating malignant cells
as well as malignant masses. Hematological malignancies are types
of cancers that affect the blood, bone marrow, and/or lymph nodes.
Examples of hematological malignancies that may be treated using
romidepsin include, but are not limited to: acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL),
hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma,
cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma
(PTCL), multiple myeloma, and myelodysplastic syndromes. In certain
embodiments, the inventive combination is used to treat multiple
myeloma. In certain particular embodiments, the cancer is relapsed
and/or refractory multiple myeloma. In other embodiments, the
inventive combination is used to treat chromic lymphocytic leukemia
(CLL). In certain particular embodiments, the cancer is relapsed
and/or refractory CLL. In other embodiments, the inventive
combination is used to treat chromic myelogenous leukemia (CML). In
certain embodiments, the inventive combination is used to treat
acute lymphoblastic leukemia (ALL). In certain embodiments, the
inventive combination is used to treat acute myelogenous leukemia
(AML). In certain embodiments, the cancer is cutaneous T-cell
lymphoma (CTCL). In other embodiments, the cancer is peripheral
T-cell lymphoma (PTCL). In certain embodiments, the cancer is a
myelodysplastic syndrome.
[0096] Other cancers besides hematological malignancies may also be
treated using DAC inhibitors. In certain embodiments, the cancer is
a solid tumor.
[0097] Exemplary cancers that may be treated using DAC inhibitor
therapy, including combination therapy, include colon cancer, lung
cancer, bone cancer, pancreatic cancer, stomach cancer, esophageal
cancer, skin cancer, brain cancer, liver cancer, ovarian cancer,
cervical cancer, uterine cancer, testicular cancer, prostate
cancer, bladder cancer, kidney cancer, neuroendocrine cancer,
etc.
[0098] In certain embodiments, a DAC inhibitor is used to treat
pancreatic cancer. In certain embodiments, a DAC inhibitor is used
to treat prostate cancer. In certain specific embodiments, the
prostate cancer is hormone refractory prostate cancer. In certain
embodiments, a DAC inhibitor is administered in combination with
one or more additional therapeutic agents, e.g., another cytotoxic
agent. Exemplary cytotoxic agents that may be administered in
combination with a DAC inhibitor include gemcitabine, decitabine,
and flavopiridol. In other embodiments, a DAC inhibitor is
administered in combination with an anti-inflammatory agent such as
aspirin, ibuprofen, acetaminophen, etc., pain reliever, anti-nausea
medication, or anti-pyretic. In certain other embodiments, a DAC
inhibitor is administered in combination with a steroidal agent
(e.g., dexamethasone). In certain embodiments, a DAC inhibitor is
administered in combination with an agent to treat gastrointestinal
disturbances such as nausea, vomiting, and diarrhea. These
additional agents may include anti-emetics, anti-diarrheals, fluid
replacement, electrolyte replacement, etc. In other embodiments, a
DAC inhibitor is administered in combination with electrolyte
replacement or supplementation such as potassium, magnesium, and
calcium, in particular, potassium and magnesium. In certain
embodiments, a DAC inhibitor is administered in combination an
anti-arrhythmic agent. In certain embodiments, a DAC inhibitor is
administered in combination with a platelet booster, for example,
an agent that increases the production of platelets. In certain
embodiments, a DAC inhibitor is administered in combination with an
agent to boost the production of blood cells such as
erythropoietin. In certain embodiments, a DAC inhibitor is
administered in combination with an agent to prevent hyperglycemia.
In certain embodiments, a DAC inhibitor is not administered with
another HDAC or DAC inhibitor.
Combination Therapy
[0099] The present invention demonstrates the particular utility of
administering a combination of a DAC inhibitor and gemcitabine. In
some particular embodiments of the present invention, the DAC
inhibitor is romidepsin (aka depsipeptide, FK228, FR901228). In
other particular embodiments, the DAC inhibitor is selected from
the group consisting of CRA-024781 (Celera Genomics),
phenylbutarate. PXD-101 (CuraGene), LAQ-824 (Novartis AG). LBH-589
(Novartis AG), MGCD0103 (MethylGene), MS-275 (Schering AG),
romidepsin (Gloucester Pharmaceuticals), SAHA (Alton Pharma/Merck),
and combinations thereof. In some particular embodiments of the
present invention, the DAC inhibitor is romidepsin (aka
depsipeptide, FK228, FR901228). In some particular embodiments, the
DAC inhibitor is SAHA. In some particular embodiments, the DAC
inhibitor is phenylbutyrate. In some particular embodiments, the
DAC inhibitor comprises a combination of DAC inhibitors.
[0100] The present invention demonstrates the particular utility of
administering a combination of a DAC inhibitor and gemcitabine.
Without wishing to be bound by any particular theory, the inventors
note that such a combination may increase apoptosis in
recipients.
[0101] As will be appreciated by those of skill in the art, and as
is otherwise addressed herein, either or both of the DAC inhibitor
and gemcitabine may be provided in any useful form including, for
example, as a salt, ester, active metabolite, prodrug, etc.
Similarly, either or both agents (or salts, esters, or prodrugs
thereof) may be provided as a pure isomer stereoisomer or as a
combination of stereoisomers, including a racemic combination, so
long as relevant activity is present. Comparably, either or both
agents (or salts, esters or prodrugs thereof) may be provided in
crystalline form, whether a pure polymorph or a combination of
polymorphs, or in amorphous form, so long as relevant activity is
present.
[0102] As addressed above, combination therapy of DAC inhibitors
and gemcitabine will typically involve administration of multiple
individual doses spaced out in time. In some embodiments,
individual DAC inhibitor doses and gemcitabine doses will be
administered together, according to the same schedule. In other
embodiments, DAC inhibitor doses and gemcitabine doses will be
administered according to different schedules.
[0103] The total daily dose of any particular active agent
administered to a human or other animal in single or in divided
doses in accordance with the present invention can be in amounts,
for example, from 0.01 to 50 mg/kg body weight or more usually from
0.1 to 25 mg/kg body weight. Single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose. In
general, treatment regimens according to the present invention
comprise administration to a patient in need of such treatment from
about 10 mg to about 1000 mg of the compound(s) of this invention
per day in single or multiple doses. In certain embodiments, about
10-100 mg of the compound is administered per day in single or
multiple doses. In certain embodiments, about 100-500 mg of the
compound is administered per day in single or multiple doses. In
certain embodiments, about 250-500 mg of the compound is
administered per day in single or multiple doses. In certain
embodiments, about 500-750 mg of the compound is administered per
day in single or multiple doses.
[0104] In the treatment of neoplasms such as cancer in a subject, a
DAC inhibitor is typically dosed at 1-30 mg/m.sup.2. In certain
embodiments, a DAC inhibitor is dosed at 1-15 mg/m.sup.2. In
certain embodiments, a DAC inhibitor is dosed at 5-15 mg/m.sup.2.
In certain particular embodiments, a DAC inhibitor is dosed at 4,
6, 8, 10, 12, 14, 16, 18, or 20 mg/m.sup.2.
[0105] A DAC inhibitor is typically administered in a 28 day cycle
with the agent being administered on days 1, 8 and 15. In certain
embodiments, the DAC is administered on days 1 and 15 with day 8
being skipped. As would be appreciated by one of skill in the art,
the dosage and timing of administration of the dosage of the DAC
inhibitor may vary depending on the patient and condition being
treated. For example, adverse side effects may call for lowering
the dosage of DAC inhibitor administered.
[0106] Typical dosing schedules have been established for certain
exemplary DAC inhibitors (e.g., HDAC inhibitors). For example, SAHA
is commonly administered within a range of about 300-400 mg daily
orally; PXD101 is commonly administered within a range of about up
to 2000 mg/m.sup.2/day intravenously (e.g., on days 1 to 5 of a 21
day cycle), and may possibly be administered orally; MGCD0103 is
commonly administered at doses up to about 27 mg/m.sup.2 given
orally (e.g., daily for about 14 days); LBH589 is commonly
administered at doses up to about 14 mg/m.sup.2 as an intravenous
infusion (e.g., on days 1-7 of a 21 day cycle); MS-275 is commonly
administered within a dose range of about 2-12 mg/m.sup.2
intravenously (e.g., every 14 days).
[0107] In the treatment of neoplasms such as cancer in a subject,
romidepsin is typically dosed at 1-28 mg/m.sup.2. In certain
embodiments, romidepsin is dosed at 1-15 mg/m.sup.2. In certain
embodiments, romidepsin is dosed at 5-14 mg/m.sup.2. In certain
particular embodiments, romdiepsin is dosed at 8, 10, 12, or 14
mg/m.sup.2. Romidepsin is typically administered in a 28 day cycle
with romidepsin being administered on days 1, 8 and 15. In certain
embodiments, romidepsin is administered on days 1 and 15 with day 8
being skipped.
[0108] Acceptable dosing schedules have also been established for
gemcitabine for at least pancreatic, non-small cell lung, breast,
and ovarian cancers. For example, for pancreatic cancer,
gemcitabine is typically administered by intravenous infusion at a
dose of 1000 mg/m.sup.2 over 30 minutes once weekly for up to 7
weeks (or until toxicity necessitates reducing or holding a dose),
followed by a week of rest from treatment. Subsequent cycles
typically consist of infusions once weekly for 3 consecutive weeks
out of every 4 weeks.
[0109] For non-small cell lung cancer, where it is typically given
in combination, gemcitabine is often administered either on a
4-week schedule that involves intravenous dosing at 1000 mg/m.sup.2
over 30 minutes on Days 1, 8, and 15 of each 28-day cycle, or on a
3-week schedule, where it is administered intravenously at 1250
mg/m.sup.2 over 30 minutes on Days 1 and 8 of each 21-day
cycle.
[0110] For breast cancer, where it is typically also given in
combination, gemcitabine is often administered intravenously at a
dose of 1250 mg/m.sup.2 over 30 minutes on Days 1 and 8 of each
21-day cycle.
[0111] For ovarian cancer, where it is typically also given in
combination, gemcitabine is often administered intravenously at a
dose of 1000 mg/m.sup.2 over 30 minutes on Days 1 and 8 of each
21-day cycle.
[0112] As would be appreciated by one of skill in the art, the
dosage and timing of administration of any particular DAC inhibitor
or gemcitabine dose, or the dosage amount and schedule generally
may vary depending on the patient and condition being treated. For
example, adverse side effects may call for lowering the dosage of
one or the other agent, or of both agents, being administered.
[0113] Moreover, those of ordinary skill in the art will readily
appreciate that the dosage schedule (i.e., amount and timing of
individual doses) by which any particular DAC inhibitor is
administered may be different for inventive combination therapy
with gemcitabine than it is alone. Comparably, the dosage schedule
for gemcitabine may be different according to inventive combination
therapy regimens than would be utilized in gemcitabine monotherapy
(even for the same disorder, disease or condition).
[0114] To give but one example, in some embodiments, a DAC
inhibitor (e.g., romidepsin) and gemcitabine are each dosed on days
1 and 15 of a 28 day cycle. Those of ordinary skill in the art will
appreciate that any of a variety of other dosing regimens are
within the scope of the invention. Commonly, dosing is adjusted
based on a patient's response to therapy, and particularly to
development of side effects.
[0115] In some embodiments of the present invention, inventive
combination therapy with one or more DAC inhibitors and gemcitabine
is further combined with administration of one or more other
agents.
[0116] In some embodiments, subjects receiving inventive
combination therapy with one or more DAC inhibitors and gemcitabine
further receive electrolyte supplementation for example as is
described in co-pending U.S. Provisional Patent application Ser.
No. 60/909,780 entitled "DEACETYLASE INHIBITOR THERAPY", filed Apr.
3, 2007.
[0117] For example, as described in that application, an individual
with a potassium serum concentration below about 3.5 mmol/L (3.5
mEq/L) and/or a serum magnesium concentration below about 0.8 mml/L
(1.95 mEq/L) suffers an increased risk of developing cardiac
repolarization effects and/or dysrhythmias.
[0118] Serum concentrations of potassium are generally considered
to be "normal" when they are within the range of about 3.5-5.5
mEq/L or about 3.5-5.0 mEq/L. According to the present invention,
it is often desirable to ensure that an individuals' serum
potassium concentration is within this range prior to (and/or
during) administration of DAC inhibitor therapy.
[0119] Serum concentrations of magnesium are generally considered
to be "normal" when they are within the range of about 1.5-2.5
mEq/L or about 1.5-2.2 mEq/L or about 1.25-2.5 mEq/L or about
1.25-2.2 mEq/L. According to the present invention, it is often
desirable to ensure that an individual's serum magnesium
concentration is within this range prior to (and/or during)
administration of DAC inhibitor therapy.
[0120] In some embodiments of the invention, an individual's serum
potassium and/or magnesium concentration(s) is/are at the high end
of the normal range prior to (and/or during) administration of DAC
inhibitor therapy. For example, in some embodiments, an
individual's serum potassium concentration is at least about 3.8,
3.9, 4.0 mEq/L, or more prior to and/or during administration of
DAC inhibitor therapy. In some embodiments, care is taken not to
increase serum potassium concentration above about 5.0, 5.2, or 5.5
mEq/L. In some embodiments, an individual's serum magnesium
concentration is at least about 1.9 mEq/L or more prior to and/or
during administration of DAC inhibitor therapy. In some
embodiments, care is taken not to increase magnesium concentration
above about 2.5 mEq/L.
[0121] In some embodiments of the present invention, an
individual's serum potassium concentration is at least about 3.5
mEq (in some embodiments at least about 3.8, 3.9, 4.0 mEq/L or
above) and the individual's serum magnesium concentration is at
least about 1.85 mEq/L (in some embodiments at least about 1.25,
1.35, 1.45, 1.55, 1.65, 1.75, 1.85, 1.95, etc) prior to and/or
during administration of DAC inhibitor therapy.
[0122] In some embodiments of the invention, electrolyte levels
(e.g., potassium and/or magnesium levels, optionally calcium
levels) are assessed more than once during the course of DAC
inhibitor therapy; in some embodiments, different assessments are
separated by a regular interval (e.g., 0.5 days or less, 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10
days, 11 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3
months, 4 months, 5 months, 6 months, etc.). In some embodiments,
electrolyte levels are assessed prior to each administration of DAC
inhibitor.
Pharmaceutical Compositions
[0123] DAC inhibitors and/or gemcitabine for use in accordance with
the present invention are often administered as pharmaceutical
compositions comprising amounts of DAC inhibitor and gemcitabine,
respectively, that are useful in inventive combination therapy
(which amounts may be different from, including less than, amounts
required for either agent to be effective alone). In some
embodiments, a DAC inhibitor and gemcitabine are present together
in a single pharmaceutical composition; in some embodiments these
agents are provided in separate pharmaceutical compositions.
[0124] In some embodiments, inventive pharmaceutical compositions
are prepared in unit dosage forms. In general, a pharmaceutical
composition of the present invention includes one or more active
agents (i.e., one or more DAC inhibitors and/or gemcitabine)
formulated with one or more pharmaceutically acceptable carriers or
excipients.
[0125] In some embodiments, the pharmaceutically acceptable carrier
is selected from the group consisting of sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions; non-toxic compatible lubricants such as sodium lauryl
sulfate and magnesium stearate; coloring agents; releasing agents;
coating agents; sweetening, flavoring and perfuming agents;
preservatives and antioxidants; and combinations thereof. In some
embodiments, the pH of the ultimate pharmaceutical formulation may
be adjusted with pharmaceutically acceptable acids, bases or
buffers to enhance the stability of the formulated compound or its
delivery form.
[0126] Pharmaceutical compositions of this invention may be
administered can be administered by any appropriate means
including, for example, orally, parenterally, by inhalation spray,
topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques. In many embodiments, pharmaceutical compositions are
administered orally or by injection in accordance with the present
invention.
[0127] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
agent(s), liquid dosage forms of pharmaceutical compositions may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0128] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. A sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0129] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0130] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from a site of
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of the
drug then depends upon its rate of dissolution, which, in turn, may
depend upon crystal size and crystalline form.
[0131] Alternatively, delayed absorption of a parenterally
administered drug form can be accomplished by dissolving or
suspending the drug in an oil vehicle. Injectable depot forms can
be made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of drug to polymer and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations can also be
prepared by entrapping the drug in liposomes or microemulsions that
are compatible with body tissues.
[0132] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing the active
agents with suitable non-irritating excipients or carriers such as
cocoa butter, polyethylene glycol or a suppository wax which are
solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
active compound.
[0133] Solid dosage forms for oral administration include, for
example, capsules, tablets, pills, powders, and granules. In such
solid dosage forms, the active agent(s) is/are typically mixed with
at least one inert, pharmaceutically acceptable excipient or
carrier such as sodium citrate or dicalcium phosphate and/or: a)
fillers or extenders such as starches, lactose, sucrose, glucose,
mannitol, and silicic acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, c) humectants such as glycerol, d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f)
absorption accelerators such as quaternary ammonium compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, h) absorbents such as kaolin and bentonite clay, and
i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form may also comprise butffering agents, permeation enhancers,
and/or other agents to enhance absorption of the active
agent(s).
[0134] Solid compositions of a similar type may also be employed as
tillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0135] Solid dosage forms such as tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells such
as enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions that can be used include
polymeric substances and waxes.
[0136] In certain embodiments, oral dosage forms are prepared with
coatings or by other means to control release of active agent (e.g.
DAC inhibitor and/or gemcitabine) over time and/or location within
the gastrointestinal tract. A variety of strategies to achieve such
controlled (or extended) release are well known in the art, and are
within the scope of the present invention.
[0137] Dosage forms for topical or transdermal administration
include ointments, pastes, creams, lotions, gels, powders,
solutions, sprays, inhalants or patches. In general, such
preparations are prepared by admixing active agent(s) under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required.
[0138] Ophthalmic formulation, ear drops, eye ointments, powders
and solutions are also contemplated as being within the scope of
this invention.
[0139] Ointments, pastes, creams and gels may contain, in addition
to active agent(s), excipients such as animal and vegetable fats,
oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0140] Powders and sprays can contain, in addition to active
agent(s), excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants such as chlorofluorohydrocarbons.
[0141] Transdermal patches have often can provide controlled
delivery of a compound to the body. Such dosage forms can be made
by dissolving or dispensing the compound in the proper medium.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate can be controlled by either
providing a rate controlling membrane or by dispersing the compound
in a polymer matrix or gel.
[0142] For pulmonary delivery, active agent(s) is/are formulated
and administered to the patient in solid or liquid particulate form
by direct administration e.g., inhalation into the respiratory
system. Solid or liquid particulate forms of the active agent(s)
prepared for practicing the present invention include particles of
respirable size: that is, particles of a size sufficiently small to
pass through the mouth and larynx upon inhalation and into the
bronchi and alveoli of the lungs. Delivery of aerosolized
therapeutics, particularly aerosolized antibiotics, is known in the
art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et
al., U.S. Pat. No. 5,508,269 to Smith et al., and WO 98/43,650 by
Montgomery, all of which are incorporated herein by reference). A
discussion of pulmonary delivery of antibiotics is also found in
U.S. Pat. No. 6,014,969, incorporated herein by reference.
