U.S. patent application number 16/776139 was filed with the patent office on 2020-05-28 for dosing of cabozantinib formulations.
The applicant listed for this patent is Exelixis, Inc.. Invention is credited to Dana T. Aftab, Steven Lacy, Sriram Naganathan, Wei Xu.
Application Number | 20200163954 16/776139 |
Document ID | / |
Family ID | 52811227 |
Filed Date | 2020-05-28 |
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United States Patent
Application |
20200163954 |
Kind Code |
A1 |
Aftab; Dana T. ; et
al. |
May 28, 2020 |
Dosing of Cabozantinib Formulations
Abstract
The invention relates to administration of various
pharmaceutical formulations of
N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N'-(4-fluorophenyl)cyc-
lopropane-1,1-dicarboxamide, (cabozantinib) a c-Met inhibitor, and
its metabolites, to achieve desirable pharmacokinetic and
pharmacodynamic effects.
Inventors: |
Aftab; Dana T.; (San Rafael,
CA) ; Naganathan; Sriram; (San Jose, CA) ; Xu;
Wei; (Danville, CA) ; Lacy; Steven; (San
Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Exelixis, Inc. |
Alameda |
CA |
US |
|
|
Family ID: |
52811227 |
Appl. No.: |
16/776139 |
Filed: |
January 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16190869 |
Nov 14, 2018 |
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16776139 |
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15126767 |
Sep 16, 2016 |
10159666 |
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PCT/US2015/021072 |
Mar 17, 2015 |
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16190869 |
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61954352 |
Mar 17, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 35/00 20180101; A61K 31/47 20130101; A61P 1/16 20180101; A61P
15/00 20180101; A61P 11/00 20180101; A61P 1/04 20180101; A61P 17/00
20180101; A61K 9/0053 20130101; A61P 13/12 20180101; A61P 1/18
20180101; A61P 35/02 20180101; A61K 9/2054 20130101 |
International
Class: |
A61K 31/47 20060101
A61K031/47; A61K 9/00 20060101 A61K009/00; A61K 9/20 20060101
A61K009/20 |
Claims
1. A pharmaceutical formulation comprising a physiologically
effective amount of cabozantinib, wherein oral administration of
said pharmaceutical formulation to a selected human subject group
produces in said selected human subject group: an average
cabozantinib plasma area under the curve (average AUC) of at least
60,000 ngh/mL per each 140 mg dosage of cabozantinib delivered; an
average maximum cabozantinib blood plasma concentration (average
C.sub.max) of at least 1000 ng/mL per each 140 mg dosage of
cabozantinib delivered; and a ratio of AUC (cabozantinib) to the
AUC of the sum of cabozantinib plus measured cabozantinib
metabolites: AUC (Cabozantinib)/AUC (Cabozantinib+Measured
Metabolites) of at least 0.25; wherein the AUC is measured from
time zero to the time of last measurable concentration.
2. A pharmaceutical formulation comprising a physiologically
effective amount of cabozantinib, wherein oral administration of
said pharmaceutical formulation to a selected human subject group
produces in said selected human subject group: a steady state area
under the curve for cabozantinib (average AUC) of at least 35,000
ngh/mL for a formulation comprising 100 mg of cabozantinib
delivered once daily; an average maximum cabozantinib blood plasma
concentration (average C.sub.max) of at least 2400 ng/mL per each
100 mg dosage of cabozantinib delivered; an average minimum
cabozantinib blood plasma concentration (average C.sub.max) of at
least 1100 ng/mL per each 100 mg dosage of cabozantinib delivered;
and a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites: AUC
(Cabozantinib)/AUC (Cabozantinib+Measured Metabolites) of at least
0.35; wherein the AUC is measured on day 22.
3. The pharmaceutical formulation of claims 1-2, wherein the
measured metabolites are selected from the group consisting of:
##STR00064## ##STR00065## ##STR00066## wherein GA is a glucuronic
acid moiety,
4. The pharmaceutical formulation of claims 1-2, wherein the
measured metabolites comprise one or more of: ##STR00067##
5. A method of treating cancer in a human patient, comprising
orally administering to said patient a pharmaceutical formulation
comprising a physiologically effective amount of cabozantinib at
such a rate to achieve: an average cabozantinib plasma area under
the curve (average AUC) of at least 60,000 ngh/mL per each 140 mg
dosage of cabozantinib delivered; an average maximum cabozantinib
blood plasma concentration (average C.sub.max) of at least 1000
ng/mL per each 140 mg dosage of cabozantinib delivered; and a ratio
of AUC (cabozantinib) to the AUC of the sum of cabozantinib plus
measured cabozantinib metabolites: AUC (Cabozantinib)/AUC
(Cabozantinib+Measured Metabolites) of at least 0.25; wherein the
AUC is measured from time zero to the time of last measurable
concentration.
6. A method of treating cancer in a human patient, comprising
orally administering to said patient a pharmaceutical formulation
comprising a physiologically effective amount of cabozantinib at
such a rate to achieve: a steady state area under the curve for
cabozantinib (average AUC) of at least 35,000 ngh/mL for a
formulation comprising 100 mg of cabozantinib delivered once daily;
an average maximum cabozantinib blood plasma concentration (average
C.sub.max) of at least 2400 ng/mL per each 100 mg dosage of
cabozantinib delivered; an average minimum cabozantinib blood
plasma concentration (average C.sub.max) of at least 1100 ng/mL per
each 100 mg dosage of cabozantinib delivered; and a ratio of AUC
(cabozantinib) to the AUC of the sum of cabozantinib plus measured
cabozantinib metabolites: AUC (Cabozantinib)/AUC
(Cabozantinib+Measured Metabolites) of at least 0.35; wherein the
AUC is measured on day 22.
7. The method of claims 5-6, wherein the measured metabolites are
selected from the group consisting of: ##STR00068## ##STR00069##
##STR00070## wherein GA is a glucuronic acid moiety,
8. The method of wherein the measured metabolites comprise one or
more of: ##STR00071##
9. The method of claims 5-8, wherein the cancer is selected from
ovarian cancer, lung cancer, medullary thyroid cancer, liver
cancer, gastrointestinal cancer, pancreatic cancer, bone cancer,
hematologic cancer, skin cancer, kidney cancer, breast cancer,
colon cancer, and fallopian tube cancer.
Description
PRIORITY CLAIM
[0001] This application claims priority to U.S. Application Ser.
No. 61/954,352, filed Mar. 17, 2014. The entire contents of the
aforementioned application are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The invention relates to administration of various
pharmaceutical formulations of
N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N'-(4-fluorophenyl)cyc-
lopropane-1,1-dicarboxamide, (cabozantinib) a c-Met inhibitor, and
its metabolites, to achieve desirable pharmacokinetic and
pharmacodynamic effects.