[0143] Pharmaceutical compositions for use in accordance with the
present invention can, for example, be administered by injection,
intravenously, intraarterially, subdermally, intraperitoneally,
intramuscularly, or subcutaneously; or orally, buccally, nasally,
transmucosally, topically, in an ophthalmic preparation, or by
inhalation, for example with a dosage ranging from about 0.1 to
about 500 mg/kg of body weight, alternatively dosages between 1 mg
and 1000 mg/dose, every 4 to 120 hours, or according to the
requirements of the particular drug.
[0144] The methods herein contemplate administration of an
effective amount of active agent or pharmaceutical composition
sufficient for a desired or stated effect. Typically, the
pharmaceutical compositions of this invention will be administered
from about 1 to about 6 times per day or alternatively, as a
continuous infusion. Such administration can be used as a chronic
or acute therapy.
[0145] The amount of any particular active agent that may be
combined with pharmaceutically acceptable excipients or carriers to
produce a single dosage form may vary depending upon the host
treated and the particular mode of administration. A typical
preparation will contain from about 5% to about 95% active compound
(w/w). Alternatively, such preparations may contain from about 20%
to about 80% active compound. For romidepsin, preparations may
commonly contain about 20-50%, 25-45%, 30-40%, or approximately
32%, 33%, 34%, or 35% active compound; for gemcitabine, the
compound is typically provided in 200 mg or 1 gram vials as a
lyophilized powder. Drug product is reconstituted with either 5 ml
(for the 200 mg vial) or 25 ml (for the 1 g vial) using sodium
chloride for injection. Both dilutions give a 38 mg/ml solution
(including displacement volume). This solution can be diluted down
to 0.1 mg/ml-0.4 mg/ml for administration.
[0146] Lower or higher doses than those recited above may be
required. Specific dosage and treatment regimens for any particular
patient will depend upon a variety of factors, including the
activity of the specific compound employed, the age, body weight,
general health status, sex, diet, time of administration, rate of
excretion, drug combination, the severity and course of the
disease, condition or symptoms, the patient's disposition to the
disease, condition or symptoms, and the judgment of the treating
physician.
[0147] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level. Patients may, however, require intermittent treatment on a
long-term basis upon any recurrence of disease symptoms.
[0148] When pharmaceutical compositions contain two or more active
agents, it is generally the case that each agent is present at
dosage levels of between about 1 to 100%, for example about 5 to
95%, of the level normally administered in a monotherapy regimen.
Unless otherwise defined, all technical and scientific terms used
herein are accorded the meaning commonly known to one of ordinary
skill in the art. All publications, patents, published patent
applications, and other references mentioned herein are hereby
incorporated by reference in their entirety. The embodiments of the
invention should not be deemed to be mutually exclusive and can be
combined.
[0149] Unless otherwise defined, all technical and scientific terms
used herein are accorded the meaning commonly known to one of
ordinary skill in the art. All publications, patents, published
patent applications, and other references mentioned herein are
hereby incorporated by reference in their entirety. The embodiments
of the invention should not be deemed to be mutually exclusive and
can be combined.
EXEMPLIFICATION
[0150] The present invention will be better understood in
connection with the following Examples, which are intended as an
illustration only and not limiting of the scope of the invention.
Various changes and modifications to the disclosed embodiments will
be apparent to those skilled in the art and such changes and
modifications including, without limitation, those relating to the
chemical structures, substituents, derivatives, formulations and/or
methods of the invention may be made without departing from the
spirit of the invention and the scope of the appended claims.
Example 1
Depsipeptide (FK228-) Alone and in Combination with Gemcitabine in
In Vivo Mouse Xenograft Model of Ras-Expressing Pancreatic
Tumor
[0151] The present Example demonstrates that both depsipeptide
(FK228; FR901228; romidepsin) and gemcitabine can effectively
inhibit tumor growth in a mouse xenograft model, and further
demonstrates a surprising synergistic effect of the
combination.
[0152] Panc-1, obtained from the ATCC, is a pancreas tumor cell
line (oncogenic K-ras) originating from a 56 year old Caucasian
male. In this study, female nude mice were implanted subcutaneously
(SC) by trocar with Panc-1 tumor fragments harvested from SC
growing tumors in nude mice hosts. When tumors reaches
approximately 140 mm.sup.3, animals were pair matched by tumor size
into treatment and control groups (N=9 mice per group). The day of
treatment initiation was specified as Day 1. Vehicle control and
FK228 were administered intravenously on a Q4Dx3 schedule (Days 1,
5, and 9). Gemcitabine was administered by an intraperitoneal
injection on a Q3Dx4 schedule (Days 1, 4, 7, 10). Tumors were
measured by Vernier calipers twice weekly.
[0153] Treatment with depsipeptide (FK228, FR901228, romidepsin)
and gemcitabine as single agents both resulted in consistently
smaller tumors than vehicle control treated animals, with tumor
growth inhibitions of 26% and 50%, respectively.
[0154] In addition, a potential synergistic effect of depsipeptide
and gemcitabine was observed in combination with a significant
tumor growth inhibition of 101%. Three animals in the combination
group exhibited evidence of tumor regression. These results
indicate depsipeptide has clear antitumor activity against the
Panc-1 human pancreas tumors in an in vivo xenograft model.
Furthermore, depsipeptide has the potential to synergize with other
approved chemotherapeutics, and specifically with gemcitabine. The
effects of this synergy, including tumor regression, are
particularly significant given the known aggressiveness of
pancreatic tumors, and their susceptibility to developing
resistance. The present invention demonstrates tumor regression
after dosing with a combination of romidepsin and gemcitabine. Note
that no regression was observed with gemcitabine alone, the current
standard therapy for pancreatic tumors, yet regression was observed
with the combination.
[0155] We note that no synergistic effect was observed with the
combination of romidepsin and gemcitabine in another cell line
(Bx-PC-3) that had normal Ras.
Example 2
Combination of FK228 and Gemcitabine is More Effective than Either
Agent Alone in a Ras-Transformed Pancreatic Adenocarcinoma
Model
[0156] The present Example demonstrates that the combination of
FK228 and gemcitabine is more effective than either agent alone in
a pancreatic adenocarcinoma model.
[0157] Abstract: To examine activity and mechanism of FK228,
antitumor efficacy was tested in PANC-1 pancreatic adenocarcinoma
model representing transformed Ras, either as a single agent or in
combination with gemcitabine. Following PANC-1 study completion,
tumor and sera were obtained from:
[0158] the vehicle control;
[0159] FK228 dosed at 5 mg/kg once every four days for three
treatments (Q4Dx3);
[0160] Gemcitabine at 80 mg/kg (Q3Dx4); and
[0161] the drug combination.
Expression of c-Myc, acetylated histones 3 and 4, and p21.sup.waf1
was compared between control and FK228 groups by immunoblotting and
was quantified following actin normalization. Serum levels of
putative tumor products b-FGF and MMP-2 were quantified by
human-specific ELISA.
[0162] Highly significant (p<0.0001) downregulation of c-Myc was
observed in all treatment groups, most dramatically in the
combination group. Acetylated histone 3 levels were not affected in
FK228 alone or in combination with gemcitabine. Upregulation of
acetylated histone 4 by the drug combination was highly
significant. Treatment with gemcitabine alone significantly
(p<0.05) downregulated p21.sup.waf; however, this effect was not
reported in combination groups.
[0163] These results suggest activity of FK228 is Ras-transformed
malignancies and demonstrate combinatorial effects with gemcitabine
at least in pancreatic adenocarcinoma. Surprising long-term effects
of FK228 in combination with gemcitabine on c-Myc and acetylated
histone 4 might suggest tumor phenotypic changes consistent with
downregulation of HDAC activity.
[0164] Materials and Methods
[0165] Specimen Collection: FK228 antitumor efficacy was tested in
PANC-1 pancreatic adenocarcinoma model representing transformed
Ras, either as a single agent or in combination with gemcitabine.
Tumor xenograft tissue and serum specimens were obtained from in
vivo studies performed by the Preclinical Research Laboratory at
the completion of the experiments. The following tumor and sera
specimens were obtained:
[0166] Vehicle control (n-9)
[0167] FK228 at 5 mg/kg once every four days for three treatments
(Q4Dx3) (n=9)
[0168] Gemcitabine at 80 mg/kg (Q3Dx4) (n=9)
[0169] FK228 at 5 mg/kg plus gemcitabine at 80 mg/kg (Q3Dx4)
(n=6)
Tumors were dissected from the animals, rinsed in cold phosphate
buffered saline, and snap frozen in liquid nitrogen. Serum was
obtained from whole blood and stored frozen at -70.degree. C.
[0170] Tissue Biomarkers: Tumor levels of acetylated histone-4,
histone-4, c-Myc, p21waf and .beta.-actin were quantified by
immunoblotting as described. Briefly, frozen tissue was pulverized
under liquid nitrogen and homogenized in hypotonic lysis buffer.
Small aliquots of the extracts were used for analysis of protein
concentration by micro-BCA assay with bovine serum albumin as a
protein standard/An equal amount of extracts containing about 20-50
.mu.g protein was electrophoresed in SDS polyacrylamide gels.
Proteins were transferred to ImmunoBlot PVDF membrane and were
probed with appropriate primary and secondary antibodies. The
chemiluminescence signal was captured by autoradiography,
quantified by densitometry and expressed as a ratio of actin in
each sample lane. For each biomarker, means and standard errors
were calculated in each treatment group. The data were analyzed by
two-sided t-tests to determine if measured end points are
significantly affected by drug treatment.
[0171] Serum Biomarkers: Serum levels of b-FGF and VEGF were
quantified by ELISA using human-specific kits from R&D Systems.
Minneapolis, Minn., according to supplier's instructions. All
assays were performed in duplicate. For each biomarker, means and
standard errors were calculated in each treatment group. The data
were analyzed by two-tailed Student t-tests to determine if
measured endpoints are significantly affected by drug
treatment.
Results
[0172] Western blot detection of acetylated histone 3, histone 4,
c-Myc, p21.sup.waf and a housekeeping gene product .beta.-actin as
an internal control were performed. Uniform expression of
.beta.-actin was noted in all samples. Following quantitative
analysis of biomarker levels in each sample, the results were
normalized for b-actin and expressed as percentages of untreated
controls. Group averages were compared by t-test.
[0173] When compared with the controls, the expression of c-Myc was
inhibited in all groups. The extent of inhibition (50%) was similar
in the gemcitabine and FK228 monotherapy treatment groups and
greater (60%) in the combination group. The inhibition of c-Myc
expression was highly significant (p<0.0001) in all cases.
[0174] Acetylation of histone 3 was significantly inhibited by
gemcitabine, but not affected by FK228 alone or in combination with
gemcitabine.
[0175] The levels of acetylated histone 4 were on the control level
in the gemcitabine group, FK228 treatment induced over 2-fold
increase of acetylated histone 4, but in comparison with the
control group the increase was not significant. On the other hand,
over 3-fold up-regulation of acetylated histone 4 by the drug
combination was highly significant (p=0.00003).
[0176] Treatment with gemcitabine alone significantly (p<0.05)
downregulated p21.sup.waf; however, this effect was not observed in
the combination groups.
[0177] Quantitative analysis of b-FGF and VEGF in serum was also
performed. The levels of b-FGF were highly variable but not
significantly different in any treatment groups in comparison with
the controls. VEGF was under the detection limits of the assay.
[0178] The effects of FK228 on expression of c-Myc and acetylated
histone 4 are unexpected considering that these endpoints were
assessed at the end of a long-term in vivo treatment with the drug.
Historically, the effects of DAC inhibitors such as FK228 on target
gene or protein expression were assessed in a time scale of hours
(not days) following drug treatment. For example, a study on the
effects of FK228 on tumor growth and expression of p21 and c-myc
genes in vivo over a period of 2 to 24 hours demonstrated induction
of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive
to FK228, while opposite effects on p21 and c-myc mRNA were seen in
tumor xenograft less sensitive to FK228.
[0179] Myc genes are key regulators of cell proliferation, and
their deregulation contributes to the genesis of most human tumors.
Transcriptional regulation by Myc-family proteins includes
recruitment of HDACs in tumors, some of which exhibit dependence
(addition) to c-mnyc. Even a brief inhibition of c-myc expression
may be sufficient to completely stop tumor growth and induce
regression of tumors. It is conceivable that biological activity of
FK228 could be partly due to inhibition of c-myc and other genes
under its control, including HDACs.
[0180] In conclusion, these results demonstrate at least additive
combinatorial effects with gemcitabine on the expression of c-Myc
and acetylation of histone 4 in pancreatic adenocarcinoma.
Surprising effects of FK228 in combination with gemcitabine might
suggest tumor phenotypic changes consistent with downregulation of
HDAC activity.
[0181] Specifically, weeks after the end of treatment, the cells
are phenotypically different from those that were initially
injected, suggesting some form of cellular transformation, possible
to a less aggressive phenotype.
EQUIVALENTS
[0182] The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those skilled in the art
will recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Those of ordinary skill in the art
will appreciate that various changes and modifications to this
description may be made without departing from the spirit or scope
of the present invention, as defined in the following claims.
[0183] To give but a few examples, in the claims articles such as
"a", "an", and "the" may mean one or more than one unless indicated
to the contrary or otherwise evident from the context. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention includes
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
The invention also includes embodiments in which more than one, or
all of the group members are present in, employed in, or otherwise
relevant to a given product or process. Furthermore, it is to be
understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, descriptive terms, etc., from one or more of the
claims or from relevant portions of the description is introduced
into another claim. For example, any claim that is dependent on
another claim can be modified to include one or more limitations
found in any other claim that is dependent on the same base
claim.
[0184] Furthermore, where the claims recite a composition, it is to
be understood that methods of using the composition for any of the
purposes disclosed herein are included, and methods of making the
composition according to any of the methods of making disclosed
herein or other methods known in the art are included, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. In addition, the invention encompasses compositions
made according to any of the methods for preparing compositions
disclosed herein.
[0185] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It is also noted that the term "comprising" is intended
to be open and permits the inclusion of additional elements or
steps. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, steps, etc., certain
embodiments of the invention or aspects of the invention consist,
or consist essentially of, such elements, features, steps, etc. For
purposes of simplicity those embodiments have not been specifically
set forth in haec verba herein. Thus for each embodiment of the
invention that comprises one or more elements, features, steps,
etc., the invention also provides embodiments that consist or
consist essentially of those elements, features, steps, etc.
[0186] Where ranges are given, endpoints are included unless
otherwise indicated. Furthermore, it is to be understood that
unless otherwise indicated or otherwise evident from the context
and/or the understanding of one of ordinary skill in the art,
values that are expressed as ranges can assume any specific value
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise. It is also to be understood
that unless otherwise indicated or otherwise evident from the
context and/or the understanding of one of ordinary skill in the
art, values expressed as ranges can assume any subrange within the
given range, wherein the endpoints of the subrange are expressed to
the same degree of accuracy as the tenth of the unit of the lower
limit of the range.
[0187] In addition, it is to be understood that any particular
embodiment of the present invention may be explicitly excluded from
any one or more of the claims. Any embodiment, element, feature,
application, or aspect of the compositions and/or methods of the
invention can be excluded from any one or more claims. For example,
in certain embodiments of the invention the biologically active
agent is not an anti-proliferative agent. For purposes of brevity,
all of the embodiments in which one or more elements, features,
purposes, or aspects is excluded are not set forth explicitly
herein.
EXHIBIT A
Treatment of Ras-Expressing Tumors
BACKGROUND OF THE INVENTION
[0188] Dysregulation or loss of control of cell division can result
in the development of any of a variety of cell proliferative
disorders, many of which are debilitating or deadly. Although much
has been learned about mechanisms involved in cell proliferation,
and therefore about common biological principles underlying a
variety of different disorders, there remains a need for the
development of new and/or improved therapies for the treatment of
such conditions.
[0189] There is a particular need for the development of improved
therapies for the treatment of tumors that express the Ras
oncogene. Ras-expressing tumors are often more resistant to
standard therapies. Furthermore, many of the most deadly cancers
involve Ras-expressing tumors. For example, 90-95% of pancreatic
tumors are Ras-expressing. Similarly, 40-45% of colorectal tumors,
40% of bladder tumors, 15-20% of non small cell lung carcinomas
express Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS),
which are not themselves cancer but are bone marrow disorders
characterized by abnormal cell maturation that typically progress
to cancer (AML), also express Ras. There is a profound need for the
development of therapies for these and other Ras-expressing
diseases and disorders.
SUMMARY OF THE INVENTION
[0190] The present invention encompasses the finding that DAC
inhibitors can show selective potency against Ras-expressing
tumors. In certain embodiments, the DAC inhibitor is romidepsin.
The present invention provides methods of treating tumors that
express the Ras oncogene by administering a DAC inhibitor. In some
embodiments, such methods involve determining that a tumor
expresses the Ras oncogene, and then, administering a DAC
inhibitor. Determination that a tumor expresses the Ras oncogene
can involve testing for expression of the Ras oncogene and/or can
involve determining that the tumor is of a type that typically
expresses the Ras oncogene.
[0191] The present invention also demonstrates that combinations of
DAC inhibitors with gemcitabine are particularly effective in the
treatment of Ras-expressing tumors. In certain particular
embodiments, combination therapy with romidepsin and gemcitabine is
provided, for example for use in the treatment of tumors expressing
the Ras oncogene.
[0192] The present invention provides combination regimens, and
unit dosages of pharmaceutical compositions useful in such
regimens. The present invention further provides kits for treatment
of Ras-expressing tumors with at least one DAC inhibitor (e.g.,
romidepsin).
DEFINITIONS
[0193] Alicyclic: The term "alicyclic," as used herein, denotes a
monovalent group derived from a monocyclic or bicyclic saturated
carbocyclic ring compound by the removal of a single hydrogen atom.
Examples include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and
bicyclo[2.2.2]octyl. Such alicyclic groups may be further
substituted.
[0194] Aliphatic: An "aliphatic group" is a non-aromatic moiety
that may contain any combination of carbon atoms, hydrogen atoms,
halogen atoms, oxygen, nitrogen or other atoms, and optionally
contain one or more units of unsaturation, e.g., double and/or
triple bonds. An aliphatic group may be straight chained, branched
or cyclic and preferably contains between about 1 and about 24
carbon atoms, more typically between about 1 and about 12 carbon
atoms. In addition to aliphatic hydrocarbon groups, aliphatic
groups include, for example, polyalkoxyalkyls, such as polyalkylene
glycols, polyamines, and polyimines, for example. Such aliphatic
groups may be further substituted.
[0195] Aryl: The term "aryl," as used herein, refers to a mono- or
polycyclic carbocyclic ring system having one or two aromatic rings
including, but not limited to, phenyl, naphthyl,
tetrahydronaphthyl, indanyl, idenyl and the like. In accordance
with the invention, any of the aryls, substituted aryls,
heteroaryls and substituted heteroaryls described herein, can be
any aromatic group. Aromatic groups can be substituted or
unsubstituted.