BACKGROUND
[0003] Traditionally, dramatic improvements in the treatment of
cancer are associated with identification of therapeutic agents
acting through novel mechanisms. One mechanism that can be
exploited in cancer treatment is the modulation of protein kinase
activity because signal transduction through protein kinase
activation is responsible for many of the characteristics of tumor
cells. Protein kinase signal transduction is of particular
relevance in, for example, thyroid, gastric, head and neck, lung,
breast, prostate, and colorectal cancers, as well as in the growth
and proliferation of brain tumor cells.
[0004] Protein kinases can be categorized as receptor type or
non-receptor type. Receptor-type tyrosine kinases are comprised of
a large number of transmembrane receptors with diverse biological
activity. For a detailed discussion of the receptor-type tyrosine
kinases, see Plowman et al., DN&P 7(6): 334-339, 1994. Since
protein kinases and their ligands play critical roles in various
cellular activities, deregulation of protein kinase enzymatic
activity can lead to altered cellular properties, such as
uncontrolled cell growth associated with cancer. In addition to
oncological indications, altered kinase signaling is implicated in
numerous other pathological diseases, including, for example,
immunological disorders, cardiovascular diseases, inflammatory
diseases, and degenerative diseases. Therefore, protein kinases are
attractive targets for small molecule drug discovery. Particularly
attractive targets for small-molecule modulation with respect to
antiangiogenic and antiproliferative activity include receptor type
tyrosine kinases Ret, c-Met, and VEGFR2.
[0005] The kinase c-Met is the prototypic member of a subfamily of
heterodimeric receptor tyrosine kinases (RTKs) which include Met,
Ron, and Sea. The endogenous ligand for c-Met is the hepatocyte
growth factor (HGF), a potent inducer of angiogenesis. Binding for
c-Met is the hepatocyte growth factor (HGF), a potent inducer of
angiogenesis. Binding of HGF to c-Met induces activation of the
receptor via autophosphorylation resulting in an increase of
receptor dependent signaling, which promotes cell growth and
invasion. Anti-HGF antibodies or HGF antagonists have been shown to
inhibit tumor metastasis in vivo (See Maulik et al, Cytokine &
Growth Factor Reviews, 2002, 13, 41-59). c-Met, VEGFR2, and/or Ret
overexpression has been demonstrated on a wide variety of tumor
types, including breast, colon, renal, lung, squamous cell myeloid
leukemia, hemangiomas, melanomas, and astrocytic tumor (which
includes glioblastoma, giant cell glioblastoma, gliosarcoma, and
glioblastoma with oligodendroglial components). The Ret protein is
a transmembrane receptor with tyrosine kinase activity. Ret is
mutated in most familial forms of medullary thyroid cancer. These
mutations activate the kinase function of Ret and convert it into
an oncogenic form.
[0006] Accordingly, small-molecule compounds that specifically
inhibit, regulate, and/or modulate the signal transduction of
kinases, particularly including Ret, c-Met, and VEGFR2 described
above, are particularly desirable as a means to treat or prevent
disease states associated with abnormal cell proliferation and
angiogenesis. One such small-molecule is XL 184, known variously as
N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N'-(4-fluorophenyl)cyc-
lopropane-1,1-dicarboxamide, cabozantinib, and as COMETRIQ.TM. (the
S-malate salt of cabozantinib). Cabozantinib has the chemical
structure:
##STR00001##
[0007] In November, 2012, cabozantinib achieved regulatory approval
in the United States for the treatment of progressive metastatic
medullary thyroid cancer. Other clinical trials of cabozantinib are
ongoing.
[0008] WO 2005/030140, incorporated herein by reference, describes
the synthesis of cabozantinib (Example 48) and also discloses the
therapeutic activity of this molecule to inhibit, regulate, and/or
modulate the signal transduction of kinases, (Assays, Table 4,
entry 289). Example 48 is on paragraph [0353] in WO
2005/030140.
[0009] A need remains for identifying pharmaceutical formulations
and dosing schedules for cabozantinib for the treatment of
cancer.
SUMMARY OF THE INVENTION
[0010] These and other needs are met by the present invention,
which is directed to various of pharmaceutical formulations of
N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N'-(4-fluorophenyl)cyc-
lopropane-1,1-dicarboxamide, (cabozantinib) a c-Met inhibitor, and
its metabolites, to achieve desired pharmacokinetic and
pharmacodynamic effects.
[0011] In one aspect, the invention relates to a pharmaceutical
formulation comprising a physiologically effective amount of
cabozantinib, wherein oral administration of said pharmaceutical
formulation to a selected human subject group produces in said
selected human subject group:
[0012] an average cabozantinib plasma area under the curve (average
AUC) of at least 60,000 ngh/mL per each 140 mg dosage of
cabozantinib delivered;
[0013] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1000 ng/mL per each 140 mg dosage
of cabozantinib delivered; and
[0014] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.25;
[0015] wherein the AUC is measured from time zero to the time of
last measurable concentration.
[0016] In another aspect, the invention relates to a pharmaceutical
formulation comprising a physiologically effective amount of
cabozantinib, wherein oral administration of said pharmaceutical
formulation to a selected human subject group produces in said
selected human subject group:
[0017] a steady state area under the curve for cabozantinib
(average AUC) of at least 35,000 ngh/mL for a formulation
comprising 100 mg of cabozantinib delivered once daily;
[0018] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 2400 ng/mL per each 100 mg dosage
of cabozantinib delivered;
[0019] an average minimum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1100 ng/mL per each 100 mg dosage
of cabozantinib delivered; and
[0020] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.35;
[0021] wherein the AUC is measured on day 22.
[0022] In a further aspect, the invention relates to a method of
treating cancer in a human patient, comprising orally administering
to said patient a pharmaceutical formulation comprising a
physiologically effective amount of cabozantinib at such a rate to
achieve:
[0023] an average cabozantinib plasma area under the curve (average
AUC) of at least 60,000 ngh/mL per each 140 mg dosage of
cabozantinib delivered;
[0024] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1000 ng/mL per each 140 mg dosage
of cabozantinib delivered; and
[0025] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.25;
[0026] wherein the AUC is measured from time zero to the time of
last measurable concentration.
[0027] In another aspect, the invention relates to a method of
treating cancer in a human patient, comprising orally administering
to said patient a pharmaceutical formulation comprising a
physiologically effective amount of cabozantinib at such a rate to
achieve:
[0028] a steady state area under the curve for cabozantinib
(average AUC) of at least 35,000 ngh/mL for a formulation
comprising 100 mg of cabozantinib delivered once daily;
[0029] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 2400 ng/mL per each 100 mg dosage
of cabozantinib delivered;
[0030] an average minimum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1100 ng/mL per each 100 mg dosage
of cabozantinib delivered; and
[0031] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.35;
[0032] wherein the AUC is measured on day 22.