[0196] Cell Proliferative Disorder, Disease, or Condition: The term
"cell proliferative disease or condition" is meant to refer to any
condition characterized by aberrant cell growth, preferably
abnormally increased cellular proliferation.
[0197] Combination Therapy: According to some embodiments of the
present invention, a DAC inhibitor may desirably be administered in
combination with one or more other therapeutic agents. Such therapy
will commonly involve administration of multiple individual doses
of a DAC inhibitor and/or of other agent, spaced out over time.
Doses of a DAC inhibitor and other agent may be administered in the
same amounts and/or according to the same schedule or alternatively
may be administered in different amounts and/or according to
different schedules.
[0198] DAC Inhibitor: In general, any agent that specifically
inhibits a deacetylase is considered to be a DAC inhibitor. Any
agent that specifically inhibits a histone deacetylase is
considered to be an HDAC inhibitor. Those of ordinary skill in the
art will appreciate that, unless otherwise set forth herein or
known in the art, DAC inhibitors may be administered in any form
such as, for example, salts, esters, prodrugs, metabolites, etc.
Furthermore. DAC inhibitors that contain chiral centers may be
administered as single stereoisomers or as mixtures, including
racemic mixtures, so long as the single stereoisomer or mixture has
DAC inhibitor activity.
[0199] DAC Inhibitor Therapy: As used herein, the phrase "DAC
inhibitor therapy" refers to the regimen by which a DAC inhibitor
is administered to an individual. Commonly, DAC inhibitor therapy
will involve administration of multiple individual doses of a DAC
inhibitor, spaced out over time. Such individual doses may be of
different amounts or of the same amount. Furthermore, those of
ordinary skill in the art will readily appreciate that different
dosing regimens (e.g., number of doses, amount(s) of doses, spacing
of doses) are typically employed with different DAC inhibitors.
[0200] Electrolyte: In general, the term "electrolyte", as used
herein, refers to physiologically relevant free ions.
Representative such free ions include, but are not limited to
sodium (Na.sup.+), potassium (K.sup.+), calcium (Ca.sup.2+),
magnesium (Mg.sup.2+), chloride (Cl--), phosphate
(PO.sub.4.sup.3--), and bicarbonate (HCO.sub.3--).
[0201] Electrolyte Supplementation: The term "electrolyte
supplementation", as used herein, refers to administration to a
subject of a composition comprising one or more electrolytes in
order to increase serum electrolyte levels in the subject. For
purposes of the present invention, when electrolyte supplementation
is administered "prior to, during, or after" combination therapy,
it may be administered prior to initiation of combination therapy
inhibitor therapy (i.e., prior to administration of any dose) or
prior to, concurrently with, or after any particular dose or
doses.
[0202] Halogen: The term "halogen", as used herein, refers to an
atom selected from fluorine, chlorine, bromine, and iodine.
[0203] Heleroaryl: The term "heteroaryl", as used herein, refers to
a mono- or polycyclic (e.g. bi-, or tri-cyclic or more) aromatic
radical or ring having from five to ten ring atoms of which one or
more ring atom is selected from, for example, S, O and N; zero, one
or two ring atoms are additional heteroatoms independently selected
from, for example, S, O and N; and the remaining ring atoms are
carbon, wherein any N or S contained within the ring may be
optionally oxidized. Heteroaryl includes, but is not limited to
pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,
thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,
benzooxazolyl, quinoxalinyl, and the like.
[0204] Heterocyclic: The term "heterocyclic" as used herein, refers
to a non-aromatic 5-, 6- or 7-membered ring or a bi- or tri-cyclic
group fused system, where (i) each ring contains between one and
three heteroatoms independently selected from oxygen, sulfur and
nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and
each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen
and sulfur heteroatoms may optionally be oxidized, (iv) the
nitrogen heteroatom may optionally be quaternized, (iv) any of the
above rings may be fused to a benzene ring, and (v) the remaining
ring atoms are carbon atoms which may be optionally
oxo-substituted. Representative heterocycloalkyl groups include,
but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,
pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,
piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,
thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and
tetrahydrofuryl. Such heterocyclic groups may be further
substituted.
[0205] Initiation: As used herein, the term "initiation" when
applied to therapy can refer to a first administration of an active
agent (e.g., a DAC inhibitor) to a patient who has not previously
received the active agent. Alternatively or additionally, the term
"initiation" can refer to administration of a particular dose of a
DAC inhibitor during therapy of a patient.
[0206] Pharmaceutically acceptable carrier or excipient: As used
herein, the term "pharmaceutically acceptable carrier or excipient"
means a non-toxic, inert solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0207] Pharmaceutically acceptable ester: As used herein, the term
"pharmaceutically acceptable ester" refers to esters which
hydrolyze in vivo and include those that break down readily in the
human body to leave the parent compound or a salt thereof. Suitable
ester groups include, for example, those derived from
pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic
acids, in which each alkyl or alkenyl moiety advantageously has not
more than 6 carbon atoms. Examples of particular esters include,
but are not limited to, formates, acetates, propionates, butyrates,
acrylates and ethylsuccinates.
[0208] Pharmaceutically acceptable prodrug: The term
"pharmaceutically acceptable prodrugs" as used herein refers to
those prodrugs of the compounds of the present invention which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, and the like, commensurate
with a reasonable benefit/risk ratio, and effective for their
intended use, as well as the zwitterionic forms, where possible, of
the compounds of the present invention. "Prodrug", as used herein
means a compound which is convertible in vivo by metabolic means
(e.g. by hydrolysis) to a compound of the invention. Various forms
of prodrugs are known in the art, for example, as discussed in
Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et
al. (ed.), Methods in Enyvmology, vol. 4. Academic Press (1985);
Krogsgaard-Larsen, et al., (ed). "Design and Application of
Prodrugs. Textbook of Drug Design and Development, Chapter 5,
113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews,
8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et
seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug
Delivery Systems, American Chemical Society (1975); and Bernard
Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug
Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and
Sons, Ltd. (2002).
[0209] Pharmaceutically acceptable salt: As used herein, the term
"pharmaceutically acceptable salt" refers to those salts which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response and the like, and are
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example. S. M.
Berge, et al. describes pharmaceutically acceptable salts in detail
in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be
prepared in situ during the final isolation and purification of the
compounds of the invention, or separately by reacting the free base
function with a suitable organic acid. Examples of pharmaceutically
acceptable salts include, but are not limited to nontoxic acid
addition salts are salts of an amino group formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, phosphoric acid,
sulfuric acid and perchloric acid or with organic acids such as
acetic acid, maleic acid, tartaric acid, citric acid, succinic acid
or malonic acid or by using other methods used in the art such as
ion exchange. Other pharmaceutically acceptable salts include, but
are not limited to adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
[0210] Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6
carbon atoms, sulfonate and aryl sulfonate.
[0211] Stable: The term "stable", as used herein, refers to
compounds which possess stability sufficient to allow manufacture
and which maintains the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein (e.g.,
therapeutic or prophylactic administration to a subject). In
general, combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds.
[0212] Substituted: The terms "substituted aryl", "substituted
heteroaryl", or "substituted aliphatic," as used herein, refer to
aryl, heteroaryl, or aliphatic groups as previously defined,
substituted by independent replacement of one, two, or three or
more of the hydrogen atoms thereon with substituents including, but
not limited to, --F, --CI, --Br, --I, --OH, protected hydroxyl,
--NO.sub.2, --CN, --C.sub.1-C.sub.12-alkyl optionally substituted
with, for example, halogen, C.sub.2-C.sub.12-alkenyl optionally
substituted with, for example, halogen, --C.sub.2-C.sub.12-alkynyl
optionally substituted with, for example, halogen, --NH.sub.2,
protected amino, --NH--C.sub.1-C.sub.12-alkyl,
--NH--C.sub.2-C.sub.12-alkenyl, --NH--C.sub.2-C.sub.12-alkenyl,
--NH--C.sub.3-C.sub.12-cycloalkyl, --NH-aryl, --NH-heteroaryl,
--NH-heterocycloalkyl, -dialkylamino, -diarylamino,
-diheteroarylamino, --O--C.sub.1-C.sub.12-alkyl,
--O--C.sub.2-C.sub.12-alkenyl, --O--C.sub.2-C.sub.12-alkenyl,
--O--C.sub.3-C.sub.12-cycloalkyl, --O-aryl, --O-heteroaryl,
--O-heterocycloalkyl, --C(O)--C.sub.1-C.sub.12-alkyl,
--C(O)--C.sub.2-C.sub.12-alkenyl, --C(O)--C.sub.2-C.sub.12-alkenyl,
--C(O)--C.sub.3-C.sub.12-cycloalkyl, --C(O)-aryl,
--C(O)-heteroaryl, --C(O)-heterocycloalkyl, --CONH.sub.2,
--CONH--C.sub.1-C.sub.12-alkyl, --CONH--C.sub.2-C.sub.12-alkenyl,
--CONH--C.sub.2-C.sub.12-alkenyl, --CONH--C.sub.3-C,2-cycloalkyl,
--CONH-aryl, --CONH-heteroaryl, --CONH-heterocycloalkyl,
--OCO.sub.2--C.sub.1-C.sub.12-alkyl,
--OCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--OCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--OCO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --OCO.sub.2-aryl,
--OCO.sub.2-heteroaryl, --OCO.sub.2-heterocycloalkyl,
--OCONH.sub.2, --OCONH--C.sub.1-C.sub.12-alkyl,
--OCONH--C.sub.2-C.sub.12-alkenyl,
--OCONH--C.sub.2-C.sub.12-alkenyl,
--OCONH--C.sub.3-C.sub.12-cycloalkyl, --OCONH-aryl,
--OCONH-heteroaryl, --OCONH-heterocycloalkyl,
--NHC(O)--C.sub.1-C.sub.12-alkyl,
--NHC(O)--C.sub.2-C.sub.12-alkenyl,
--NHC(O)--C.sub.2-C.sub.12-alkenyl,
--NHC(O)--C.sub.3-C.sub.12-cycloalkyl, --NHC(O)-aryl,
--NHC(O)-heteroaryl, --NHC(O)-heterocycloalkyl,
--NHCO.sub.2--C.sub.1-C.sub.12-alkyl,
--NHCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NHCO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NHCO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --NHCO.sub.2-aryl,
--NHCO.sub.2-heteroaryl, --NHCO.sub.2-heterocycloalkyl,
--NHC(O)NH.sub.2, --NHC(O)NH--C.sub.1-C.sub.12-alkyl,
--NHC(O)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(O)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(O)NH--C.sub.3-C.sub.12-cycloalkyl, --NHC(O)NH-aryl,
--NHC(O)NH-heteroaryl, --NHC(O)NH-heterocycloalkyl,
--NHC(S)NH.sub.2, --NHC(S)NH--C.sub.1-C.sub.12-alkyl,
--NHC(S)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(S)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(S)NH--C.sub.3-C.sub.12-cycloalkyl, --NHC(S)NH-aryl,
--NHC(S)NH-heteroaryl, --NHC(S)NH-heterocycloalkyl,
--NHC(NH)NH.sub.2, --NHC(NH)NH--C.sub.1-C.sub.12-alkyl,
--NHC(NH)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)NH--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)NH--C.sub.3-C.sub.12-cycloalkyl, --NHC(NH)NH-aryl,
--NHC(NH)NH-heteroaryl, --NHC(NH)NH-heterocycloalkyl,
--NHC(NH)--C.sub.1-C.sub.12-alkyl,
--NHC(NH)--C.sub.2-C.sub.,2-alkenyl,
--NHC(NH)--C.sub.2-C.sub.12-alkenyl,
--NHC(NH)--C.sub.3-C.sub.12-cycloalkyl, --NHC(NH)-aryl,
--NHC(NH)-heteroaryl, --NHC(NH)-heterocycloalkyl,
--C(NH)NH--C.sub.1-C.sub.12-alkyl,
--C(NH)NH--C.sub.2-C.sub.12-alkenyl,
--C(NH)NH--C.sub.2-C.sub.12-alkenyl,
--C(NH)NH--C.sub.3-C.sub.12-cycloalkyl, --C(NH)NH-aryl,
--C(NH)NH-heteroaryl, --C(NH)NH-heterocycloalkyl,
--S(O)--C.sub.1-C.sub.12-alkyl, --S(O)--C.sub.2-C.sub.12-alkenyl,
--S(O)--C.sub.2-C.sub.12-alkenyl,
--S(O)--C.sub.3-C.sub.12-cycloalkyl, --S(O)-aryl,
--S(O)-heteroaryl, --S(O)-heterocycloalkyl, --SO.sub.2NH.sub.2,
--SO.sub.2NH--C.sub.1-C.sub.12-alkyl,
--SO.sub.2NH--C.sub.2-C.sub.12-alkenyl,
--SO.sub.2NH--C.sub.2-C.sub.12-alkenyl,
--SO.sub.2NH--C.sub.3-C.sub.12-cycloalkyl, --SO.sub.2NH-aryl,
--SO.sub.2NH-heteroaryl, --SO.sub.2NH-heterocycloalkyl,
--NHSO.sub.2--C.sub.1-C.sub.12-alkyl,
--NHSO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NHSO.sub.2--C.sub.2-C.sub.12-alkenyl,
--NHSO.sub.2--C.sub.3-C.sub.12-cycloalkyl, --NHSO.sub.2-aryl,
--NHSO.sub.2-heteroaryl, --NHSO.sub.2-heterocycloalkyl,
--CH.sub.2NH.sub.2, --CH.sub.2SO.sub.2CH.sub.3, -aryl, -arylalkyl,
-heteroaryl, -heteroarylalkyl, -heterocycloalkyl,
--C.sub.3-C.sub.12-cycloalkyl, polyalkoxyalkyl, polyalkoxy,
-methoxymethoxy, -methoxyethoxy, --SH, --S--C.sub.1-C.sub.12-alkyl,
--S--C.sub.2-C.sub.12-alkenyl, --S--C.sub.2-C.sub.12-alkenyl,
--S--C.sub.3-C.sub.12-cycloalkyl, --S-aryl, --S-heteroaryl,
--S-heterocycloalkyl, or methylthiomethyl. It is understood that
the aryls, heteroaryls, alkyls, and the like can be further
substituted.
[0213] Susceptible to: The term "susceptible to", as used herein
refers to an individual having higher risk (typically based on
genetic predisposition, environmental factors, personal history, or
combinations thereof) of developing a particular disease or
disorder, or symptoms thereof, than is observed in the general
population.
[0214] Therapeutically effective amount: The term "therapeutically
effective amount" of an active agent or combination of agents is
intended to refer to an amount of agent(s) which confers a
therapeutic effect on the treated subject, at a reasonable
benefit/risk ratio applicable to any medical treatment. The
therapeutic effect may be objective (i.e., measurable by some test
or marker) or subjective (i.e., subject gives an indication of or
feels an effect). An effective amount of a particular agent may
range from about 0.1 mg/Kg to about 500 mg/Kg, preferably from
about 1 to about 50 mg/Kg. Effective doses may also vary depending
on route of administration, as well as the possibility of co-usage
with other agents. It will be understood, however, that the total
daily usage of any particular active agent utilized in accordance
with the present invention will be decided by the attending
physician within the scope of sound medical judgment. The specific
therapeutically effective dose level for any particular patient
will depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or contemporaneously with the
specific compound employed; and like factors well known in the
medical arts.
[0215] Therapeutic agent: As used herein, the phrase "therapeutic
agent" refers to any agent that, when administered to a subject,
has a therapeutic effect and/or elicits a desired biological and/or
pharmacological effect.
[0216] Treatment: As used herein, the term "treatment" (also
"treat" or "treating") refers to any administration of a
biologically active agent that partially or completely alleviates,
ameliorates, relives, inhibits, delays onset of, reduces severity
of and/or reduces incidence of one or more symptoms or features of
a particular disease, disorder, and/or condition. Such treatment
may be of a subject who does not exhibit signs of the relevant
disease, disorder and/or condition and/or of a subject who exhibits
only early signs of the disease, disorder, and/or condition.
Alternatively or additionally; such treatment may be of a subject
who exhibits one or more established signs of the relevant disease,
disorder and/or condition.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0217] As indicated, the present invention demonstrates that DAC
inhibitors are specifically effective in inhibiting growth of cells
that express Ras. According to the present invention, therefore,
DAC inhibitors are useful in the treatment of cell proliferative
disorders, diseases, or conditions that are associated with Ras
expression. According to the present invention. DAC inhibitors are
particularly useful in the treatment of Ras-expressing tumors.
Cell Proliferative Disorders, Diseases, or Conditions
[0218] In some embodiments, the invention provides methods for
treating cell proliferative disorders, diseases or conditions, in
particular where cells express the Ras oncogene.
[0219] In general, cell proliferative disorders, diseases or
conditions encompass a variety of conditions characterized by
aberrant cell growth, preferably abnormally increased cellular
proliferation. For example, cell proliferative disorders, diseases,
or conditions include, but are not limited to cancer,
immune-mediated responses and diseases (e.g., transplant rejection,
graft vs host disease, immune reaction to gene therapy, autoimmune
diseases, pathogen-induced immune dysregulation, etc.), certain
circulatory diseases, and certain neurodegenerative diseases.
[0220] In certain embodiments, the invention relates to methods of
treating cancer. In general, cancer is a group of diseases which
are characterized by uncontrolled growth and spread of abnormal
cells. Examples of such diseases are carcinomas, sarcomas,
leukemias, lymphomas and the like.
[0221] For example, cancers include, but are not limited to
leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL),
peripheral T-cell lymphomas, lymphomas associated with human T-cell
lymphotropic virus (HTLV) such as adult T-cell leukemia/lymphoma
(ATLL). B-cell lymphoma, acute lymphocytic leukemia, acute
nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic
myelogenous leukemia, acute myelogenous leukemia, Hodgkin's
disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic
syndrome, mesothelioma, common solid tumors of adults such as head
and neck cancers (e.g., oral, laryngeal and esophageal),
genitourinary cancers (e.g., prostate, bladder, renal, uterine,
ovarian, testicular, rectal and colon), lung cancer, breast cancer,
pancreatic cancer, melanoma and other skin cancers, stomach cancer,
brain tumors, liver cancer and thyroid cancer, and/or childhood
solid tumors such as brain tumors, neuroblastoma, retinoblastoma,
Wilms' tumor, bone tumors, and soft-tissue sarcomas.
[0222] In some embodiments, the invention relates to treatment of
leukemias. For example, in some embodiments, the invention relates
to treatment of chronic lymphocytic leukemia, chronic myelogenous
leukemia, acute lymphocytic leukemia, acute myelogenous leukemia,
and/or adult T cell leukemia/lymphoma. In certain embodiments, the
invention relates to the treatment of AML. In certain embodiments,
the invention relates to the treatment of ALL. In certain
embodiments, the invention relates to the treatment of CML. In
certain embodiments, the invention relates to the treatment of
CLL.