[0033] In these aspects, "metabolite" refers to a compound of
formula Ia
##STR00002##
having one or more of the following attributes: [0034] a) one of
R.sub.1 or R.sub.2 is H, SO.sub.3H, or a glucuronic acid moiety,
and the other is Me; [0035] b) R.sub.3 is OH or OSO.sub.3H; [0036]
c) R.sub.4 is O, provided that when R.sub.4 is O.sup.-, N is
N.sup.+; [0037] d) R.sub.5 is OH, or OSO.sub.3H; and [0038] e)
R.sub.6 is OH or OSO.sub.3H.
[0039] "Metabolite" also refers to a compound of a compound of
formula Ib
##STR00003##
wherein: [0040] a) R.sub.1 or R.sub.2 are Me; or one of R.sub.1 or
R.sub.2 is H, SO.sub.3H, or a glucuronic acid moiety, and the other
is Me; [0041] b) R.sub.3 is H, OH, or OSO.sub.3H; [0042] c) R.sub.4
is absent or is O.sup.-, provided that when R.sub.4 is O.sup.-, N
is N.sup.+; and [0043] d) R.sub.6 is H or Me.
[0044] "Metabolite" also refers to a compound of a compound of
formula Ic
##STR00004##
wherein: [0045] a) R.sub.5 is OH or OSO.sub.3H; and [0046] b)
R.sub.6 is OH or OSO.sub.3H; and [0047] c) R.sub.7 is H, SO.sub.3H,
or a glucuronic acid moiety.
[0048] Specifically the metabolites include:
##STR00005## ##STR00006## ##STR00007##
wherein GA is a glucuronic acid moiety such as in, for example,
##STR00008##
DETAILED DESCRIPTION OF THE INVENTION
[0049] The metabolites described herein may be referred to
hereinafter as "human metabolites." Human metabolites of
cabozantinib include metabolites of cabozantinib that were formed
in the bodies of human subjects after ingestion or application of
cabozantinib according to clinical protocols regarding dosing and
monitoring, including those described herein. In various
embodiments, the term encompasses molecular species formed in vivo,
whether or not the species is even detected or analyzed in a
particular trial. It is also contemplated that some metabolites are
unique to particular individuals, reflecting different genetic
make-up and the presence and activity of various enzymes, including
cytochrome P450 and UGT enzymes which are involved in metabolism.
Thus, human metabolites cover all such metabolites formed in the
human body.
[0050] Some human metabolites are depicted in Scheme 1. These human
metabolites were identified during clinical studies of
cabozantinib, which appears as compound I in Scheme 1, by metabolic
profiling, particularly from human plasma, urine, and feces.
##STR00009## ##STR00010##
[0051] The cabozantinib metabolites described herein, including
those depicted in Scheme 1, are isolated from body tissues and
fluids, and/or are prepared synthetically according to methods
available to the skilled artisan. A variety of separation processes
can be carried out on tissue and fluid samples to provide samples
for further analysis, such as nuclear magnetic resonance, gas
chromatography (GC), liquid chromatography (LC), and mass
spectrometry. In such samples, the metabolites are contained in
compositions that are essentially lacking in the presence of any of
the other metabolites. The presence of the metabolites can be
quantified by physical methods, such as the measurement of nuclear
decay from radioactive isotopes, measurement of index of
refraction, flame ionization, ionization and deflection in magnetic
fields, ultraviolet (UV absorption), and the like.
[0052] The human metabolites can be provided in crystalline or
solution forms that have considerable degrees of purity. Organic
synthetic routes are available for preparing the compounds in
relative pure form, for example, in purities of 80 percent or
greater, 90 percent or greater, 95 percent or greater, or 99
percent or greater. Recrystallization and other purification
methods can be carried out to provide compounds that are
essentially 100 percent pure. Such synthetic methods and
purification techniques are known in the art.
[0053] The metabolites can be provided in substantially pure form.
"Substantially pure" means that metabolites are pure enough for FDA
approval and contain essentially no contaminants or other
materials. Alternatively, "substantially pure" means a level of
impurity that does not adversely or unacceptably affect the
properties of the compounds with respect to safety, effectiveness,
stability, and other desirable properties.
[0054] The isolated metabolites depicted in Scheme 1 include:
##STR00011## ##STR00012## ##STR00013##
wherein GA is a glucuronic acid.
[0055] More particularly, the metabolites include:
##STR00014## ##STR00015##
[0056] In particular, the isolated metabolite is
##STR00016##
or a pharmaceutically acceptable salt thereof.
[0057] In particular, the isolated metabolite is
##STR00017##
or a pharmaceutically acceptable salt thereof.
[0058] In particular, the isolated metabolite is
##STR00018##
or a pharmaceutically acceptable salt thereof.
[0059] In particular, the isolated metabolite is
##STR00019##
or a pharmaceutically acceptable salt thereof.
[0060] In particular, the isolated metabolite is
##STR00020##
or a pharmaceutically acceptable salt thereof.
[0061] In particular, the isolated metabolite is
##STR00021##
or a pharmaceutically acceptable salt thereof.
[0062] In particular, the isolated metabolite is
##STR00022##
or a pharmaceutically acceptable salt thereof.
[0063] In particular, the isolated metabolite is
##STR00023##
or a pharmaceutically acceptable salt thereof.
[0064] The pharmaceutical formulations contemplated in the
invention can be selected from one of the following:
TABLE-US-00001 Ingredient (% w/w) Cabozantinib 31.68
Microcrystalline Cellulose 38.85 Lactose anhydrous 19.42
Hydroxypropyl Cellulose 3.00 Croscarmellose Sodium 3.00 Total
Intra-granular 95.95 Silicon dioxide, Colloidal 0.30 Croscarmellose
Sodium 3.00 Magnesium Stearate 0.75 Total 100.00 Cabozantinib
25.0-33.3 Microcrystalline Cellulose q.s Hydroxypropyl Cellulose 3
Poloxamer 0-3 Croscarmellose Sodium 6.0 Colloidal Silicon Dioxide
0.5 Magnesium Stearate 0.5-1.0 Total 100 Theoretical Quantity
Ingredient (mg/unit dose) Cabozantinib 100.0 Microcrystalline
Cellulose PH-102 155.4 Lactose Anhydrous 60M 77.7 Hydroxypropyl
Cellulose, EXF 12.0 Croscarmellose Sodium 24 Colloidal Silicon
Dioxide 1.2 Magnesium Stearate (Non-Bovine) 3.0 Opadry Yellow 16.0
Total 416 Weight/Weight Component Percent Cabozantinib 25-29
Microcrystalline Cellulose q.s. Lactose Anhydrous 40-44
Hydroxypropyl Cellulose 2-4 Croscarmellose Sodium 2-8 Colloidal
Silicon Dioxide 0.1-0.4 Magnesium Stearate 0.7-0.9 Total 100
Ingredient % w/w Cabozantinib 12.67 MCC 51.52 Lactose 25.76
Hydroxypropyl cellulose 3.0 Croscarmellose Sodium 6.0 Colloidal
Silicon Dioxide 0.3 Magnesium Stearate 0.75 Total 100 Ingredient
mg/unit dose Cabozantinib 25 Silicified Microcrystalline Cellulose
196.75 Croscarmellose sodium 12.5 Sodium starch glycolate 12.5
Fumed Silica 0.75 Stearic acid 2.5 Total Fill Weight 250
Cabozantinib 100 Silicified Microcrystalline Cellulose 75.40
Croscarmellose sodium 10.00 Sodium Starch Glycolate 10.00 Fumed
silica 0.6 Stearic Acid 4.0 Total Fill Weight 200 mg/unit dose
Ingredient 50 mg Cabozantinib 63.35 Microcrystalline Cellulose
95.39 Croscarmellose sodium 9.05 Sodium starch glycolate 9.05 Fumed
Silica 0.54 Stearic acid 3.62 Total Fill Weight 181.00 mg/unit dose
Ingredient 60 mg Cabozantinib 73.95 Microcrystalline Cellulose
114.36 Croscarmellose sodium 10.85 Sodium starch glycolate 10.85
Fumed Silica 0.65 Stearic acid 4.34 Total Fill Weight 217.00
Ingredient % w/w Cabozantinib 31.7 Microcrystalline Cellulose 38.9
(Avicel PH-102) Lactose Anhydrous (60M) 19.4 Hydroxypropyl
Cellulose (EXF) 3.0 Croscarmellose Sodium (Ac-Di-Sol) 6.0 Colloidal
Silicon Dioxide, 0.3 Magnesium Stearate 0.75 Opadry Yellow Film
Coating which 4.00 includes: HPMC 2910 /Hypromellose 6 cp Titanium
dioxide Triacetin Iron Oxide Yellow
[0065] The invention also relates to a pharmaceutical formulation
comprising one or more cabozantinib isolated metabolites that
inhibits CMet or that have other beneficial attributes. The
isolated metabolite is selected from:
##STR00024## ##STR00025##
or a pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier.
[0066] The pharmaceutical formulations can be used to treat cancer
when administered as described herein. In typical embodiments,
administration is oral, once daily, but can be adjusted as required
by the situation. Tablets or capsules containing different amounts
of cabozantinib can be combined to achieve the desired dose. For
example, a 140 mg dose of cabozantinib would require administration
of one tablet or capsule containing 80 mg cabozantinib and three
tablets or capsules containing 20 mg of cabozantinib. A 140 mg dose
of cabozantinib would require administration of one tablet or
capsule containing 80 mg cabozantinib and one tablet or capsule
containing 20 mg of cabozantinib.
[0067] Thus, in a further aspect, the invention relates to a method
of treating cancer in a human patient, comprising orally
administering to said patient a pharmaceutical formulation
comprising a physiologically effective amount of cabozantinib at
such a rate to achieve:
[0068] an average cabozantinib plasma area under the curve (average
AUC) of at least 60,000 ngh/mL per each 140 mg dosage of
cabozantinib delivered;
[0069] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1000 ng/mL per each 140 mg dosage
of cabozantinib delivered; and
[0070] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.25;
[0071] wherein the AUC is measured from time zero to the time of
last measurable concentration.
[0072] In another aspect, the invention relates to a method of
treating cancer in a human patient, comprising orally administering
to said patient a pharmaceutical formulation comprising a
physiologically effective amount of cabozantinib at such a rate to
achieve:
[0073] a steady state area under the curve for cabozantinib
(average AUC) of at least 35,000 ngh/mL for a formulation
comprising 100 mg of cabozantinib delivered once daily;
[0074] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 2400 ng/mL per each 100 mg dosage
of cabozantinib delivered;
[0075] an average minimum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1100 ng/mL per each 100 mg dosage
of cabozantinib delivered; and
[0076] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.35;
[0077] wherein the AUC is measured on day 22.
[0078] "Cancer" includes tumor types such as tumor types including
breast, colon, renal, lung, squamous cell myeloid leukemia,
hemangiomas, melanomas, astrocytomas, and glioblastomas as well as
other cellular-proliferative disease states, including but not
limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,
rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma,
lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell,
undifferentiated small cell, undifferentiated large cell,
adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hanlartoma,
inesothelioma; Gastrointestinal: esophagus (squamous cell
carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach
(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal
adenocarcinoma, insulinorna, glucagonoma, gastrinoma, carcinoid
tumors, vipoma), small bowel (adenocarcinorna, lymphoma, carcinoid
tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel (adenocarcinoma, tubular
adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary
tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],
lymphoma, leukemia, renal cell carcinoma), bladder and urethra
(squamous cell carcinoma, transitional cell carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma, small cell
carcinoma of the prostate), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;
Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant
fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant
lymphoma (reticulum cell sarcoma), malignant giant cell tumor
chordoma, osteochronfroma (osteocartilaginous exostoses), benign
chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and
giant cell tumors; Nervous system: skull (osteoma, hemangioma,
granuloma, xanthoma, osteitis defornians), meninges (meningioma,
meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,
glioma, ependymoma, germinoma [pinealoma], glioblastorna multiform,
oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),
spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological: uterus (endometrial carcinoma), cervix (cervical
carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian
carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma,
unclassified carcinoma], granulosa-thecal cell tumors,
Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (squamous cell carcinoma, intraepithelial carcinoma,
adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell
carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal
rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood
(myeloid leukemia [acute and chronic], acute lymphoblastic
leukemia, chronic lymphocytic leukemia, myeloproliferative
diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's
disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin:
malignant melanoma, basal cell carcinoma, squamous cell carcinoma,
Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,
dermatofibroma, keloids, psoriasis; and Adrenal glands:
neuroblastoma; as well as cancers of the thyroid including
medullary thyroid cancer. Thus, the term "cancerous cell," as
provided herein, includes a cell afflicted by any one of the
above-identified conditions.
[0079] In one embodiment, the cancer is selected from ovarian
cancer, prostate cancer, lung cancer, medullary thyroid cancer,
liver cancer, gastrointestinal cancer, pancreatic cancer, bone
cancer, hematologic cancer, skin cancer, kidney cancer, breast
cancer, colon cancer, and fallopian tube cancer.
[0080] In another embodiment, the disease or disorder is ovarian
cancer.
[0081] In another embodiment, the disease or disorder is prostate
cancer.
[0082] In another embodiment, the disease or disorder is lung
cancer.
[0083] In another embodiment, the disease or disorder is medullary
thyroid cancer.
[0084] In another embodiment, the disease or disorder is liver
cancer.
[0085] In another embodiment, the disease or disorder is
gastrointestinal cancer.
[0086] In another embodiment, the disease or disorder is pancreatic
cancer.
[0087] In another embodiment, the disease or disorder is bone
cancer.
[0088] In another embodiment, the disease or disorder is
hematologic cancer.
[0089] In another embodiment, the disease or disorder is skin
cancer.
[0090] In another embodiment, the disease or disorder is kidney
cancer.
[0091] In another embodiment, the disease or disorder is breast
cancer.