[0223] In some embodiments, the invention relates to treatment of
lymphomas. For example, in some embodiments, the invention relates
to treatment of Hodgkin's or non-Hodgkin's (e.g., T-cell lymphomas
such as peripheral T-cell lymphomas, cutaneous T-cell lymphomas,
etc.) lymphoma.
[0224] In some embodiments, the invention relates to the treatment
of myelomas and/or myelodysplastic syndromes. In some embodiments,
the invention relates to treatment of solid tumors. In some such
embodiments the invention relates to treatment of solid tumors such
as lung, breast, colon, liver, pancreas, renal, prostate, ovarian,
and/or brain. In some embodiments, the invention relates to
treatment of pancreatic cancer. In some embodiments, the invention
relates to treatment of renal cancer. In some embodiments, the
invention relates to treatment of prostate cancer. In some
embodiments, the invention relates to treatment of sarcomas. In
some embodiments, the invention relates to treatment of soft tissue
sarcomas. In some embodiments, the invention relates to methods of
treating one or more immune-mediated responses and diseases.
[0225] For example, in some embodiments, the invention relates to
treatment of rejection following transplantation of synthetic or
organic grafting materials, cells, organs or tissue to replace all
or part of the function of tissues, such as heart, kidney, liver,
bone marrow, skin, cornea, vessels, lung, pancreas, intestine,
limb, muscle, nerve tissue, duodenum, small-bowel,
pancreatic-islet-cell, including xeno-transplants, etc.; treatment
of graft-versus-host disease, autoimmune diseases, such as
rheumatoid arthritis, systemic lupus erythematosus, thyroiditis,
Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis,
type I diabetes uveitis, juvenile-onset or recent-onset diabetes
mellitus, uveitis. Graves' disease, psoriasis, atopic dermatitis.
Crohn's disease, ulcerative colitis, vasculitis, auto-antibody
mediated diseases, aplastic anemia, Evan's syndrome, autoimmune
hemolytic anemia, and the like; and further to treatment of
infectious diseases causing aberrant immune response and/or
activation, such as traumatic or pathogen induced immune
dysregulation, including for example, that which are caused by
hepatitis B and C infections. HIV, Staphylococcus aureus infection,
viral encephalitis, sepsis, parasitic diseases wherein damage is
induced by an inflammatory response (e.g. leprosy). In some
embodiments, the invention relates to treatment of graft vs host
disease (especially with allogenic cells), rheumatoid arthritis,
systemic lupus erythematosus, psoriasis, atopic dermatitis, Crohn's
disease, ulcerative colitis and/or multiple sclerosis.
[0226] Alternatively or additionally, in some embodiments, the
invention relates to treatment of an immune response associated
with a gene therapy treatment, such as the introduction of foreign
genes into autologous cells and expression of the encoded product.
In some embodiments, the invention relates to treatment of
circulatory diseases, such as arteriosclerosis, atherosclerosis,
vasculitis, polyarteritis nodosa and/or myocarditis.
[0227] In some embodiments, the invention relates to treatment of
any of a variety of neurodegenerative diseases, a non-exhaustive
list of which includes: [0228] I. Disorders characterized by
progressive dementia in the absence of other prominent neurologic
signs, such as Alzheimer's disease; Senile dementia of the
Alzheimer type; and Pick's disease (lobar atrophy); [0229] II.
Syndromes combining progressive dementia with other prominent
neurologic abnormalities such as A) syndromes appearing mainly in
adults (e.g., Huntington's disease, Multiple system atrophy
combining dementia with ataxia and/or manifestations of Parkinson's
disease, Progressive supranuclear palsy
(Steel-Richardson-Olszewski), diffuse Lewy body disease, and
corticodentatonigral degeneration); and B) syndromes appearing
mainly in children or young adults (e.g., Hallervorden-Spatz
disease and progressive familial myoclonic epilepsy); [0230] III.
Syndromes of gradually developing abnormalities of posture and
movement such as paralysis agitans (Parkinson's disease),
striatonigral degeneration, progressive supranuclear palsy, torsion
dystonia (torsion spasm; dystonia musculorum deformans), spasmodic
torticollis and other dyskinesis, familial tremor, and Gilles de la
Tourette syndrome; [0231] IV. Syndromes of progressive ataxia such
as cerebellar degenerations (e.g., cerebellar cortical degeneration
and olivopontocerebellar atrophy (OPCA)); and spinocerebellar
degeneration (Friedreich's ataxia and related disorders); [0232] V.
Syndromes of central autonomic nervous system failure (Shy-Drager
syndrome); [0233] VI. Syndromes of muscular weakness and wasting
without sensory changes (motorneuron disease such as amyotrophic
lateral sclerosis, spinal muscular atrophy (e.g. infantile spinal
muscular atrophy (Werdnig-Hoffman), juvenile spinal muscular
atrophy (Wohlfart-Kugelberg-Welander) and other forms of familial
spinal muscular atrophy), primary lateral sclerosis, and hereditary
spastic paraplegia; [0234] VII. Syndromes combining muscular
weakness and wasting with sensory changes (progressive neural
muscular atrophy; chronic familial polyneuropathies) such as
peroneal muscular atrophy (Charcot-Marie-Tooth), hypertrophic
interstitial polyneuropathy (Dejerine-Sottas), and miscellaneous
forms of chronic progressive neuropathy; [0235] VIII. Syndromes of
progressive visual loss such as pigmentary degeneration of the
retina (retinitis pigmentosa), and hereditary optic atrophy
(Leber's disease).
[0236] In some embodiments, the neurodegenerative disease is
Alzheimer's disease, Parkinson's disease, and/or Huntington's
disease.
[0237] In some embodiments, the invention relates to treatment of
disorders, diseases or conditions associated with chromatin
remodeling.
[0238] The present invention is particularly directed to treatment
of tumors expressing the Ras oncogene. As indicated above,
Ras-expressing tumors are often more resistant to standard
therapies. Furthermore, many of the most deadly cancers involve
Ras-expressing tumors. For example, 90-95% of pancreatic tumors are
Ras-expressing. Similarly, 40-45% of colorectal tumors, 40% of
bladder tumors, 15-20% of non small cell lung carcinomas express
Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS), which are
not themselves cancer but are bone marrow disorders characterized
by abnormal cell maturation that typically progress to cancer, also
express Ras. There is a profound need for the development of
therapies for these and other Ras-expressing diseases and
disorders.
DAC Inhibitors
[0239] Deacetylase inhibitors, as that term is used herein, are
compounds which are capable of inhibiting the deacetylation of
proteins in vivo, in vitro or both. In many embodiments, the
invention relates to HDAC inhibitors, which inhibit the
deacetylation of histones. However, those of ordinary skill in the
art will appreciate that HDAC inhibitors often have a variety of
biological activities, at least some of which may well be
independent of histone deacetylase inhibition.
[0240] As indicated, DAC inhibitors inhibit the activity of at
least one deacetylase. Where the DAC inhibitor is an HDAC
inhibitor, an increase in acetylated histones occurs and
accumulation of acetylated histones is a suitable biological marker
for assessing the activity of HDAC inhibitors. Therefore,
procedures which can assay for the accumulation of acetylated
histones can be used to determine the HDAC inhibitory activity of
agents of interest. Analogous assays can determine DAC inhibitory
activity.
[0241] It is understood that agents which can inhibit deacetylase
activity (e.g., histone deacetylase activity) typically can also
bind to other substrates and as often can inhibit or otherwise
regulate other biologically active molecules such as enzymes.
[0242] Suitable DAC or HDAC inhibitors according to the present
invention include, for example, 1) hydroxamic acid derivatives; 2)
Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4)
benzamides; 5) electrophilic ketones; and/or any other class of
compounds capable of inhibiting histone deacetylase. Examples of
such DAC inhibitors include, but are not limited to: [0243] A)
HYDROXAMIC ACID DERIVATIVES such as Suberoylanilide Hydroxamic Acid
(SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95:3003, 1998);
M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Richon et al.,
supra), pyroxamide; CBHA; Trichostatin analogues such as
Trichostatin A (TSA) and Trichostatin C (Koghe et al. Biochem.
Pharmacol. 56:1359, 1998); Salicylihydroxamic Acid (SBHA) (Andrews
et al., International J. Parasitology 30:761, 2000); Azelaic
Bishydroxamic Acid (ABHA) (Andrews et al., supra);
Azelaic-1-Hydroxamate-9-Anilide (AAHA) (Qiu et al., Mol. Biol. Cell
11:2069, 2000); 6-(3-Chlorophenylureido) carpoic Hydroxamic Acid
(3C1-UCHA), Oxamflatin [(2E)-5-[3-[(phenylsulfonyl-)amino
phenyl]-pent-2-en-4-ynohydroxamic acid (Kim et al. Oncogene, 18:
2461, 1999); A-161906, Scriptaid (Su et al. Cancer Research,
60:3137, 2000); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews
et al., supra), and MW2996 (Andrews et al., supra). [0244] B)
CYCLIC TETRAPEPTIDES such as Trapoxin A (TPX)-Cyclic Tetrapeptide
(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10--
epoxy decanoyl)) (Kijima et al., J. Biol. Chem. 268:22429, 1993);
FR901228 (FK 228, FR901228. Depsipeptide, Romidepsin) (Nakajima et
al., Er. Cell Res. 241:12, 1998); FR225497 Cyclic Tetrapeptide
(Mori et al., PCT Application WO 00/08048, Feb. 17, 2000); Apicidin
Cyclic Tetrapeptide [cyclo
(NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipe-colinyl-L-2-amino-8-
-oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA
93:13143, 1996); Apicidin Ia. Apicidin Ib, Apicidin Ic, Apicidin
IIa, and Apicidin IIb (P. Dulski et al., PCT Application WO
97/11366); CHAP, HC-Toxin Cyclic Tetrapeptide (Bosch et al., Plant
Cell 7:1941, 1995); WF27082 Cyclic Tetrapeptide (PCT Application WO
98/48825); and Chiamydocin (Bosch et al., supra). [0245] C) SHORT
CHAIN FATTY ACID (SCFA) DERIVATIVES such as: Sodium Butyrate
(Cousens et al, J. Biol. Chem. 254:1716, 1979); Isovalerate (McBain
et al., Biochem. Pharm. 53:1357, 1997); Valerate (McBain et al.,
supra); 4 Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer
Research, 15:879, 1995); Phenylbutyrate (PB) (Wang et al., Cancer
Research. 59:2766, 1999); Propionate (McBain et al., supra);
Butyramide (Lea and Tulsyan, supra), Isobutyramide (Lea and
Tulsyan, supra). Phenylacetate (Lea and Tulsyan, supra);
3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et
al., Cancer Research, 60:749, 2000); Valproic acid and Valproate.
[0246] D) BENZAMIDE DERIVATIVES such as CI-994; MS-275
[N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamid-
e] (Saito et al., Proc. Natl. Acad. Sci. USA 96:4592, 1999;
3'-amino derivative of MS-27-275 (Saito et al., supra); MGCD0103
(MethylGene), or related compounds. [0247] E) ELECTROPHILIC KETONE
DERIVATIVES such as trifluoromethyl ketones (Frey et al, Bioorganic
& Med. Chem. Lett. 12:3443, 2002; U.S. Pat. No. 6,511,990) and
.alpha.-keto amides such as N-methyl-.alpha.-ketoamides. [0248] F)
OTHER DAC Inhibitors such as Depudecin (Kwon et al., Proceedings of
the National Academy of Sciences USA, 95:3356, 1998), and other
compounds.
[0249] Suitable DAC inhibitors for use in accordance with the
present invention particularly include, for example. CRA-024781
(Celera Genomics), PXD-101 (CuraGene), LAQ-824 (Novartis AG),
LBH-589 (Novartis AG), MGCD0103 (MethylGene), MS-275 (Schering AG),
romidepsin (Gloucester Pharmaceuticals), and/or SAHA (Alton
Pharma/Merck).
[0250] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (I):
##STR00019##
wherein [0251] m is 1, 2, 3 or 4; [0252] n is 0, 1, 2 or 3; [0253]
p and q are independently 1 or 2; [0254] X is O, NH, or NR.sub.8;
[0255] R.sub.1, R.sub.2, and R.sub.3 are independently hydrogen;
unsubstituted or substituted, branched or unbranched, cyclic or
acyclic aliphatic; unsubstituted or substituted, branched or
unbranched, cyclic or acyclic heteroaliphatic; unsubstituted or
substituted aryl; or unsubstituted or substituted heteroaryl;
[0256] R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
independently hydrogen; or substituted or unsubstituted, branched
or unbranched, cyclic or acyclic aliphatic; and pharmaceutically
acceptable forms thereof. In certain embodiments, m is 1. In
certain embodiments, n is 1. In certain embodiments, p is 1. In
certain embodiments, q is 1. In certain embodiments. X is O. In
certain embodiments. R.sub.1, R.sub.2, and R.sub.3 are
unsubstituted, or substituted, branched or unbranched, acyclic
aliphatic. In certain embodiments, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are all hydrogen.
[0257] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (II):
##STR00020##
wherein: [0258] m is 1, 2, 3 or 4; [0259] n is 0, 1, 2 or 3; [0260]
q is 2 or 3; [0261] X is O, NH, or NR.sub.8; [0262] Y is ORa, or
SR; [0263] R.sub.2 and R.sub.3 are independently hydrogen;
unsubstituted or substituted, branched or unbranched, cyclic or
acyclic aliphatic; unsubstituted or substituted, branched or
unbranched, cyclic or acylic heteroaliphatic; unsubstituted or
substituted aryl; or unsubstituted or substituted heteroaryl;
[0264] R.sub.4, R.sub.5, R.sub.5, R.sub.7 and R.sub.8 are
independently selected from hydrogen; or substituted or
unsubstituted, branched or unbranched, cyclic or acyclic aliphatic;
and pharmaceutically acceptable forms thereof. In certain
embodiments, m is 1. In certain embodiments, n is 1. In certain
embodiments, q is 2. In certain embodiments, X is O. In other
embodiments, X is NH. In certain embodiments, R.sub.2 and R.sub.3
are unsubstituted or substituted, branched or unbranched, acyclic
aliphatic. In certain embodiments. R.sub.4, R.sub.5, R.sup.6, and
R.sub.7 are all hydrogen.
[0265] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (III):
##STR00021##
wherein [0266] A is a moiety that is cleaved under physiological
conditions to yield a thiol group and includes, for example, an
aliphatic or aromatic acyl moiety (to form a thioester bond); an
aliphatic or aromatic thioxy (to form a disulfide bond); or the
like; and pharmaceutically acceptable forms thereof. Such aliphatic
or aromatic groups can include a substituted or unsubstituted,
branched or unbranched, cyclic or acyclic aliphatic group; a
substituted or unsubstituted aromatic group; a substituted or
unsubstituted heteroaromatic group; or a substituted or
unsubstituted heterocyclic group. A can be, for example,
--COR.sub.1, --SC(.dbd.O)--O--R.sub.1, or --SR.sub.2. R.sub.1 is
independently hydrogen; substituted or unsubstituted amino;
substituted or unsubstituted, branched or unbranched, cyclic or
acyclic aliphatic; substituted or unsubstituted aromatic group;
substituted or unsubstituted heteroaromatic group; or a substituted
or unsubstituted heterocyclic group. In certain embodiments,
R.sub.1 is hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl,
isobutyl, benzyl, or bromobenzyl. R.sub.2 is a substituted or
unsubstituted, branched or unbranched, cyclic or acyclic aliphatic
group; a substituted or unsubstituted aromatic group; a substituted
or unsubstituted heteroaromatic group; or a substituted or
unsubstituted heterocyclic group. In certain embodiments, R.sub.2
is methyl, ethyl, 2-hydroxyethyl, isobutyl, fatty acids, a
substituted or unsubstituted benzyl, a substituted or unsubstituted
aryl, cysteine, homocysteine, or glutathione.
[0267] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (IV) or
(IV'):
##STR00022##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same or
different and represent an amino acid side chain moiety, each
R.sub.6 is the same or different and represents hydrogen or
C.sub.1-C.sub.4 alkyl, and Pr.sup.1 and Pr.sup.2 are the same or
different and represent hydrogen or thiol-protecting group. In
certain embodiments, the amino acid side chain moieties are those
derived from natural amino acids. In other embodiments, the amino
acid side chain moieties are those derived from unnatural amino
acids. In certain embodiments, each amino acid side chain is a
moiety selected from --H, --C.sub.1-C.sub.6 alkyl,
--C.sub.2-C.sub.6 alkenyl, -L-O--C(O)--R', -L-C(O)--O--R'', -L-A,
-L-NR''R'', -L-Het-C(O)--Het-R'', and -L-Het-R''', wherein L is a
C.sub.1-C.sub.6 alkylene group. A is phenyl or a 5- or 6-membered
heteroaryl group, each R' is the same or different and represents
C.sub.1-C.sub.4 alkyl, each R'' is the same or different and
represent H or C.sub.1-C.sub.6 alkyl, each -Het- is the same or
different and is a heteroatom spacer selected from --O--,
--N(R''')--, and --S--, and each R''' is the same or different and
represents H or C.sub.1-C.sub.4 alkyl. In certain embodiments,
R.sub.6 is --H. In certain embodiments, Pr.sup.1 and Pr.sup.2 are
the same or different and are selected from hydrogen and a
protecting group selected from a benzyl group which is optionally
substituted by C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 acyloxy,
hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl,
diphenylmethyl, phenyl, t-butyl, adamanthyl, C.sub.1-C.sub.6
acyloxymethyl, C.sub.1-C.sub.6 alkoxymethyl, tetrahydropyranyl,
benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl,
benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its
derivatives, benzoyl and its derivatives, carbamoyl,
phenylcarbamoyl, and C.sub.1-C.sub.6 alkylcarbamoyl. In certain
embodiments, Pr.sup.1 and Pr.sup.2 are hydrogen. Various romidepsin
derivatives of formula (IV) and (IV') are disclosed in published
PCT application WO 2006/129105, published Dec. 7, 2006; which is
incorporated herein by reference.
[0268] In some embodiments, the DAC or HDAC inhibitor used in the
method of the invention is represented by formula (V):
##STR00023##
wherein [0269] B is a substituted or unsubstituted, saturated or
unsaturated aliphatic group, a substituted or unsubstituted,
saturated or unsaturated alicyclic group, a substituted or
unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, or a substituted or unsubstituted
heterocyclic group; R.sub.20 is hydroxylamino, hydroxyl, amino,
alkylamino, dialkylamino, or alkyloxy group; R.sub.21 and R.sub.22
are independently selected from hydrogen, hydroxyl, a substituted
or unsubstituted, saturated or unsaturated aliphatic group, a
substituted or unsubstituted, saturated or unsaturated alicyclic
group, a substituted or unsubstituted aromatic group, a substituted
or unsubstituted heteroaromatic group, or a substituted or
unsubstituted heterocyclic group. In a particular embodiment of
Formula V, R.sub.20 is a hydroxylamino, hydroxyl, amino,
methylamino, dimethylamino or methyloxy group and B is a
C.sub.6-alkyl. In yet another embodiment of Formula IV, R.sub.21 is
a hydrogen atom, R.sub.22 is a substituted or unsubstituted phenyl
and B is a C.sub.6-alkyl. In further embodiments of Formula IV,
R.sub.21 is hydrogen and R.sub.22 is an .alpha.-, .beta.-, or
.gamma.-pyridine.