[0092] In another embodiment, the disease or disorder is colon
cancer.
[0093] In another embodiment, the disease or disorder is fallopian
cancer.
[0094] In another embodiment, the disease or disorder is liver
cancer, wherein the liver cancer is hepatocellular carcinoma,
cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular
adenoma, or hemagioma.
[0095] In another embodiment, the disease or disorder is
gastrointestinal cancer, wherein the gastrointestinal cancer is
cancer of the esophagous which is squamous cell carcinoma,
adenocarcinoma, or leiomyosarcoma; cancer of the stomach which is
carcinoma, or lymphoma; cancer of the pancreas, which is ductal
adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid
tumors, or vipoma; cancer of the small bowel, which is
adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,
leiomyoma, hemagioma, lipoma, or cancer of the large bowel, which
is adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, or
leiomyoma.
[0096] In another embodiment, the disease or disorder is cancer of
the pancreas, wherein the cancer of the pancreas is ductal
adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid
tumors, or vipoma.
[0097] In another embodiment, the disease or disorder is bone
cancer, wherein the bone cancer is osteosarcoma, fibrosarcoma,
malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,
malignant reticulum cell sarcoma, multiple myeloma, malignant giant
cell tumor chordoma, osteocartiliginous exostoses, chondroblastoma,
chondromyxofibroma, or osteoid osteoma.
[0098] In another embodiment, the disease or disorder is
hematologic cancer, wherein the hematologic cancer is myeloid
leukemia, acute lymphoblastic leukemia, chronic lymphocytic
leukemia, myeloproliferative diseases, multiple myeloma, or
myelodysplastic syndrome.
[0099] In another embodiment, the disease or disorder is skin
cancer, wherein the skin cancer is malignant melanoma, basal cell
carcinoma, squamous cell carcinoma, or Karposi's sarcoma.
[0100] In another embodiment, the disease or disorder is a renal
tumor or renal cell carcinoma.
[0101] In another embodiment, the disease or disorder is breast
cancer.
[0102] In another embodiment, the disease or disorder is a colon
cancer tumor.
[0103] In another embodiment, the disease or disorder is fallopian
tube carcinoma.
Preparation of Isolated Metabolites
[0104] The isolated metabolites disclosed herein can be made
according to methods available to the skilled practitioner. For
example, as depicted in Scheme 2, peptide chemistry can be used to
make the phenols C-1 and C-2 from the corresponding amines and
carboxylic acids. A variety of processes and reagents are available
for achieving such transformations and are described, for instance,
in Tetrahedron 61 (2005) 10827-10852. A representative example is
depicted in Scheme 2, wherein the activating agent is thionyl
chloride, oxalyl chloride, or the like. The corresponding acid
chloride reacts with compound A or B, respectively, to provide
phenol C-1 or C-2. Subsequent reaction of phenol C-1 or C-2 with a
sulfating agent, such as chlorosulfonic acid or sulfur
trioxide-trimethylamine complex, in the presence of a base, such as
triethylamine, alkali metal hydroxide or the like, can provide the
corresponding hydrogen sulfate 2b or 2a, respectively.
##STR00026##
[0105] Compound A was prepared according to Scheme 3. Benzylation
of A-1 using a benzyl halide or the like (BnX, where X is Cl, Br,
or I, methanesulofonate (OMs), trifluoromethansulfonate (OTf) or
toluenesulfonate (OTs)) provides benzyl-protected A-2. Nitration of
A-2 using a mixture of nitric acid and sulfuric acid provides A-3.
Reduction of the nitro group in A-3 to the amine A-4, may be
accomplished using standard nitro reduction conditions, such as
iron and ammonium acetate. Cyclization of A-4 with ethyl formate
and an alkoxide such as sodium methoxide provides the A-5.
Conversion of A-5 to the corresponding chloride using phosphorous
oxychloride provides A-6. Reaction of A-6 with 4-amino phenol
provides A-7, which is deprotected with methane sulfonic acid to
provide compound A.
##STR00027## ##STR00028##
[0106] Similarly, compound B was prepared according to Scheme 4.
Demethylation of B-1 provides B-2. Benzylation of B-2 using a
benzyl halide or the like (BnX, where X is Cl, Br, or I,
methanesulofonate (OMs), trifluoromethansulfonate (OTf) or
toluenesulfonate (OTs)). Nitration of B-3 using a mixture of nitric
acid and sulfuric acid provides B-4. Reduction of nitro group in
B-4 to the amine B-5, may be accomplished using standard nitro
reduction conditions, such as iron and ammonium acetate.
Cyclization of B-5 with ethyl formate and an alkoxide such as
sodium methoxide provides the B-6. Conversion of B-6 to the
corresponding chloride using phosphorous oxychloride provides B-7.
Reaction of B-7 with 4-amino phenol provides B-8, which was
deprotected with methane sulfonic acid to provide compound B.
##STR00029## ##STR00030##
[0107] Phenols 13 and 16 can be similarly prepared from compound 7,
the synthesis of which is disclosed in WO 2005/030140 as Example
73. Thus, in Scheme 5, coupling of 7 with 2-amino-5-fluorophenal
(CAS Reg. No. 53981-24-1) provides
##STR00031##
Coupling of 7 with 5-amino-2-fluorophenol (CAS Reg. No.
100367-48-4) provides
##STR00032##
##STR00033##
[0108] Phenols 13 and 16 can be readily converted to the
corresponding sulfates 9, and 12 depicted in Scheme 1 using, for
example, a sulfating agent, such as sulfur trioxide trimethyl amine
complex, in the presence of a strong hydroxide, such as potassium
hydroxide, sodium hydroxide, or the like, or using chlorosulfonic
acid in the presence of an amine base such as triethylamine.
[0109] The phenols 15a and 15b can be prepared by employing the
similar method that is disclosed in WO 2005/030140 for the
preparation of Example 43. Thus, in Scheme 6, coupling of phenol C
(Example 38 in WO 2005/030140) with triflate D (Example 33 in WO
2005/030140), or chloride A-6 (Example 32 in WO 2005/030140)
provides E, which is then deprotected under Pd-catalyzed
hydrogenolysis condition to yield compound 15. Similarly, reaction
of phenol C with triflate F or chloride B-7 provides G, which is
subjected to O-benzyl deportection to provide compound 15b.
##STR00034##
[0110] The N-oxide 19 can be prepared by the reaction of
cabozantinib with an oxidizing agent, such as, for example a
peroxide, a peracid, or the like. In one embodiment, the oxidizing
agent is sodium perborate tetrahydrate.
[0111] The following non-limiting examples are meant to illustrate
the invention.
EMBODIMENTS
Embodiment 1
[0112] A pharmaceutical formulation comprising a physiologically
effective amount of cabozantinib, wherein oral administration of
said composition to a selected human subject group produces in said
selected human subject group:
[0113] an average cabozantinib plasma area under the curve (average
AUC) of at least 60,000 ngh/mL per each 140 mg dosage of
cabozantinib delivered;
[0114] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1000 ng/mL per each 140 mg dosage
of cabozantinib delivered; and
[0115] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.25;
[0116] wherein the AUC is measured from time zero to the time of
last measurable concentration.