[0270] Other examples of DAC or HDAC inhibitors can be found in,
for example, U.S. Pat. Nos. 5,369,108, issued on Nov. 29, 1994,
5,700,811, issued on Dec. 23, 1997, 5,773,474, issued on Jun. 30,
1998, 5,932,616 issued on Aug. 3, 1999 and 6,511,990, issued Jan.
28, 2003 all to Breslow et al.; U.S. Pat. Nos. 5,055,608, issued on
Oct. 8, 1991, 5,175,191, issued on Dec. 29, 1992 and 5,608,108,
issued on Mar. 4, 1997 all to Marks et al.; U.S. Provisional
Application No. 60/459,826, filed Apr. 1, 2003 in the name of
Breslow et al.; as well as, Yoshida. M. et al., Bioassays 17,
423-430 (1995); Saito, A., et al., PNAS USA 96, 4592-4597, (1999);
Furamai, R. et al., PNAS USA 98 (1), 87-92 (2001); Komatsu. Y. et
al. Cancer Res. 61(11), 4459-4466 (2001); Su. G. H., et al., Cancer
Res. 60, 3137-3142 (2000); Lee, B. I. et al., Cancer Res. 61(3),
931-934; Suzuki, T., et al., J. Med. Chem. 42(15), 3001-3003
(1999); published PCT Application WO 01/18171 published on Mar. 15,
2001 Sloan-Kettering Institute for Cancer Research and The Trustees
of Columbia University; published PCT Application WO02/246144 to
Hoffmann-La Roche; published PCT Application WO02/22577 to
Novartis; published PCT Application WO02/30879 to Prolifix;
published PCT Applications WO 01/38322 (published May 31, 2001). WO
01/70675 (published on Sep. 27, 2001) and WO 00/71703 (published on
Nov. 30, 2000) all to Methylgene, Inc.; published PCT Application
WO 00/21979 published on Oct. 8, 1999 to Fujisawa Pharmaceutical
Co., Ltd.; published PCT Application WO 98/40080 published on Mar.
11, 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current
patent status of histone deacetylase inhibitors Expert Opin. Ther.
Patents (2002) 12(9): 1375-1384 and references cited therein).
[0271] Specific non-limiting examples of DAC or HDAC inhibitors are
provided in the Table below. It should be noted that the present
invention encompasses any compounds which both are structurally
similar to the compounds represented below and are capable of
inhibiting histone deacetylases.
TABLE-US-00002 Title MS-275 ##STR00024## DEPSIPEPTIDE ##STR00025##
Cl-994 ##STR00026## Apicidin ##STR00027## A-161906 ##STR00028##
Scriptaid ##STR00029## PXD-101 ##STR00030## CHAP ##STR00031##
LAQ-824 ##STR00032## Butyric Acid ##STR00033## Depudecin
##STR00034## Oxamflatin ##STR00035## Trichostatin C
##STR00036##
[0272] DAC or HDAC inhibitors for use in accordance with the
present invention may be prepared by any available means including,
for example, synthesis, semi-synthesis, or isolation from a natural
source.
[0273] DAC or HDAC inhibitors for use in accordance with the
present invention may be isolated or purified. For example,
synthesized compounds can be separated from a reaction mixture, and
natural products can be separated from their natural source, by
methods such as column chromatography, high pressure liquid
chromatography and/or recrystallization.
[0274] A variety of synthetic methodologies for preparing DAC or
HDAC inhibitors are known in the art. As can be appreciated by the
skilled artisan, further methods of synthesizing the compounds of
the formulae herein will be evident to those of ordinary skill in
the art. Additionally, the various synthetic steps may be performed
in an alternate sequence or order to give the desired compounds.
Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein are known in the art and include,
for example, those such as described in R. Larock, Comprehensive
Organic Transformations, VCH Publishers (1989); T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis. 2d. Ed., John
Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995), and subsequent editions thereof.
[0275] DAC or HDAC inhibitors for use in accordance with the
present invention may be modified as compared with presently known
DAC or HDAC inhibitors, for example, by appending appropriate
functionalities to enhance selective biological properties. Such
modifications are known in the art and may include those which
increase biological penetration into a given biological system
(e.g., blood, lymphatic system, central nervous system), increase
oral availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion.
[0276] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in
accordance with the present invention may contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric forms that may be defined,
in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)-
or (L)-for amino acids. The present invention encompasses all such
possible isomers, as well as their racemic and optically pure forms
to the extent that they have DAC inhibitory activity.
[0277] In general, optical isomers may be prepared from their
respective optically active precursors by the procedures described
above, or by resolving the racemic mixtures. The resolution can be
carried out in the presence of a resolving agent, by chromatography
or by repeated crystallization or by some combination of these
techniques which are known to those skilled in the art. Further
details regarding resolutions can be found in Jacques, et al.,
Enantiomers, Racemates, and Resolutions (John Wiley & Sons,
1981).
[0278] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in
accordance with the present invention may contain olefinic double
bonds, other unsaturation, or other centers of geometric asymmetry.
The present invention encompasses both E and Z geometric isomers or
cis- and trans-isomers to the extent that they have DAC inhibitory
activity. The of geometric asymmetry. The present invention
encompasses both E and Z geometric isomers or cis- and
trans-isomers to the extent that they have DAC inhibitory activity.
The present invention likewise encompasses all tautomeric forms
that have DAC inhibitory activity. In general, where a chemical
structure is presented, the configuration of any carbon-carbon
double bond appearing herein is selected for convenience only and
is not intended to designate a particular configuration unless the
text so states or it is otherwise clear from context; thus a
carbon-carbon double bond or carbon-heteroatom double bond depicted
arbitrarily herein as trans may be cis, trans, or a mixture of the
two in any proportion.
[0279] DAC inhibitors (e.g., HDAC inhibitors) are particularly
useful in the treatment of neoplasms in vivo. However, they may
also be used in vitro for research or clinical purposes (e.g.,
determining the susceptibility of a patient's disease to a
particular DAC inhibitor). In certain embodiments, the neoplasm is
a benign neoplasm. In other embodiments, the neoplasm is a
malignant neoplasm. Any cancer may be treated using a DAC inhibitor
alone or in combination with another pharmaceutical agent.
[0280] In certain embodiments, the malignancy is a hematological
malignancy. Manifestations can include circulating malignant cells
as well as malignant masses. Hematological malignancies are types
of cancers that affect the blood, bone marrow, and/or lymph nodes.
Examples of hematological malignancies that may be treated using
romidepsin include, but are not limited to: acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL),
hairy cell leukemia. Hodgkin's lymphoma, non-Hodgkin's lymphoma,
cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma
(PTCL), multiple myeloma, and myelodysplastic syndromes. In certain
embodiments, the inventive combination is used to treat multiple
myeloma. In certain particular embodiments, the cancer is relapsed
and/or refractory multiple myeloma. In other embodiments, the
inventive combination is used to treat chronic lymphocytic leukemia
(CLL). In certain particular embodiments, the cancer is relapsed
and/or refractory CLL. In other embodiments, the inventive
combination is used to treat chronic myelogenous leukemia (CML). In
certain embodiments, the inventive combination is used to treat
acute lymphoblastic leukemia (ALL). In certain embodiments, the
inventive combination is used to treat acute myelogenous leukemia
(AML). In certain embodiments, the cancer is cutaneous T-cell
lymphoma (CTCL). In other embodiments, the cancer is peripheral
T-cell lymphoma (PTCL). In certain embodiments, the cancer is a
myelodysplastic syndrome.
[0281] Other cancers besides hematological malignancies may also be
treated using DAC inhibitors. In certain embodiments, the cancer is
a solid tumor.
[0282] Exemplary cancers that may be treated using DAC inhibitor
therapy, including combination therapy, include colon cancer, lung
cancer, bone cancer, pancreatic cancer, stomach cancer, esophageal
cancer, skin cancer, brain cancer, liver cancer, ovarian cancer,
cervical cancer, uterine cancer, testicular cancer, prostate
cancer, bladder cancer, kidney cancer, neuroendocrine cancer,
etc.
[0283] In certain embodiments, a DAC inhibitor is used to treat
pancreatic cancer. In certain embodiments, a DAC inhibitor is used
to treat prostate cancer. In certain specific embodiments, the
prostate cancer is hormone refractory prostate cancer.
Combination Therapy
[0284] DAC inhibitors in accordance with the present invention may
be administered in combination with one or more other therapeutic
agents to treat a disease or disorder associated with Ras
expression, or to treat one or more symptoms of such a disease or
disorder. To give one specific example, the present invention
demonstrates the particular utility of administering a combination
of a DAC inhibitor and gemcitabine. In some particular embodiments
of the present invention, the DAC inhibitor is romidepsin (aka
depsipeptide, FK228, FR901228). In some particular embodiments, the
DAC inhibitor is SAHA. In some particular embodiments, the DAC
inhibitor is phenylbutyrate. In some particular embodiments, the
DAC inhibitor comprises a combination of DAC inhibitors.
[0285] Useful agents that can be administered in combination with
DAC inhibitors (e.g., romidepsin) include, for example, other
chemotherapeutic agents, pain relievers, antipsychotics,
anti-inflammatories, anti-infectives, hormones, immunomodulators,
hematopoietic agents, anticoagulants, steroids, thrombolytics,
antiplatelet drugs, drugs that affect gastrointestinal function,
diuretics, antihypertensives, antiarrhythmials, or other drugs
affecting renal and/or cardiovascular function, etc. Alternatively
or additionally, DAC inhibitors may be administered in combination
with vitamins, electrolytes, etc.
[0286] In certain embodiments, a DAC inhibitor is administered in
combination with one or more additional therapeutic agents, e.g.,
another cytotoxic agent. Exemplary cytotoxic agents that may be
administered in combination with a DAC inhibitor include
gemcitabine, decitabine, and flavopiridol.
[0287] In other embodiments, a DAC inhibitor is administered in
combination with an anti-inflammatory agent such as aspirin,
ibuprofen, acetaminophen, etc., pain reliever, anti-nausea
medication, or anti-pyretic.
[0288] In certain other embodiments, a DAC inhibitor is
administered in combination with a steroidal agent (e.g.
dexamethasone).
[0289] In certain embodiments, a DAC inhibitor is administered in
combination with an agent to treat gastrointestinal disturbances
such as nausea, vomiting, and diarrhea. These additional agents may
include anti-emetics, anti-diarrheals, fluid replacement,
electrolyte replacement, etc.
[0290] In other embodiments, a DAC inhibitor is administered in
combination with electrolyte replacement or supplementation such as
potassium, magnesium, and calcium, in particular, potassium and
magnesium.
[0291] In certain embodiments, a DAC inhibitor is administered in
combination with an anti-arrhythmic agent.
[0292] In certain embodiments, a DAC inhibitor is administered in
combination with a platelet booster, for example, an agent that
increases the production of platelets.
[0293] In certain embodiments, a DAC inhibitor is administered in
combination with an agent to boost the production of blood cells
such as erythropoietin.
[0294] In certain embodiments, a DAC inhibitor is administered in
combination with an agent to prevent hyperglycemia.
[0295] In certain embodiments, a DAC inhibitor is not administered
with another HDAC or DAC inhibitor.
[0296] As will be appreciated by those of skill in the art, and as
is otherwise addressed herein, either or both of the DAC inhibitor
and other agent may be provided in any useful form including, for
example, as a salt, ester, active metabolite, prodrug, etc.
Similarly, either or both agents (or salts, esters, or prodrugs
thereof) may be provided as a pure isomer stereoisomer or as a
combination of stereoisomers, including a racemic combination, so
long as relevant activity is present. Comparably, either or both
agents (or salts, esters or prodrugs thereof) may be provided in
crystalline form, whether a pure polymorph or a combination of
polymorphs, or in amorphous form, so long as relevant activity is
present.
[0297] As addressed above, combination therapy of DAC inhibitors
and other agent(s) will typically involve administration of
multiple individual doses spaced out in time. In some embodiments,
individual DAC inhibitor doses and other agent doses will be
administered together, according to the same schedule. In other
embodiments, DAC inhibitor doses and other agent doses will be
administered according to different schedules.
[0298] The total daily dose of any particular active agent
administered to a human or other animal in single or in divided
doses in accordance with the present invention can be in amounts,
for example, from 0.01 to 50 mg/kg body weight or more usually from
0.1 to 25 mg/kg body weight. Single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose. In
general, treatment regimens according to the present invention
comprise administration to a patient in need of such treatment from
about 10 mg to about 1000 mg of the compound(s) of this invention
per day in single or multiple doses. In certain embodiments, about
10-100 mg of the compound is administered per day in single or
multiple doses. In certain embodiments, about 100-500 mg of the
compound is administered per day in single or multiple doses. In
certain embodiments, about 250-500 mg of the compound is
administered per day in single or multiple doses. In certain
embodiments, about 500-750 mg of the compound is administered per
day in single or multiple doses.
[0299] In the treatment of neoplasms such as cancer in a subject, a
DAC inhibitor is typically dosed at 1-30 mg/m.sup.2. In certain
embodiments, a DAC inhibitor is dosed at 1-15 mg/m.sup.2. In
certain embodiments, a DAC inhibitor is dosed at 5-15 mg/m.sup.2.
In certain particular embodiments, a DAC inhibitor is dosed at 4,
6, 8, 10, 12, 14, 16, 18, or 20 mg/m.sup.2.
[0300] A DAC inhibitor is typically administered in a 28 day cycle
with the agent being administered on days 1, 8 and 15. In certain
embodiments, the DAC is administered on days 1 and 15 with day 8
being skipped. As would be appreciated by one of skill in the art,
the dosage and timing of administration of the dosage of the DAC
inhibitor may vary depending on the patient and condition being
treated. For example, adverse side effects may call for lowering
the dosage of DAC inhibitor administered.
[0301] Typical dosing schedules have been established for certain
exemplary DAC inhibitors (e.g. HDAC inhibitors). For example, SAHA
is commonly administered within a range of about 300-400 mg daily
orally; PXD 101 is commonly administered within a range of about up
to 2000 mg/m.sup.2/day intravenously (e.g., on days 1 to 5 of a 21
day cycle), and may possibly be administered orally; MGCD0103 is
commonly administered at doses up to about 27 mg/m.sup.2 given
orally (e.g., daily for about 14 days); LBH589 is commonly
administered at doses up to about 14 mg/m.sup.2 as an intravenous
infusion (e.g., on days 1-7 of a 21 day cycle); MS-275 is commonly
administered within a dose range of about 2-12 mg/m.sup.2
intravenously (e.g., every 14 days).
[0302] In the treatment of neoplasms such as cancer in a subject,
romidepsin is typically dosed at 1-28 mg/m.sup.2. In certain
embodiments, romidepsin is dosed at 1-15 mg/m.sup.2. In certain
embodiments, romidepsin is dosed at 5-14 mg/m.sup.2. In certain
particular embodiments, romdiepsin is dosed at 8, 10, 12, or 14
mg/m.sup.2. Romidepsin is typically administered in a 28 day cycle
with romidepsin being administered on days 1, 8 and 15. In certain
embodiments, romidepsin is administered on days 1 and 15 with day 8
being skipped.
[0303] As would be appreciated by one of skill in the art, the
dosage and timing of administration of any particular DAC inhibitor
or other agent dose, or the dosage amount and schedule generally
may vary depending on the patient and condition being treated. For
example, adverse side effects may call for lowering the dosage of
one or the other agent, or of both agents, being administered.
[0304] Moreover, those of ordinary skill in the art will readily
appreciate that the dosage schedule (i.e., amount and timing of
individual doses) by which any particular DAC inhibitor is
administered may be different for inventive combination therapy
than it is alone.
[0305] To give but one example, in some embodiments, a DAC
inhibitor (e.g., romidepsin) and other agent are each dosed on days
1 and 15 of a 28 day cycle. Those of ordinary skill in the art will
appreciate that any of a variety of other dosing regimens are
within the scope of the invention. Commonly, dosing is adjusted
based on a patient's response to therapy, and particularly to
development of side effects.
[0306] In some embodiments of the present invention, a DAC
inhibitor is administered in combination with gemcitabine for
example as is described in co-pending United States Patent
Publication application number US 2009/0305956 published Dec. 10,
2009, entitled "GEMCITABINE COMBINATION THERAPY", and attached
hereto in its entirety as Exhibit A.
Pharmaceutical Compositions
[0307] DAC inhibitors and/or other agents for use in accordance
with the present invention are often administered as pharmaceutical
compositions comprising amounts of DAC inhibitor and/or other agent
that are useful in inventive therapy. In some embodiments, a DAC
inhibitor and another agent are present together in a single
pharmaceutical composition; in some embodiments these agents are
provided in separate pharmaceutical compositions.
[0308] In some embodiments, inventive pharmaceutical compositions
are prepared in unit dosage forms. In general, a pharmaceutical
composition of the present invention includes one or more active
agents (i.e., one or more DAC inhibitors, such as romidepsin)
formulated with one or more pharmaceutically acceptable carriers or
excipients.
[0309] In some embodiments, the pharmaceutically acceptable carrier
is selected from the group consisting of sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions; non-toxic compatible lubricants such as sodium lauryl
sulfate and magnesium stearate; coloring agents; releasing agents;
coating agents; sweetening, flavoring and perfuming agents;
preservatives and antioxidants; and combinations thereof. In some
embodiments, the pH of the ultimate pharmaceutical formulation may
be adjusted with pharmaceutically acceptable acids, bases or
buffers to enhance the stability of the formulated compound or its
delivery form.
[0310] Pharmaceutical compositions of this invention may be
administered by any appropriate means including, for example,
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term parenteral as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques. In
many embodiments, pharmaceutical compositions are administered
orally or by injection in accordance with the present
invention.
[0311] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
agent(s), liquid dosage forms of pharmaceutical compositions may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, com, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0312] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. A sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0313] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0314] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from a site of
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of the
drug then depends upon its rate of dissolution, which, in turn, may
depend upon crystal size and crystalline form.
[0315] Alternatively, delayed absorption of a parenterally
administered drug form can be accomplished by dissolving or
suspending the drug in an oil vehicle. Injectable depot forms can
be made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of drug to polymer and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations can also be
prepared by entrapping the drug in liposomes or microemulsions that
are compatible with body tissues.
[0316] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing the active
agents with suitable non-irritating excipients or carriers such as
cocoa butter, polyethylene glycol or a suppository wax which are
solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
active compound.