Embodiment 2
[0117] A pharmaceutical formulation comprising a physiologically
effective amount of cabozantinib, wherein oral administration of
said pharmaceutical formulation to a selected human subject group
produces in said selected human subject group:
[0118] a steady state area under the curve for cabozantinib
(average AUC) of at least 35,000 ngh/mL for a formulation
comprising 100 mg of cabozantinib delivered once daily;
[0119] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 2400 ng/mL per each 100 mg dosage
of cabozantinib delivered;
[0120] an average minimum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1100 ng/mL per each 100 mg dosage
of cabozantinib delivered; and
[0121] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.35;
[0122] wherein the AUC is measured on day 22.
Embodiment 3
[0123] The pharmaceutical formulation of embodiments 1-2, wherein
the measured metabolites are selected from the group consisting
of:
##STR00035## ##STR00036## ##STR00037##
wherein GA is a glucuronic acid moiety,
Embodiment 4
[0124] The pharmaceutical formulation of claims 1-2, wherein the
measured metabolites comprise one or more of:
##STR00038##
Embodiment 5
[0125] A method of treating cancer in a human patient, comprising
orally administering to said patient a pharmaceutical formulation
comprising a physiologically effective amount of cabozantinib at
such a rate to achieve:
[0126] an average cabozantinib plasma area under the curve (average
AUC) of at least 60,000 ngh/mL per each 140 mg dosage of
cabozantinib delivered;
[0127] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1000 ng/mL per each 140 mg dosage
of cabozantinib delivered; and
[0128] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.25;
[0129] wherein the AUC is measured from time zero to the time of
last measurable concentration.
Embodiment 6
[0130] A method of treating cancer in a human patient, comprising
orally administering to said patient a pharmaceutical formulation
comprising a physiologically effective amount of cabozantinib at
such a rate to achieve:
[0131] a steady state area under the curve for cabozantinib
(average AUC) of at least 35,000 ngh/mL for a formulation
comprising 100 mg of cabozantinib delivered once daily;
[0132] an average maximum cabozantinib blood plasma concentration
(average C.sub.max) of at least 2400 ng/mL per each 100 mg dosage
of cabozantinib delivered;
[0133] an average minimum cabozantinib blood plasma concentration
(average C.sub.max) of at least 1100 ng/mL per each 100 mg dosage
of cabozantinib delivered; and
[0134] a ratio of AUC (cabozantinib) to the AUC of the sum of
cabozantinib plus measured cabozantinib metabolites:
AUC (Cabozantinib)/AUC (Cabozantinib+Measured Metabolites)
of at least 0.35;
[0135] wherein the AUC is measured on day 22.
Embodiment 7
[0136] The method of embodiments 5-6, wherein the measured
metabolites are selected from the group consisting of:
##STR00039## ##STR00040## ##STR00041##
wherein GA is a glucuronic acid moiety,
Embodiment 8
[0137] The method of embodiments 5-7 wherein the measured
metabolites comprise one or more of:
##STR00042##
Embodiment 9
[0138] The method of embodiments 5-8, wherein the cancer is
selected from ovarian cancer, lung cancer, medullary thyroid
cancer, liver cancer, gastrointestinal cancer, pancreatic cancer,
bone cancer, hematologic cancer, skin cancer, kidney cancer, breast
cancer, colon cancer, and fallopian tube cancer.
Embodiment 10
[0139] The pharmaceutical formulation of embodiments 1-4 or the
embodiments of claims 5-9, wherein the administration is once
daily.
Examples
Identification of Cabozantinib Metabolites
[0140] The objective of this study was to profile and identify
metabolites of cabozantinib in human plasma, urine, and feces. The
plasma, urine and fecal samples were collected from a mass balance
study of cabozantinib following a single 175 mg oral administration
of cabozantinib (L-malate salt) containing [.sup.14C] cabozantinib
(100 .mu.Ci) in healthy male subjects.
[0141] Study Design and Plasma, Urine, and Feces Sampling
[0142] Eight male subjects participated in the study, and each
subject received a single oral dose of 175 mg of cabozantinib
(L-malate salt) containing [.sup.14C]-cabozantinib (100 .mu.Ci).
The plasma, urine, and fecal samples were collected from the 8
subjects for the metabolite profiling.
[0143] Plasma samples were collected at pre-dose, 0.5, 1, 2, 3, 4,
5, 8, 14, 24, 72, 168, 336, 504 and 648 hours post-dose; urine
samples were collected at pre-dose, 0-8 hours, 8-24 hours, at
24-hour intervals to 480 hours post-dose, and at greater than
48-hour intervals from 504 to 1152 hours post-dose; and feces
samples were collected at pre-dose, at 24-hour intervals to 480
hours post-dose, and at greater than 48-hour intervals from 504 to
1152 hours post-dose. All samples were shipped to QPS LLC (Newark,
Del.) and stored at -70.degree. C. HPLC/tandem MS coupled with a
radio flow-through detector (RFD) was used for metabolite profiling
and identification for samples with sufficient levels of
radioactivity.
[0144] HPLC fraction collection followed by counting with TopCount
NXT.TM. was used for radioquantitation of plasma samples with
sufficient levels of radioactivity. Three (3) HPLC methods were
used in this study to separate cabozantinib and its metabolites.
HPLC Method 1 was used for the analysis of pooled urine and fecal
samples and individual plasma samples from different time points.
HPLC Method 2 was used for the analysis of plasma samples from a
drug-drug interaction study to search for possible metabolites that
may co-elute with cabozantinib sulfate. HPLC Method 3 was used for
pooled plasma samples.
[0145] Selected samples for plasma, urine, and feces from 6
subjects were analyzed for cabozantinib and metabolites and
reported.
[0146] Samples from 2 subjects were used for the investigation
study.
[0147] Test Article
[0148] The test article for this study was a mixture of [.sup.14C]
cabozantinib and cabozantinib. The asterisk indicates the position
of the [.sup.14C] label. [.sup.14C] labeled cabozantinib was
prepared as provided in WO 2005/030140, except that [.sup.14C]
labeled 4-amino phenol was used instead of unlabeled 4-amino
phenol. [.sup.14C] labeled 4-amino phenol is commercially available
as the hydrochloride salt, for instance, from Hartmann Analytic,
American Radiolabeled Chemicals, or Fisher Scientific.
##STR00043##
[0149] General Chemicals and Reference Standards
[0150] Formic acid and ammonium acetate were obtained from
Sigma-Aldrich Chemical Co. (St. Louis, Mo.). Acetonitrile (B &
J brand, carbonyl free, for applications sensitive to trace
aldehyde and ketone), water (B & J brand, for GC, HPLC and
spectrophotometry), and methanol (HPLC grade) were purchased from
Fisher Scientific (Pittsburgh, Pa.). Type I water was generated
using an Elgastat UHQ PS water purification system.