[0317] Solid dosage forms for oral administration include, for
example, capsules, tablets, pills, powders, and granules. In such
solid dosage forms, the active agent(s) is/are typically mixed with
at least one inert, pharmaceutically acceptable excipient or
carrier such as sodium citrate or dicalcium phosphate and/or: a)
fillers or extenders such as starches, lactose, sucrose, glucose,
mannitol, and silicic acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, c) humectants such as glycerol, d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f)
absorption accelerators such as quaternary ammonium compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, h) absorbents such as kaolin and bentonite clay, and
i) lubricants such as talc, calcium stearate, magnesium-stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form may also comprise buffering agents, permeation enhancers,
and/or other agents to enhance absorption of the active
agent(s).
[0318] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0319] Solid dosage forms such as tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells such
as enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions that can be used include
polymeric substances and waxes.
[0320] In certain embodiments, oral dosage forms are prepared with
coatings or by other means to control release of active agent
(e.g., DAC inhibitor and/or gemcitabine) over time and/or location
within the gastrointestinal tract. A variety of strategies to
achieve such controlled (or extended) release are well known in the
art, and are within the scope of the present invention.
[0321] Dosage forms for topical or transdermal administration
include ointments, pastes, creams, lotions, gels, powders,
solutions, sprays, inhalants or patches. In general, such
preparations are prepared by admixing active agent(s) under sterile
conditions with a pharmaceutically acceptable carrier and any
needed preservatives or buffers as may be required.
[0322] Ophthalmic formulation, ear drops, eye ointments, powders
and solutions are also contemplated as being within the scope of
this invention.
[0323] Ointments, pastes, creams and gels may contain, in addition
to active agent(s), excipients such as animal and vegetable fats,
oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0324] Powders and sprays can contain, in addition to active
agent(s), excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants such as chlorofluorohydrocarbons.
[0325] Transdermal patches can provide controlled delivery of a
compound to the body. Such dosage forms can be made by dissolving
or dispensing the compound in the proper medium. Absorption
enhancers can also be used to increase the flux of the compound
across the skin. The rate can be controlled by either providing a
rate controlling membrane or by dispersing the compound in a
polymer matrix or gel.
[0326] For pulmonary delivery, active agent(s) is/are formulated
and administered to the patient in solid or liquid particulate form
by direct administration e.g., inhalation into the respiratory
system. Solid or liquid particulate forms of the active agent(s)
prepared for practicing the present invention include particles of
respirable size: that is, particles of a size sufficiently small to
pass through the mouth and larynx upon inhalation and into the
bronchi and alveoli of the lungs. Delivery of aerosolized
therapeutics, particularly aerosolized antibiotics, is known in the
art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et
al., U.S. Pat. No. 5,508,269 to Smith et al., and WO 98/43,650 by
Montgomery, all of which are incorporated herein by reference). A
discussion of pulmonary delivery of antibiotics is also found in
U.S. Pat. No. 6,014,969, incorporated herein by reference.
[0327] Pharmaceutical compositions for use in accordance with the
present invention can, for example, be administered by injection,
intravenously, intraarterially, subdermally, intraperitoneally,
intramuscularly, or subcutaneously; or orally, buccally, nasally,
transmucosally, topically, in an ophthalmic preparation, or by
inhalation, for example with a dosage ranging from about 0.1 to
about 500 mg/kg of body weight, alternatively dosages between 1 mg
and 1000 mg/dose, every 4 to 120 hours, or according to the
requirements of the particular drug.
[0328] The methods herein contemplate administration of an
effective amount of active agent or pharmaceutical composition
sufficient for a desired or stated effect. Typically, the
pharmaceutical compositions of this invention will be administered
from about 1 to about 6 times per day or alternatively, as a
continuous infusion. Such administration can be used as a chronic
or acute therapy.
[0329] The amount of any particular active agent that may be
combined with pharmaceutically acceptable excipients or carriers to
produce a single dosage form may vary depending upon the host
treated and the particular mode of administration. A typical
preparation will contain from about 5% to about 95% active compound
(w/w). Alternatively, such preparations may contain from about 20%
to about 80% active compound. For romidepsin, preparations may
commonly contain about 20-50%, 25-45%, 30-40%, or approximately
32%, 33%, 34%, or 35% active compound; for gemcitabine, the
compound is typically provided in 200 mg or 1 gram vials as a
lyophilized powder. Drug product is reconstituted with either 5 ml
(for the 200 mg vial) or 25 ml (for the 1 g vial) using sodium
chloride for injection. Both dilutions give a 38 mg/ml solution
(including displacement volume). This solution can be diluted down
to 0.1 mg/ml-0.4 mg/ml for administration.
[0330] Lower or higher doses than those recited above may be
required. Specific dosage and treatment regimens for any particular
patient will depend upon a variety of factors, including the
activity of the specific compound employed, the age, body weight,
general health status, sex, diet, time of administration, rate of
excretion, drug combination, the severity and course of the
disease, condition or symptoms, the patient's disposition to the
disease, condition or symptoms, and the judgment of the treating
physician.
[0331] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level. Patients may, however, require intermittent treatment on a
long-term basis upon any recurrence of disease symptoms.
[0332] When pharmaceutical compositions contain two or more active
agents, it is generally the case that each agent is present at
dosage levels of between about 1 to 100%, for example about 5 to
95%, of the level normally administered in a monotherapy
regimen.
[0333] Unless otherwise defined, all technical and scientific terms
used herein are accorded the meaning commonly known to one of
ordinary skill in the art. All publications, patents, published
patent applications, and other references mentioned herein are
hereby incorporated by reference in their entirety. The embodiments
of the invention should not be deemed to be mutually exclusive and
can be combined.
EXEMPLIFICATION
[0334] The present invention will be better understood in
connection with the following Examples, which are intended as an
illustration only and not limiting of the scope of the invention.
Various changes and modifications to the disclosed embodiments will
be apparent to those skilled in the art and such changes and
modifications including, without limitation, those relating to the
chemical structures, substituents, derivatives, formulations and/or
methods of the invention may be made without departing from the
spirit of the invention and the scope of the appended claims.
Example 1
Depsipeptide (FK228) Alone and in Combination with Gemcitabine in
In Vivo Mouse Xenograft Model of Ras-Expressing Pancreatic
Tumor
[0335] The present Example demonstrates that both depsipeptide
(FK228; romidepsin) and gemcitabine can effectively inhibit tumor
growth in a mouse xenograft model, and further demonstrates a
surprising synergistic effect of the combination.
[0336] Panc-1, obtained from the ATCC, is a pancreas tumor cell
line (oncogenic K-ras) originating from a 56 year old Caucasian
male. In this study, female nude mice were implanted subcutaneously
(SC) by trocar with Panc-1 tumor fragments harvested from SC
growing tumors in nude mice hosts. When tumors reached
approximately 140 mm.sup.3, animals were pair matched by tumor size
into treatment and control groups (N=9 mice per group). The day of
treatment initiation was specified as Day 1. Vehicle control and
FK228 were administered intravenously on a Q4Dx3 schedule (Days 1,
5, and 9). Gemcitabine was administered by an intraperitoneal
injection on a Q3Dx4 schedule (Days 1, 4, 7, 10). Tumors were
measured by Vernier calipers twice weekly.
[0337] Treatment with depsipeptide (FK228, FR901228, romidepsin)
and gemcitabine as single agents both resulted in consistently
smaller tumors than vehicle control treated animals, with tumor
growth inhibitions of 26% and 50%, respectively.
[0338] In addition, a potential synergistic effect of depsipeptide
and gemcitabine was observed in combination with a significant
tumor growth inhibition of 101%. Three animals in the combination
group exhibited evidence of tumor regression. These results
indicate depsipeptide has clear antitumor activity against the
Panc-1 human pancreas tumors in an in vivo xenograft model.
Furthermore, depsipeptide has the potential to synergize with other
approved chemotherapeutics, and specifically with gemcitabine. The
effects of this synergy, including tumor regression, are
particularly significant given the known aggressiveness of
pancreatic tumors, and their susceptibility to developing
resistance. The present invention demonstrates tumor regression
after dosing with a combination of romidepsin and gemcitabine. Note
that no regression was observed with gemcitabine alone, the current
standard therapy for pancreatic tumors, yet regression was observed
with the combination.
[0339] We note that no synergistic effect was observed with the
combination of romidepsin and gemcitabine in another cell line
(Bx-PC-3) that had normal Ras (See Example 3, below).
Example 2
Combination of FK228 and Gemcitabine is More Effective than Either
Agent Alone in a Ras-Transformed Pancreatic Adenocarcinoma
Model
[0340] The present Example demonstrates that the combination of
FK228 and gemcitabine is more effective than either agent alone in
a pancreatic adenocarcinoma model.
[0341] Abstract: To examine activity and mechanism of FK228,
antitumor efficacy was tested in PANC-1 pancreatic adenocarcinoma
model representing transformed Ras, either as a single agent or in
combination with gemcitabine. Following PANC-1 study completion,
tumor and sera were obtained from:
[0342] the vehicle control;
[0343] FK228 dosed at 5 mg/kg once every four days for three
treatments (Q4Dx3);
[0344] Gemcitabine at 80 mg/kg (Q3Dx4); and
[0345] the drug combination.
Expression of c-Myc, acetylated histones 3 and 4, and p21.sup.waf1
was compared between control and FK228 groups by immunoblotting and
was quantified following actin normalization. Serum levels of
putative tumor products b-FGF and MMP-2 were quantified by
human-specific ELISA.
[0346] Highly significant (p<0.0001) downregulation of c-Myc was
observed in all treatment groups, most dramatically in the
combination group. Acetylated histone 3 levels were not affected in
FK228 alone or in combination with gemcitabine. Upregulation of
acetylated histone 4 by the drug combination was highly
significant. Treatment with gemcitabine alone significantly
(p<0.05) downregulated p21.sup.waf; however, this effect was not
reported in combination groups.
[0347] These results suggest activity of FK228 is Ras-transformed
malignancies and demonstrate combinatorial effects with gemcitabine
at least in pancreatic adenocarcinoma. Surprising long-term effects
of FK228 in combination with gemcitabine on c-Myc and acetylated
histone 4 might suggest tumor phenotypic changes consistent with
downregulation of HDAC activity.
[0348] Materials and Methods
[0349] Specimen Collection: FK228 antitumor efficacy was tested in
PANC-1 pancreatic adenocarcinoma model representing transformed
Ras, either as a single agent or in combination with gemcitabine.
Tumor xenograft tissue and serum specimens were obtained from in
vivo studies performed by the Preclinical Research Laboratory at
the completion of the experiments. The following tumor and sera
specimens were obtained:
[0350] Vehicle control (n-9)
[0351] FK228 at 5 mg/kg once every four days for three treatments
(Q4Dx3) (n=9)
[0352] Gemcitabine at 80 mg/kg (Q3Dx4) (n=9)
[0353] FK228 at 5 mg/kg plus gemcitabine at 80 mg/kg (Q3Dx4)
(n=6)
Tumors were dissected from the animals, rinsed in cold phosphate
buffered saline, and snap frozen in liquid nitrogen. Serum was
obtained from whole blood and stored frozen at -70.degree. C.
[0354] Tissue Biomarkers: Tumor levels of acetylated histone-4,
histone-4, c-Myc, p21.sup.waf and .beta.-actin were quantified by
immunoblotting as described. Briefly, frozen tissue was pulverized
under liquid nitrogen and homogenized in hypotonic lysis buffer.
Small aliquots of the extracts were used for analysis of protein
concentration by micro-BCA assay with bovine serum albumin as a
protein standard. An equal amount of extracts containing about
20-50 .mu.g protein was electrophoresed in SDS polyacrylamide gels.
Proteins were transferred to ImmunoBlot PVDF membrane and were
probed with appropriate primary and secondary antibodies. The
chemiluminescence signal was captured by autoradiography,
quantified by densitometry and expressed as a ratio of actin in
each sample lane. For each biomarker, means and standard errors
were calculated in each treatment group. The data were analyzed by
two-sided t-tests to determine if measured end points are
significantly affected by drug treatment.
[0355] Serum Biomarkers: Serum levels of b-FGF and VEGF were
quantified by ELISA using human-specific kits from R&D Systems,
Minneapolis, Min., according to supplier's instructions. All assays
were performed in duplicate. For each biomarker, means and standard
errors were calculated in each treatment group. The data were
analyzed by two-sided t-tests to determine if measured endpoints
are significantly affected by drug treatment.
[0356] Results
[0357] Western blot detection of acetylated histone 3, histone 4,
c-Myc, p21.sup.waf and a housekeeping gene product .beta.-actin as
an internal control were performed. Uniform expression of
.beta.-actin was noted in all samples. Following quantitative
analysis of biomarker levels in each sample, the results were
normalized for .beta.-actin and expressed as percentages of
untreated controls. Group averages were compared by t-test.
[0358] When compared with the controls, the expression of c-Myc was
inhibited in all groups. The extent of inhibition (50%) was similar
in the gemcitabine and FK228 monotherapy treatment groups and
greater (60%) in the combination group. The inhibition of c-Myc
expression was highly significant (p<0.0001) in all cases.
[0359] Acetylation of histone 3 was significantly inhibited by
gemcitabine, but not affected by FK228 alone or in combination with
gemcitabine.
[0360] The levels of acetylated histone 4 were at the control level
in the gemcitabine group, FK228 treatment induced over 2-fold
increase of acetylated histone 4, but in comparison with the
control group the increase was not significant. On the other hand,
over 3-fold up-regulation of acetylated histone 4 by the drug
combination was highly significant (p=0.00003).
[0361] Treatment with gemcitabine alone significantly (p<0.05)
downregulated p21.sup.waf; however, this effect was not observed in
the combination groups.
[0362] Quantitative analysis of b-FGF and VEGF in serum was
performed. The levels of b-FGF were highly variable but not
significantly different in any treatment groups in comparison with
the controls. VEGF was under the detection limits of the assay.
[0363] The effects of FK228 on expression of c-Myc and acetylated
histone 4 are unexpected considering that these endpoints were
assessed at the end of a long-term in vivo treatment with the drug.
Historically, the effects of DAC inhibitors such as FK228 on target
gene or protein expression were assessed in a time scale of hours
(not days) following drug treatment. For example, a study on the
effects of FK228 on tumor growth and expression of p21 and c-myc
genes in vivo over a period of 2 to 24 hours demonstrated induction
of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive
to FK228, while opposite effects on p21 and c-myc mRNA were seen in
tumor xenograft less sensitive to FK228.
[0364] Myc genes are key regulators of cell proliferation, and
their deregulation contributes to the genesis of most human tumors.
Transcriptional regulation by Myc-family proteins includes
recruitment of HDACs in tumors, some of which exhibit dependence
(addition) to c-myc. Even a brief inhibition of c-myc expression
may be sufficient to completely stop tumor growth and induce
regression of tumors. It is conceivable that biological activity of
FK228 could be partly due to inhibition of c-myc and other genes
under its control, including HDACs.
[0365] In conclusion, these results demonstrate at least additive
combinatorial effects with gemcitabine on the expression of c-Myc
and acetylation of histone 4 in pancreatic adenocarcinoma.
Surprising effects of FK228 in combination with gemcitabine might
suggest tumor phenotypic changes consistent with downregulation of
HDAC activity.
[0366] Specifically, weeks after the end of treatment, the cells
are phenotypically different from those that were initially
injected, suggesting some form of cellular transformation, possibly
to a less aggressive phenotype.
Example 3
Depsipeptide (FK228) Alone and in Combination with Gemcitabine in
In Vivo Mouse Xenograft Model of Ras-Expressing Pancreatic Tumor as
Compared with Non-Ras-Expressing Tumor
[0367] The present Example demonstrates a specific effect of
depsipeptide and gemcitabine on Ras-expressing tumors (PANC-1) as
compared with non-Ras-expressing tumors (BxPC03).
[0368] Materials and Methods
[0369] Model Information--Female nude mice (nu/nu) between 5 and 6
weeks of age weighing approximately 20 grams were obtained from
Harlan, Inc. (Madison, Wis.). PANC-1, obtained from the ATCC, is a
pancreas tumor cell line originating from a 56 year-old Caucasian
male. BxPC-3, obtained from the American Type Culture Collection
(ATCC), is a pancreas tumor cell line originating from a 61
year-old female. PANC-1 Study: Animals were implanted
subcutaneously (SC) by trocar with fragments of PANC-1 harvested
from SC growing tumors in nude mice hosts. When tumors grew to
approximately 135 cubic millimeters (mm.sup.3) in size (17 days
following implantation), animals were pair-matched by tumor size
into treatment and control groups; each treatment group contained
nine mice.
[0370] BxPC-3 Study: Animals were implanted subcutaneously (SC) by
trocar with fragments of BxPC-3 harvested from SC growing tumors in
nude mice hosts. When tumors grew to approximately 85 cubic
millimeters (mm.sup.3) in size (19 days following implantation),
animals were pair-matched by tumor size into treatment and control
groups; each treatment group contained nine mice. Animals in both
studies were ear-tagged and followed individually throughout the
experiment.
[0371] Study Design and Dosing--Initial doses were administered on
Day 1 following pair-matching; both experiments were carried out as
tumor growth inhibition (TGI) studies. Animals were dosed
intravenously (IV) via tail vein with FK228 once every four days
for three treatments (Q4Dx3) or by intraperitoneal (IP) injection
with gemcitabine once every three days for four treatments (Q3Dx4),
either alone or in combination, at the doses listed below (Table
1). To serve as a negative control, FK228 vehicle (2% ethanol, 8%
propylene glycol, 90% 0.9% saline) was injected on a Q4Dx3
schedule.
TABLE-US-00003 TABLE 1 Study Design (PANC-1 and BxPC-3 Xenograft
Studies) Group # Animals Compound Dose (mg/kg) Route/Schedule 1 8-9
Vehicle -- IV; Q4Dx3 2 8-9 FK228 2.5 IV; Q4Dx3 3 8-9 FK228 5 IV;
Q4Dx3 4 8-9 Gemcitabine 40 IP; Q3Dx4 5 8-9 Gemcitabine 80 IP; Q3Dx4
FK228 2.5 IV; Q4Dx3 6 8-9 Gemcitabine 40 IV; Q4Dx3 FK228 5 IV;
Q4Dx3 7 8-9 Gemcitabine 70 IP; Q3Dx4 FK228 2.5 IV; Q4Dx3 8 8-9
Gemcitabine 80 IP; Q3Dx4 FK228 5 IV; Q4Dx3 9 8-9 Gemcitabine 80 IP;
Q3Dx4
Data Collection and Statistical Analysis
[0372] Animal Weights--Individual and group mean weights .+-.SD and
percent weight change through Day 25 (PANC-1) or Day 29 (BxPC-3)
were recorded twice weekly until study completion beginning Day 1.
Final group mean weights .+-.SD and group nadir values are
reported; weight data from individual animals experiencing
technical or drug-related deaths was censored from final group
calculations.