Non-radiolabeled metabolite standards (fluoroaniline cleavage
product, cabozantinib sulfate, and cabozantinib N-oxide) were
provided by Exelixis, Inc.
[0151] Biological Sample Collection
[0152] The plasma, urine, and fecal samples were collected from a
mass balance study of cabozantinib following a single 175 mg oral
administration of cabozantinib (L-malate salt) containing
[.sup.14C] cabozantinib (100 .mu.Ci) in healthy male subjects.
Samples were shipped from Celerion (Lincoln, Nebr.) to QPS LLC
(Newark, Del.) on dry ice and were stored at -70.degree. C. until
analysis. Samples from 6 subjects were used for metabolite
profiling, identification, and radio-quantitation. Plasma samples
from 2 subjects were only used in a bridging study as part of
investigation of co-eluting metabolites.
[0153] Sample Preparation and Radioactive Recovery for Human
Plasma
[0154] For metabolite profiling, identification, and
radio-quantitation, individual radiolabeled plasma samples
collected at 0.5, 1, 2, 3, 4, 5, 8, 14, 24, 72, 168, and 336 hours
post-dose were processed and analyzed for 6 subjects. For the
investigation of co-eluting metabolites, nonradiolabeled plasma
samples of six subjects were pooled, processed, and analyzed for
pre-dose, 1-7, 8-96, and 120-336 hours post-dose. To bridge the
metabolite data from non-radiolabeled to radiolabeled plasma
samples from the human mass balance study, [.sup.14C] plasma
samples from 0-168 hours post-dose for each of the six subjects
were also pooled using the Hamilton pooling method, processed, and
analyzed by radio-quantitation. [.sup.14C] Plasma samples from
1-168 hours post-dose for two subjects were pooled (equal volume),
processed, and analyzed.
[0155] Initial Method for Plasma Extraction and Recovery
[0156] Two plasma samples from a subject (4 and 72 hours post-dose)
were used for initial extraction and recovery determination. The
total radioactivity for each plasma sample in mass balance study
was provided by Exelixis, Inc., and was defined as 100%. After the
samples were thawed under a biological hood, two 0.5 mL aliquots of
each plasma sample were added to 3 volumes (1.5 mL) of MeOH:ACN
(20:80, v/v) and vortexed (5 min). The mixtures were centrifuged at
2000 rpm for 10 minutes, and the supernatants were transferred to
clean tubes. The pellets were extracted with two additional 3
volumes of MeOH:CAN (20:80, v/v). The mixtures were centrifuged,
and the supernatants were combined. Aliquots were analyzed by a
2900 TR liquid scintillation counter (LSC) (Packard Instruments,
Meridian, Conn.). The extraction recovery was calculated as the
following:
Extraction Recovery (%)=(DPM in supernatant/DPM in plasma
sample).times.100
[0157] The supernatants from the extraction were evaporated to
dryness under a stream of nitrogen in an ambient water bath. The
residues were then reconstituted in 0.35-0.5 mL of MeOH:ACN:water
(10:20:70, v/v/v). The reconstituted samples were centrifuged at
15,000 rpm for 10 minutes and aliquots were analyzed by LSC for
reconstitution recovery.
Reconstitution Recovery (%)=(DPM in reconstitution solution I/DPM
in supernatant).times.100
[0158] Plasma Sample Preparation
[0159] Radiolabeled and non-radiolabeled plasma samples were
extracted employing the same method, using 1.0-2 mL plasma samples,
depending on the volume available and radioactivity level of the
samples. The supernatants were evaporated to dryness under a stream
of nitrogen in an ambient water bath, and the residues were
reconstituted in 0.35-0.5 mL of MeOH:ACN:water (10:20:70, v/v/v).
The reconstituted samples were centrifuged at 15,000 rpm for 10
minutes. Aliquots of the supernatants were injected onto the HPLC
system for analysis.
[0160] Sample Preparation and Radioactive Recovery for Human
Urine
[0161] Pooled urine samples from a subject (0-72, 168-192, and
312-336 hours post dose) were lyophilized in triplicate (each 4
mL), and the residues were reconstituted in 1 mL of water:ACN:FA
(80:20:0.1, v/v/v). The radioactivity in pooled urine and
reconstituted solution was counted using LSC, and the
reconstitution recovery calculated. For metabolite profiling,
identification, and radio-quantitation, pre-dose and 3 pooled urine
samples (0-72 hours, 168-192 hours, and 312-336 hours post-dose)
from each of the six subjects were analyzed. Each pooled urine
sample was lyophilized, the residue was reconstituted in
water:ACN:FA (80:20:0.1, v/v/v), and the reconstituted sample was
centrifuged at 15,000 rpm for 10 min before analysis.
[0162] Sample Preparation and Radioactive Recovery for Human
Urine
[0163] To evaluate the extraction recovery of fecal samples, two
fecal homogenate samples from a subject were thawed under a
biological hood. Approximately 5.5-6 g fecal homogenate was
accurately weighed out for the extraction. Fifteen mL ACN:MeOH
(80:20) was added to the fecal homogenates. The mixtures were
vortexed for 3 minutes and centrifuged at 3000 rpm for 10 minutes.
The supernatants were transferred to clean tubes. The extraction
procedure was repeated two more times. The supernatants from all
three extractions were combined. The radioactivity in the combined
supernatants was determined by LSC. The extraction recovery was
calculated using the following formula:
Extraction Recovery (%)=(DPM in supernatant/DPM in fecal
homogenate).times.100
[0164] The supernatant was concentrated under a nitrogen stream at
ambient temperature, and the residues were reconstituted in
MeOH:ACN:water (10:20:70). Aliquots of reconstitution solution were
counted with LSC for reconstitution recovery.
Reconstitution Recovery (%)=(DPM in reconstitution solution/DPM in
supernatant).times.100
Overall Recovery (%)=Extraction Recovery (%).times.Reconstitution
Recovery (%)/100
[0165] For metabolite profiling, identification, and
radio-quantitation, pre-dose and 3 pooled fecal samples (0-72,
168-192, and 312-336 hours post-dose) from each of the six subjects
were extracted using the same procedures for extraction recovery.
The supernatants were dried under a nitrogen stream, and the
residues were reconstituted in water:ACN:FA (80:20:0.1, v/v/v). The
reconstituted samples were centrifuged at 15,000 rpm for 10 min
before analysis.
[0166] HPLC Column Recovery
[0167] HPLC column recovery was carried out to demonstrate that all
radioactive components were effectively eluted from the column
using HPLC Method I. Aliquots of urine samples (Subject 1042, 24-48
hours post-dose) were injected onto the HPLC system with or without
a column, and the eluents from 0-30 minutes were collected into
clean 50 mL centrifuge tubes. The weights of eluent from each
injection were obtained after collection, and duplicate aliquots (1
mL) were counted using LSC. The average value of the counts was
used to calculate the total radioactivity contained in the
collected eluent with or without a column installed.