[0373] Moribundity/Mortality--Animals were observed twice weekly
for general moribundity and daily for mortality. Animal deaths were
assessed as drug-related or technical based on factors including
gross observation and weight loss: reported animal death
information includes type, number, and day of death for each
group.
[0374] Tumor Volume--Individual and group mean tumor volumes
.+-.SEM through Day 25 (PANC-1) or Day 29 (BxPC-3) were recorded
twice weekly until study completion beginning Day 1. Tumor
measurements were converted to cubic millimeter tumor volume using
the formula:
Tumor Volume (mm.sup.3)=Width.sup.2(mm).times.Length
(mm).times.0.52
Final mean tumor volume .+-.SEM for each group was reported;
animals experiencing partial or complete tumor regressions or
animals experiencing technical or drug-related deaths were censored
from these calculations.
[0375] Tumor Growth Inhibition--On Day 25 (PANC-1) or Day 29
(BxPC-3), mice were weighed and caliper tumor measurements taken.
Tumor growth inhibition (TGI) values were calculated for each group
containing treated animals using the formula:
1 - Mean Final Tumor Volume ( Treated ) - Mean Initial Tumor Volume
( Treated ) Mean Final Tumor Volume ( Control ) - Mean Initial
Tumor Volume ( Control ) .times. 100 ##EQU00001##
Animals experiencing partial or complete tumor regressions or
animals experiencing technical or drug-related deaths were censored
from TGI calculations; the National Cancer Institute (NCI) criteria
for compound activity is TGI>58% TGI values for each treatment
group are reported at study completion; these calculations are
based on the final study day.
[0376] Partial/Complete Tumor Response--Individual mice possessing
tumors measuring less than on Day 1 were classified as having
partial regression (PR) and a tumor regression value is determined
using the formula:
1 - Final Tumor Volume ( mm 3 ) Initial Tumor Volume ( mm 3 )
.times. 100 % ##EQU00002##
If partial tumor regression was reported in multiple animals within
one group, a mean value was determined. Individual mice lacking
palpable tumors were classified as undergoing complete regression
(CR). Animals experiencing partial or complete tumor regressions
were censored from TGI calculations. However, data from these
animals was included in statistical analysis calculations. In
addition, weight data from these animals was included in daily and
final group calculations.
[0377] Tumor Necrosis--Degree of tumor necrosis was rated at each
tumor measurement using the following arbitrary index:
TABLE-US-00004 NO None No Visible Necrosis N1 Slight Reddened or
Inflamed; Intact Tumor N2 Mild <10% Tumor Necrosis N3 Moderate
<50% Tumor Necrosis N4 Severe >50% Tumor Necrosis
Notable differences in tumor necrosis between treated and control
groups are reported.
[0378] Statistics--Statistical analyses were carried out between
treated and control groups comparing final weight, percent weight
change, and tumor growth inhibition. For these groups, a two-tailed
One-Way Analysis of Variance (ANOVA) followed by the Dunnett
multiple comparisons test was employed. All analyses are performed
using GraphPad Prismg) software (version 4.0). Weight and tumor
data from individual animals experiencing technical or drug-related
deaths was censored from analysis. However, weight and tumor data
from animals reporting partial or complete regressions was included
in these calculations.
TABLE-US-00005 TABLE 2 Animal Weight and Drug Toxicity Results:
PANC-1 Control and Single Agent Groups (Day 25) ROUTE/ FINAL WEIGHT
DATA (DAY 25) WEIGHT NADIR DRUG DEATHS GROUP DOSE SCHEDULE MEAN(G)
.+-. SD % CHANGE % CHANGE DAY TOTAL DAY (#) Vehicle -- IV/Q4D
.times. 3 24 .+-. 3 +8 -- -- 0 -- Gemcitabine 40 mg/kg IP/Q3D
.times. 4 24 .+-. 1 +7 -2 11 0 -- Gemcitabine 80 mg/kg IP/Q3D
.times. 4 26 .+-. 3 +12 -3 11 0 -- FK228 2.5 mg/kg IV/Q4D .times. 3
23 .+-. 2 +8 -9 11 0 -- FK228 5 mg/kg IV/Q4D .times. 3 25 .+-. 2 +9
-12 11 0 -- N = 9/GRP ON DAY 1
TABLE-US-00006 TABLE 3 Tumor Volume and Efficacy Results: PANC-1
Control and Single Agent Groups (Day 25) ROUTE/ FINAL TUMOR VOLUME
(DAY 25) GROUP DOSE SCHEDULE MEAN(MM.sup.3) .+-. SEM % TGI #PR/CR %
TR Vehicle -- IV/Q4D .times. 3 2234 .+-. 402 0/0 Gemcitabine 40
mg/kg IP/Q3D .times. 4 1566 .+-. 255 31 1/0 62% Gemcitabine 80
mg/kg IP/Q3D .times. 4 1172 .+-. 234 51 0/0 FK228 2.5 mg/kg IV/Q4D
.times. 3 1688 .+-. 290 26 0/0 FK228 5 mg/kg IV/Q4D .times. 3 1690
.+-. 331 26 0/0 N = 9/GRP ON DAY 1
Vehicle Control Group (2% EtOH: 8% PG: 80% Saline; IV; Q4Dx3)
[0379] Animal Weights: A final mean weight of 24.+-.3 grams was
calculated at study completion (Day 25). No weight loss was
reported in this study. Mean animal weights and percent change from
Day 1 are reported in Table 2.
[0380] Moribundity/Mortality: 0/9 animals reported vehicle-related
toxicity or deaths (Table 2).
[0381] Tumor Volume: A final mean tumor volume of 2234.+-.402
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 3.
[0382] Tumor Growth Inhibition: N/A (Table 3)
[0383] Partial/Complete Tumor Response: No spontaneous tumor
regressions were reported (Table 3).
[0384] Tumor Necrosis: 1/9 animals reported moderate tumor
necrosis; 6/9 reported severe necrosis; this is not uncommon in the
PANC-1 model.
Single Agent Treatment Groups
[0385] I. Gemcitabine; 40 mg/kg; IP; Q3Dx4
[0386] Animal Weights: A final mean weight of 24.+-.1 grams was
calculated at study completion (Day 25). Slight weight loss was
reported (nadir=-2%, Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 2.
[0387] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 2).
[0388] Tumor Volume: A final mean tumor volume of 1566.+-.255
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 3.
[0389] Tumor Growth Inhibition: A TGI of 31% was reported versus
control in this study (Table 3): this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0390] Partial/Complete Tumor Response: 1/9 animals reported a
partial tumor response with a 62% tumor regression (Table 3).
[0391] Tumor Necrosis: 1/9 animals reported mild tumor necrosis and
4/9 reported moderate necrosis, which was unremarkable compared
with control.
II. Gemcitabine: 80 mg/kg; IP: Q3Dx4
[0392] Animal Weights: A final mean weight of 26.+-.3 grams was
calculated at study completion (Day 25). Slight weight loss was
reported (nadir=-3%, Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 2.
[0393] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 2).
[0394] Tumor Volume: A final mean tumor volume of 1172.+-.234
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 3.
[0395] Tumor Growth Inhibition: A TGI of 51% was reported versus
control in this study (Table 3); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0396] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 3).
[0397] Tumor Necrosis: 1/9 animals reported slight tumor necrosis,
2/9 reported mild tumor necrosis, and 1/9 reported moderate
necrosis; observations were unremarkable compared with control.
III. FK228; 2.5 mg/kg; IV; Q4Dx3
[0398] Animal Weights: A final mean weight of 23.+-.2 grams was
calculated at study completion (Day 25). Modest weight loss was
reported (nadir=-9%, Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 2.
[0399] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 2).
[0400] Tumor Volume: A final mean tumor volume of 1688.+-.290
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 3.
[0401] Tumor Growth Inhibition: A TGI of 26% was reported versus
control in this study (Table 3); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0402] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 3).
[0403] Tumor Necrosis: 1/9 animals reported slight tumor necrosis,
1/9 reported mild tumor necrosis, 2/9 reported moderate necrosis,
and 1/9 reported severe necrosis; observations were unremarkable
compared with control.
IV. FK228; 5 mg/kg; IV; Q4Dx3
[0404] Animal Weights: A final mean weight of 25.+-.2 grams was
calculated at study completion (Day 25). Moderate weight loss was
reported (nadir=-12%. Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 2.
[0405] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 2).
[0406] Tumor Volume: A final mean tumor volume of 1690.+-.290
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 3.
[0407] Tumor Growth Inhibition: A TGI of 26% was reported versus
control in this study (Table 3); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0408] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 3).
[0409] Tumor Necrosis: 2/9 animals reported slight tumor necrosis,
and 1/9 reported moderate necrosis; observations were unremarkable
compared with control.
[0410] Combination Treatment Groups
TABLE-US-00007 TABLE 4 Animal Weight and Drug Toxicity Results:
PANC-1 Combination Groups (Day 25) FINAL WEIGHT DATA ROUTE/ (DAY
25) WEIGHT NADIR GROUP DOSE SCHEDULE MEAN (C) .+-. SD % CHANGE %
CHANGE DAY Gemcitabine 40 mg/kg IP/Q3Dx4 24 .+-. 1 +7 -2 11
Gemcitabine 80 mg/kg IP/Q3Dx4 26 .+-. 3 +12 -3 11 FK228 2.5 mg/kg
IV/Q4Dx3 25 .+-. 2 +10 -16 11 Gemcitabine 40 mg/kg IP/Q3Dx4 FK228 5
mg/kg IV/Q4Dx3 26 .+-. 2 +12 -23 11 Gemcitabine 40 mg/kg IP/Q3Dx4
FK228 2.5 mg/kg IV/Q4Dx3 26 1 +15 -24 11 Gemcitabine 80 mg/kg
IP/Q3Dx4 FK228 5 mg/kg IV/Q4Dx3 25 2 +10 -27 11 Gemcitabine 80
mg/kg IP/Q2Dx4 N = 9/GRP ON DAY 1
TABLE-US-00008 TABLE 5 Tumor Volume and Efficacy Results: PANC-1
Combination Groups (Day 25) FINAL TUMOR VOLUME ROUTE/ (DAY 25)
GROUP DOSE SCHEDULE MEAN .+-. SEM % TGI # PR/CR Gemcitabine 40
mg/kg IP/Q3Dx4 1566 .+-. 255 31 1/0 Gemcitabine 80 mg/kg IP/Q3Dx4
1172 .+-. 234 51 0/0 *FK228 2.5 mg/kg IV/Q4Dx3 600 .+-. 129 78 0/0
Gemcitabine 40 mg/kg IP/Q3Dx4 *FK228 5 mg/kg IV/Q4Dx3 326 .+-. 70
90 1/0 Gemcitabine 40 mg/kg IP/Q3Dx4 *FK228 2.5 mg/kg IV/Q4Dx3 383
98 88 0/0 Gemcitabine 80 mg/kg IP/Q3Dx4 *FK228 5 mg/kg IV/Q4Dx3 147
63 97 3/0 Gemcitabine 80 mg/kg IP/Q2Dx4 N = 9/GRP ON DAY 1 # P <
0.001 vs. standard agent alone
I. FK228; 2.5 mg/kg; IV; Q4Dx3+Gemcitabine; 40 mg/kg; IP; Q3Dx4
[0411] Animal Weights: A final mean weight of 25.+-.2 grams was
calculated at study completion (Day 25). Significant weight loss
was reported (nadir=-16%, Day 11), which is comparable to additive
loss from single agent groups; weight was fully recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 4.
[0412] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 4).
[0413] Tumor Volume: A final mean tumor volume of 600.+-.129
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 5.
[0414] Tumor Growth Inhibition: A TGI of 78% was reported versus
control in this study (Table 5); this combination is considered
active according to NCI Standards (TGI>58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically significant
(p<0.001) compared with 40 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0415] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 5).
[0416] Tumor Necrosis: 0/9 animals reported tumor necrosis
observations were remarkable compared with control or either single
agent group at Day 25 or at similar mean tumor volume.
II. FK228; 5 mg/kg; IV; Q4Dx3+Gemcitabine; 40 mg/kg; IP; Q3Dx4
[0417] Animal Weights: A final mean weight of 26.+-.2 grams was
calculated at study completion (Day 25). Significant weight loss
was reported (nadir=-23%. Day 11), which is increased compared to
additive loss from single agent groups; weight was fully recovered
by study completion. Mean animal weights and percent change from
Day 1 are reported in Table 4.
[0418] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 4).
[0419] Tumor Volume: A final mean tumor volume of 326.+-.70
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 5.
[0420] Tumor Growth Inhibition: A TGI of 90% was reported versus
control in this study (Table 5); this combination is considered
active according to NCI Standards (TGI>58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically significant
(p<0.001) compared with 40 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0421] Partial/Complete Tumor Response: 1/9 animals reported a
partial tumor response with a 76% tumor regression (Table 5).
[0422] Tumor Necrosis: 0/9 animals reported tumor necrosis;
observations were remarkable compared with control or either single
agent group at Day 25 or at similar mean tumor volume.
III. FK228; 2.5 mg/kg; IV; Q4Dx3+Gemcitabine; 80 mg/kg; IP;
Q3Dx4
[0423] Animal Weights: A final mean weight of 26.+-.1 grams was
calculated at study completion (Day 25). Significant weight loss
was reported (nadir=-24%. Day 11), which is increased compared to
additive loss from single agent groups; weight was fully recovered
by study completion. Mean animal weights and percent change from
Day 1 are reported in Table 4.
[0424] Moribundity/Mortality: 1/9 animals reported a drug-related
death on Day 11 (Table 4).
[0425] Tumor Volume: A final mean tumor volume of 383.+-.103
mm.sup.3 was calculated at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 5.
[0426] Tumor Growth Inhibition: A TGI of 88% was reported versus
control in this study (Table 5); this combination is considered
active according to NCI Standards (TGI>58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically significant
(p<0.001) compared with 80 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0427] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 5).
[0428] Tumor Necrosis: 1/9 animals reported slight tumor necrosis;
observations were remarkable compared with control or either single
agent group at Day 25 or at similar mean tumor volume.
IV. FK228; 5 mg/kg; IV; Q4Dx3 Gemcitabine; 80 mg/kg; IP; Q3Dx4
[0429] Animal Weights: A final mean weight of 25.+-.2 grams was
calculated at study completion (Day 25). Significant weight loss
was reported (nadir=-27%. Day 11), which is increased compared to
additive loss from single agent groups; weight was fully recovered
by study completion. Mean animal weights and percent change from
Day 1 are reported in Table 4.
[0430] Moribundity/Mortality: 1/9 animals reported drug-related
deaths on Day 10 and 1/9 on Day 15 (Table 4).
[0431] Tumor Volume: A final mean tumor volume of 147.+-.63
mm.sup.3 was calculatcd at study completion (Day 25). Mean tumor
volumes beginning Day 1 are reported in Table 5.
[0432] Tumor Growth Inhibition: A TGI of 97% was reported versus
control in this study (Table 5); this combination is considered
active according to NCI Standards (TGI>58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically significant
(p<0.001) compared with 80 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (AN OVA) followed by the
Dunnett multiple comparisons test.
[0433] Partial/Complete Tumor Response: 3/9 animals reported a
partial tumor response with a 66% mean tumor regression (Table
5).
[0434] Tumor Necrosis; 0/9 animals reported tumor necrosis;
observations were remarkable compared with control or either single
agent group at Day 25 or at similar mean tumor volume.
BxPC-3 Study Results
TABLE-US-00009 [0435] TABLE 6 Animal Weight and Drug Toxicity
Results: BxPC-3 Control and Single Agent Groups (Day 29) FINAL
WEIGHT DATA ROUTE/ (DAY 29) WEIGHT NADIR DRUG DEATHS GROUP DOSE
SCHEDULE MEAN(G) .+-. SD % CHANGE % CHANGE DAY TOTAL DAY (#)
Vehicle -- IV/Q4Dx3 25 .+-. 2 +6 -- -- 0 -- Gemcitabine 40 mg/kg
IP/Q3Dx4 25 .+-. 3 +9 -- -- 0 -- Gemcitabine 80 mg/kg IP/Q3Dx4 22
.+-. 4 +1 -9 11 0 -- FK228 2.5 mg/kg IV/Q4Dx3 25 .+-. 2 +10 -5 11 0
-- FK228 5 mg/kg IV/Q4Dx3 24 .+-. 1 +7 -15 11 0 -- N = 8/GRP ON DAY
1
TABLE-US-00010 TABLE 7 Tumor Volume and Efficacy Results: BxPC-3
Control and Single Agent Groups (Day 29) FINAL TUMOR VOLUME ROUTE/
(DAY 29) GROUP DOSE SCHEDULE MEAN .+-. SEM % TGI #PR/CR % TR
Vehicle -- IV/Q4Dx3 2073 .+-. 315 -- 0/0 -- Gemcitabine 40 mg/kg
IP/Q3Dx4 1683 .+-. 426 20 0/0 -- Gemcitabine 80 mg/kg IP/Q3Dx4 1890
.+-. 237 9 0/0 -- FK228 2.5 mg/kg IV/Q4Dx3 2440 .+-. 643 -- 0/0 --
FK228 5 mg/kg IV/Q4Dx3 2620 .+-. 238 -- 0/0 -- N = 8/GRP ON DAY
1
Vehicle Control Group (2% EtOH: 8% PG: 80% Saline: IV; Q4Dx3)
[0436] Animal Weights: A final mean weight of 25.+-.2 grams was
calculated at study completion (Day 29). No weight loss was
reported in this study. Mean animal weights and percent change from
Day 1 are reported in Table 6.
[0437] Moribundity/Mortality: 0/9 animals reported vehicle-related
toxicity or deaths (Table 6).
[0438] Tumor Volume: A final mean tumor volume of 2073.+-.315
mm.sup.3 calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 7.
[0439] Tumor Growth Inhibition: N/A (Table 3)
[0440] Partial/Complete Tumor Response: No spontaneous tumor
regressions were reported (Table 7).
[0441] Tumor Necrosis: 1/9 animals reported moderate tumor
necrosis, which is not uncommon in the BxPC-3 model.
Single Agent Treatment Groups
[0442] I. Gemcitabine; 40 mg/kg; IP; Q3Dx4
[0443] Animal Weights: A final mean weight of 25.+-.3 grams was
calculated at study completion (Day 29). No weight loss was
reported in this study. Mean animal weights and percent change from
Day 1 are reported in Table 6.
[0444] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 6).
[0445] Tumor Volume: A final mean tumor volume of 1683.+-.426
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 7.
[0446] Tumor Growth Inhibition: A TGI of 20% was reported versus
control in this study (Table 7); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0447] Partial/Complete Tumor Response: 0/9 animals reported a
partial/complete response (Table 7).
[0448] Tumor Necrosis: 1/9 animals reported severe tumor necrosis;
observations were unremarkable compared with control.
II. Gemcitabine; 80 mg/kg; IP; Q3Dx4
[0449] Animal Weights: A final mean weight of 22.+-.4 grams was
calculated at study completion (Day 29). Modest weight loss was
reported (nadir=-9%, Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 6.
[0450] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 6).
[0451] Tumor Volume: A final mean tumor volume of 1890.+-.237
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 7.
[0452] Tumor Growth Inhibition: A TGI of 9% was reported versus
control in this study (Table 7); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0453] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 7).
[0454] Tumor Necrosis: 1/9 animals reported mild tumor necrosis;
observations were unremarkable compared with control.
III. FK228; 2.5 mg/kg; IV; Q4Dx3
[0455] Animal Weights: A final mean weight of 25.+-.2 grams was
calculated at study completion (Day 29). Slight weight loss was
reported (nadir=-5%, Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 6.
[0456] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 6).
[0457] Tumor Volume: A final mean tumor volume of 2440.+-.643
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 7.
[0458] Tumor Growth Inhibition: No TGI was reported versus control
in this study (Table 7); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0459] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 7).
[0460] Tumor Necrosis: 2/9 animals reported mild tumor necrosis;
observations were unremarkable compared with control.
IV. FK228; 5 mg/kg; IV; Q4Dx3
[0461] Animal Weights: A final mean weight of 24.+-.1 grams was
calculated at study completion (Day 29). Moderate weight loss was
reported (nadir=-15%. Day 11) which was recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 6.
[0462] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 6).
[0463] Tumor Volume: A final mean tumor volume of 2620.+-.238
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 7.
[0464] Tumor Growth Inhibition: No TGI was reported versus control
in this study (Table 7); this agent is considered inactive
according to NCI Standards (TGI<58%) at the evaluated dose,
schedule, and route of administration. In addition, activity of
this agent was found statistically insignificant (p>0.05)
compared with control using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunnett multiple comparisons
test.
[0465] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 7).
[0466] Tumor Necrosis: 2/9 animals reported mild tumor necrosis;
observations were unremarkable compared with control.
Combination Treatment Groups
TABLE-US-00011 [0467] TABLE 8 Animal Weight and Drug Toxicity
Results: BxPC-3 Combination Groups (Day 29) FINAL WEIGHT DATA
ROUTE/ (DAY 29) WEIGHT NADIR DRUGS DEATHS GROUP DOSE SCHEDULE MEAN
(G) .+-. SD % CHANGE % CHANGE DAY TOTAL DAY (#) Gemcitabine 40
mg/kg IP/Q3Dx4 25 .+-. 3 +9 -- -- 0 -- Gemcitabine 80 mg/kg
IP/Q3Dx4 22 + 4 +1 -9 11 -- -- FK228 2.5 mg/kg IV/Q4Dx3 Gemcitabine
40 mg/kg IP/Q3Dx4 24 .+-. 1 +9 -16 11 0 -- FK228 5 mg/kg IV/Q4Dx3
Gemcitabine 40 mg/kg IP/Q3Dx4 25 .+-. 1 +10 -18 11 0 -- FK228 2.5
mg/kg IV/Q4Dx3 Gemcitabine 80 mg/kg IP/Q3Dx4 26 .+-. 2 +9 -17 11 1
8 FK228 5 mg/kg IV/Q4Dx3 Gemcitabine 80 mg/kg IP/Q3Dx4 26 .+-. 1
+10 -29 11 1 8 N = 8/GRP ON DAY 1
TABLE-US-00012 TABLE 9 Tumor Volume and Efficacy Results: BxPC-3
Combination Groups (Day 29) FINAL TUMOR VOLUME ROUTE/ (DAY 29)
GROUP DOSE SCHEDULE MEAN .+-. SEM % TGI #PR/CR % TR Gemcitabine 40
mg/kg IP/Q3Dx4 1683 .+-. 426 20 0/0 -- Gemcitabine 80 mg/kg
IP/Q3Dx4 1890 + 237 9 0/0 -- FK228 2.5 mg/kg IV/Q4Dx3 Gemcitabine
40 mg/kg IP/Q3Dx4 1663 .+-. 322 21 0/0 -- FK228 5 mg/kg IV/Q4Dx3
Gemcitabine 40 mg/kg IP/Q3Dx4 1198 .+-. 234 44 0/0 -- FK228 2.5
mg/kg IV/Q4Dx3 Gemcitabine 80 mg/kg IP/Q3Dx4 1278 .+-. 286 40 0/0
-- FK228 5 mg/kg IV/Q4Dx3 Gemcitabine 80 mg/kg IP/Q3Dx4 1592 .+-.
304 24 0/0 -- N = 8/GRP ON DAY 1
I. FK228; 2.5 mg/kg; IV; Q4Dx3+Gemcitabine; 40 mg/kg; IP;
Q3Dx4)
[0468] Animal Weights: A final mean weight of 24.+-.1 grams was
calculated at study completion (Day 29). Significant weight loss
was reported (nadir=-16%. Day 11), which is increased compared to
additive loss from single agent groups; weight was fully recovered
by study completion. Mean animal weights and percent change from
Day 1 are reported in Table 8.
[0469] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 8).
[0470] Tumor Volume: A final mean tumor volume of 1663.+-.322
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 9.
[0471] Tumor Growth Inhibition: A TGI of 21% was reported versus
control in this study (Table 9); this combination is considered
inactive according to NCI Standards (TGI<58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically insignificant
(p>0.05) compared with 40 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0472] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 9).
[0473] Tumor Necrosis: 1/9 animals reported mild tumor necrosis;
observations were unremarkable compared with control.
II. FK228; 5 mg/kg; IV; Q4Dx3+Gemcitabine; 40 mg/kg; IP; Q3Dx4
[0474] Animal Weights: A final mean weight of 25.+-.1 grams was
calculated at study completion (Day 29). Significant weight loss
was reported (nadir=-1 8%, Day 11), which is comparable to additive
loss from single agent groups; weight was fully recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 8.
[0475] Moribundity/Mortality: 0/9 animals reported drug-related
toxicity or deaths (Table 8).
[0476] Tumor Volume: A final mean tumor volume of 1198.+-.234
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 9.
[0477] Tumor Growth Inhibition: A TGI of 44% was reported versus
control in this study (Table 9); this combination is considered
inactive according to NCI Standards (TGI<58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically insignificant
(p>0.05) compared with 40 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0478] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 9).
[0479] Tumor Necrosis: 0/9 animals reported tumor necrosis,
observations were unremarkable compared with control.
III. FK228; 2.5 mg/kg; IV; Q4Dx3+Gemcitabine; 80 mg/kg; IP;
Q3Dx4
[0480] Animal Weights: A final mean weight of 26.+-.2 grams was
calculated at study completion (Day 29). Significant weight loss
was reported (nadir=-17%. Day 11), which is increased compared to
additive loss from single agent groups; weight was fully recovered
by study completion. Mean animal weights and percent change from
Day 1 are reported in Table 8.
[0481] Moribundity/Mortality: 1/9 animals reported a drug-related
death on Day 8 (Table 8).
[0482] Tumor Volume: A final mean tumor volume of 1278.+-.286
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 9.
[0483] Tumor Growth Inhibition: A TGI of 40% was reported versus
control in this study (Table 9); this combination is considered
inactive according to NCI Standards (TGI<58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically insignificant
(p>0.05) compared with 80 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0484] Partial/Complete Tumor Response: 0/9 animals reported
partial or complete tumor responses (Table 9).
[0485] Tumor Necrosis: 3/9 animals reported mild tumor necrosis;
observations were unremarkable compared with control.
IV. FK228; 5 mg/kg; IV; Q4Dx3+Gemcitabine; 80 mg/kg; IP; Q3Dx4
[0486] Animal Weights: A final mean weight of 26.+-.1 grams was
calculated at study completion (Day 29). Significant weight loss
was reported (nadir=-29%, Day 11), which is comparable to additive
loss from single agent groups; weight was fully recovered by study
completion. Mean animal weights and percent change from Day 1 are
reported in Table 8.
[0487] Moribundity/Mortality: 1/9 animals reported a drug-related
death on Day 8 (Table 8).
[0488] Tumor Volume: A final mean tumor volume of 1592.+-.304
mm.sup.3 was calculated at study completion (Day 29). Mean tumor
volumes beginning Day 1 are reported in Table 9.
[0489] Tumor Growth Inhibition: A TGI of 24% was reported versus
control in this study (Table 9); this combination is considered
inactive according to NCI Standards (TGI<58%) at the evaluated
doses, schedules, and routes of administration. In addition,
activity of this combination was found statistically insignificant
(p>0.05) compared with 80 mg/kg gemcitabine alone using a
two-tailed One-Way Analysis of Variance (ANOVA) followed by the
Dunnett multiple comparisons test.
[0490] Partial/Complete Tumor Response. 0/9 animals reported
partial or complete tumor responses (Table 9).
[0491] Tumor Necrosis. 1/9 animals reported slight tumor necrosis;
observations were unremarkable compared with control.
Discussion
[0492] In the PANC-1 study, single agent gemcitabine resulted in
slight, dose-independent weight loss, which was recovered by study
completion. Moderate tumor growth inhibition was reported with 40
or 80 mg/kg gemcitabine; however, calculated TGI values at the
evaluated doses and schedule were less than 58% and considered
inactive in this model according to NCI standards. In addition,
single activity of gemcitabine did not reach statistical
significance in this model (p>0.05). However, one partial
response was reported in the 40 mg/kg group with a 62% tumor
regression.
[0493] Single agent FK228 treatment resulted in moderate,
dose-dependent weight loss, which was recovered by study
completion. Moderate tumor growth inhibition was reported with 2.5
or 5 mg/kg FK228; however, calculated TGI values at the evaluated
doses and schedule were less than 58% and considered inactive in
this model according to NCI standards. In addition, single activity
of FK228 did not reach statistical significance in this model
(p>0.05).
[0494] Groups co-dosed with FK228 and gemcitabine reported
significant (>15%) weight loss, which was recovered in all
groups by study completion. In addition, drug-related deaths were
reported in the high-dose gemcitabine combination groups.
Impressive, dose-dependent tumor growth inhibition was reported in
these combination groups with TGI values at the evaluated doses and
schedules greater than 58%, thus these regimens were considered
active in this model according to NCI standards; activity of these
combinations reach statistical significance in this model
(p<0.001). In addition, partial responses were reported in the
high-dose FK228 combination groups, further demonstrating activity
of this agent in combination with gemcitabine towards this model.
Finally, tumor ulceration and necrosis, common in PANC-1 was
decreased or absent in animals treated with these combinations,
demonstrating an additional effect of these agents in this
model.
[0495] In the BxPC-3 study, 80 mg/kg gemcitabine resulted in
slight, weight loss, which was recovered by study completion.
Moderate tumor growth inhibition was reported with 40 or 80 mg/kg
gemcitabine; however, calculated TGI values at the evaluated doses
and schedule were less than 58% and considered inactive in this
model according to NCI standards. In addition, single activity of
gemcitabine did not reach statistical significance in this model
(p>0.05).
[0496] Single agent FK228 treatment resulted in moderate,
dose-dependent weight loss, which was recovered by study
completion. No tumor growth inhibition was reported with 2.5 or 5
mg/kg FK228 and at the evaluated doses and schedule was considered
inactive in this model according to NCI standards.
[0497] Groups co-dosed with FK228 and gemcitabine reported
significant (>15%) weight loss, which was recovered in all
groups by study completion. In addition, drug-related deaths were
reported in the high-dose gemcitabine combination groups. Moderate
tumor growth inhibition was reported with the evaluated combination
groups; however, calculated TGI values at the evaluated doses and
schedule except one group (5 mg/kg FK228/40 mg/kg Gem) were less
than 58% and thus considered inactive in this model according to
NCI standards. In addition, activity of these combinations did not
reach statistical significance in this model (p>0.05).
[0498] In these studies, single agent and combination toxicity
occurred in these studies with 10-20% mortality with FK228 in
combination with 80 mg/kg gemcitabine. Dose-dependent weight loss
was also associated with FK228 treatment, although this effect was
transient and weight regained in all groups.
[0499] As a single agent, FK228 demonstrated some activity towards
the ras-transformed PANC-1 tumor model but was inactive in the wild
type BxPC-3 line.
[0500] In combination with gemcitabine, FK228 demonstrated
impressive, statistically significant (p<0.001) activity towards
PANC-1 but not BxPC-3, suggesting agent specificity for the
ras-transformed line.
[0501] Overall, FK228 demonstrated significant combination
antitumor activity with gemcitabine towards the ras-transformed
PANC-1 human pancreas tumor model.
REFERENCES
[0502] 1. Loor R. et al. Use of pancreas-specific antigen in
immunodiagnosis of pancreatic cancer. Clin. Lab. Med. 2: 567-578,
1982. [0503] 2. Lan M S, et al. Polypeptide core of a human
pancreatic tumor mucin antigen. Cancer Res. 50: 2997-3001, 1990.
[0504] 3. Lieber M, et al. Establishment of a continuous tumor-cell
line (panc-1) from a human carcinoma of the exocrine pancreas. Int.
J. Cancer 15:741-747, 1975. [0505] 4. Wu M C. et al. Mechanism of
sensitivity of cultured pancreatic carcinoma to asparaginase. Int.
J. Cancer 22: 728-733, 1978. [0506] 5. Yasui. Nobutaka. et al.
Tumor growth and metastasis of human colorectal cancer cell lines
in SCID mice resemble clinical metastatic behaviors. Invasion
letastasis 17: 259-69, 1997. [0507] 6. Goldin A. et al. Current
results of the screening program at the Division of Cancer
Treatment. National Cancer Institute. Eur J Cancer. 17:129-42,
1981. [0508] 7. Corbett T H et al. In vivo methods for screening
and preclinical testing. In: Teicher B, ed., Anticancer Drug
Development Guide. Totowa, N.J.: Humana. 2004: 99-123.
Example 4
Assessment of Romidepsin's Ability to Inhibit K-Ras-mediated
Transformation
[0509] The present Example is designed to reveal whether
romidepsin, in addition to inhibiting the transformed morphology
and growth of H-Ras-transformed rodent fibroblasts (e.g. NIH 3T3,
C3H10T1/2), can inhibit K-Ras-mediated transformation.
Model Cell Systems:
[0510] The following cell systems will be tested:
[0511] 1. NIH 3T3 mouse fibroblasts stably transformed by activated
forms of human H-Ras. N-Ras, or K-Ras4B; NIH 3T3 cells stably
transformed by activated B-Raf or Neu/HER2 can be used to determine
specificity for Ras.
[0512] 2. Rat RIE-1 intestinal or ROSE ovarian epithelial cells
stably transformed by activated forms of H-Ras. N-Ras or
K-Ras4B.
[0513] 3. Human embryonic kidney epithelial cells (HEK)
immortalized by telomerase (hTERT) and SV40 T/t antigen expression,
then stably transformed with activated H-Ras or K-Ras4B.
[0514] 4. Human capan-1 pancreatic or SW480 colon carcinoma cell
lines stably infected with the empty pSUPER-retro retrovirus vector
(Oligoengine) or encoding shRNA for silencing expression of
endogenous activated K-Ras(G12V).
Growth Assays:
[0515] The following growth assays will be used for model cell
systems 1-4 above:
[0516] 1. Anchorage-dependent growth on plastic and evaluation of
selectivity for tranformed versus untransformed cells--growth rate,
saturation density; MTT viability assay; morphologic reversion.
[0517] 2. Anchorage-independent growth of Ras-transformed
cells--soft agar colony formation.
Example 5
Romidepsin Inhibits Ras-Expressing Tumors
[0518] The present Example demonstrates that romidepsin inhibits
proliferation of Ras-expressing tumor cells, and also transforms
the morphology of the cells.
[0519] A series of transformed NIH-3T3 cell lines were generated
and a proliferation assay was performed in the presence of 2, 3, or
4 nM romidepsin.
[0520] As can be seen, in a 4-day proliferation assay, 2 nM
romidepsin selectively inhibited the proliferation of transformed
(H, K, N-Ras and the rat Her-2/NeuT) vs untransformed cells. Also,
romidepsin was not potent on B-Raf600E cells in this study. One
possible interpretation of these findings is that romidepsin does
not inhibit proliferation of these non-Ras-expressing transformed
cells. Another possible interpretation is that any effect on these
cells is obfuscated by other defects of the cells (e.g., slow
growth rate, etc.)
[0521] Regardless, the data presented in this Example clearly
demonstrate potent inhibition of cell proliferation and
transformation of Ras-expressing cells by romidepsin.
EQUIVALENTS
[0522] The foregoing has been a description of certain non-limiting
preferred embodiments of the invention. Those skilled in the art
will recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Those of ordinary skill in the art
will appreciate that various changes and modifications to this
description may be made without departing from the spirit or scope
of the present invention, as defined in the following claims.
[0523] To give but a few examples, in the claims articles such as
"a", "an", and "the" may mean one or more than one unless indicated
to the contrary or otherwise evident from the context. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention includes
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
The invention also includes embodiments in which more than one, or
all of the group members are present in, employed in, or otherwise
relevant to a given product or process. Furthermore, it is to be
understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, descriptive terms, etc., from one or more of the
claims or from relevant portions of the description is introduced
into another claim. For example, any claim that is dependent on
another claim can be modified to include one or more limitations
found in any other claim that is dependent on the same base
claim.
[0524] Furthermore, where the claims recite a composition, it is to
be understood that methods of using the composition for any of the
purposes disclosed herein are included, and methods of making the
composition according to any of the methods of making disclosed
herein or other methods known in the art are included, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. In addition, the invention encompasses compositions
made according to any of the methods for preparing compositions
disclosed herein.
[0525] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It is also noted that the term "comprising" is intended
to be open and permits the inclusion of additional elements or
steps. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, steps, etc., certain
embodiments of the invention or aspects of the invention consist,
or consist essentially of, such elements, features, steps, etc. For
purposes of simplicity those embodiments have not been specifically
set forth in haec verba herein. Thus for each embodiment of the
invention that comprises one or more elements, features, steps,
etc., the invention also provides embodiments that consist or
consist essentially of those elements, features, steps, etc.
[0526] Where ranges are given, endpoints are included unless
otherwise indicated. Furthermore, it is to be understood that
unless otherwise indicated or otherwise evident from the context
and/or the understanding of one of ordinary skill in the art,
values that are expressed as ranges can assume any specific value
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise. It is also to be understood
that unless otherwise indicated or otherwise evident from the
context and/or the understanding of one of ordinary skill in the
art, values expressed as ranges can assume any subrange within the
given range, wherein the endpoints of the subrange are expressed to
the same degree of accuracy as the tenth of the unit of the lower
limit of the range.
[0527] In addition, it is to be understood that any particular
embodiment of the present invention may be explicitly excluded from
any one or more of the claims. Any embodiment, element, feature,
application, or aspect of the compositions and/or methods of the
invention can be excluded from any one or more claims. For example,
in certain embodiments of the invention the biologically active
agent is not an antiproliferative agent. For purposes of brevity,
all of the embodiments in which one or more elements, features,
purposes, or aspects is excluded are not set forth explicitly
herein.
* * * * *