Column Recovery (%)=(DPM in eluent with column/DPM in eluent
without column).times.100
[0168] HPLC Method 3 was used for pooled plasma only, and the
column recovery was not performed due to limited sample volume
available.
[0169] HPLC/MS/RFD and HPLC Radio-Analysis Systems
[0170] The system for metabolite profiling and identification
(HPLC/MS/RFD) consisted of a HTC PAL autosampler, a Surveyor HPLC
pump, a LTQ linear ion trap mass spectrometer, and a .beta.-RAM
Model 3 RFD. The data obtained by mass spectrometry and RFD were
processed by Xcalibur and Laura Lite 3 software, respectively. The
HPLC eluent was split between the RFD and mass spectrometer with a
ratio of 3 to 1. The following are the summary of the conditions
for HPLC, mass spectrometer, and RFD.
[0171] HPLC/MS/RFD Method 1
TABLE-US-00002 HPLC Surveyor HPLC pump Column Type Phenominex
Synergi Polar RP, 4.6 .times. 250 mm, 4 .mu.m Mobile Phases A:
H.sub.2O with 0.1% FA B: ACN with 0.1% FA Gradient Program Time
(min) A % B % 0 80 20 2 80 20 22 30 70 23 5 95 27 5 95 28 80 20 34
80 20 Flow Rate 800 .mu.L/minutes Analysis Time 34 minutes Mass
Spectrometry: Thermo Finnigan LTQ Linear Ion Trap Sheath gas flow
rate 50 unit Auxiliary gas flow rate 20 unit Sweep gas flow rate 10
unit Ion spray voltage 5 kV for ESI+; 4.3 kV for ESI- Capillary
temperature 300.degree. C. Capillary voltage 22 V for ESI+; -9 V
for ESI- Tube lens voltage 80 V for ESI+; -96 V for ESI- Ionization
mode ESI+, ESI- Radio Flow-through .beta.-RAM Model 3 Detector:
Radionuclide .sup.14C Cell Volume 400 .mu.L Scintillation Cocktail
Ultima-Flo M. Perkin Elmer Cocktail/HPLC 3:1 flow ratio
HPLC/MS Method 2
TABLE-US-00003 [0172] HPLC Surveyor HPLC pump Column Type
Phenominex Synergi Polar RP, 4.6 .times. 250 mm, 4 .mu.m Mobile
Phases A: H.sub.2O with 0.1% FA B: ACN with 0.1% FA Gradient
Program Time (min) A % B % 0 80 20 2 80 20 40 35 65 42 5 95 47 5 95
48 80 20 55 80 20 Flow Rate 800 .mu.L/minutes Analysis Time 55
minutes Mass Spectrometry: Thermo Finnigan LTQ Linear Ion Trap
Sheath gas flow rate 50 unit Auxiliary gas flow rate 20 unit Sweep
gas flow rate 10 unit Ion spray voltage 5 kV Capillary temperature
300.degree. C. Capillary voltage 22 V Tube lens voltage 80 V
Ionization mode ESI+
HPLC/MS Method 3
TABLE-US-00004 [0173] BIEL Surveyor HPLC pump Column Type Waters
Xbridge phenyl, 4.6 .times. 150 mm, 3.5 .mu.m Mobile Phases A:
H.sub.2O with 0.1% FA B: ACN with 0.15% FA Gradient Program Time
(min) A % B % 0 80 20 2 80 20 40 30 70 42 5 95 47 5 95 48 80 20 55
80 20 Flow Rate 800 .mu.L/minutes Analysis Time 55 minutes Mass
Spectrometry: Thermo Finnigan LTQ Linear Ion Trap Sheath gas flow
rate 50 unit Auxiliary gas flow rate 20 unit Sweep gas flow rate 10
unit Ion spray voltage 5 kV Capillary temperature 300.degree. C.
Capillary voltage 22 V Tube lens voltage 80 V Ionization mode
ESI+
[0174] The HPLC-MS system for high resolution MS consisted of a
Michrom Bioresources Paradigm MS4B HPLC and a Thermo LTQ Orbitrap
Discovery mass spectrometer. Chromatographic conditions and the ion
source parameters were the same as HPLC method 1 for the LTQ
system. Data were acquired with a resolution of 30000 in centroid
mode.
[0175] An HPLC/TopCount NXT.TM. system was used for the
radio-quantitation of plasma samples. The system consisted of an
HTC PAL autosampler, two Shimadzu HPLC pumps, and a Foxy Jr.
Fraction Collector (Isco, Lincoln, Nebr.). HPLC fractions collected
in a LumaPlate.TM. 96-well plate were dried using an EZ-2plus
Personal Evaporator (Genevac, Valley Cottage, N.Y.), and the dried
samples were counted by TopCount NXT.TM. Microplate Scintillation
& Luminescence Counter (PerkinElmer.RTM.). The data were
processed using ProFSA (PerkinElmer.RTM.) software. The HPLC
methods were the same as described above.
[0176] Metabolite Identification
[0177] Metabolites that represented greater than 5% of the total
radioactivity or 5% of total AUC in the matrix were identified
according to the following process. Mass spectra (MS, MS/MS, and
MS/MS/MS) of cabozantinib and its metabolite standards, provided by
the Exelixis, Inc., were acquired on an ion trap mass spectrometer.
Major fragment patterns were proposed. Identification of these
metabolites was confirmed by matching mass spectra (MS and MS/MS)
and retention times with authentic reference standards. For other
unknown metabolites, molecular ions were searched on LC/MS
chromatograms operating in full scan positive as well as negative
ionization modes at the same retention times as those found on
LC-radio chromatogram. Product ion mass spectra and high resolution
mass spectra were then acquired for the corresponding molecular
ions. Putative metabolite structures were proposed based on the
analysis of their mass fragment patterns.
[0178] Quantitation of cabozantinib and its Metabolites
[0179] Quantitation of cabozantinib and its metabolites in pooled
or individual samples from each matrix at different time points or
time intervals was based on integration of the corresponding peaks
found on their radio-chromatograms. For plasma samples, percent of
total radioactivity in the sample for each peak at each time point
was calculated and converted to ng/mL.
[0180] For quantification of cabozantinib and its metabolites in
plasma:
ng/mL=(% of the total radioactivity).times.(total ng equivalent/mL
for the time point)/100
[0181] The values of total ng equivalent/mL were obtained from the
results of the human mass balance study.
[0182] For the pooled urine samples, percent of total radioactivity
in the pooled sample for each peak was calculated as the % of total
non-parent in the pooled samples:
% of total non-parent in the pooled samples=(total radioactivity of
the peak/total radioactivity of the non-parent peaks).times.100
[0183] For the pooled fecal samples, percent of total radioactivity
in the pooled sample for each peak was calculated as the percent of
total non-parent plus parent in the pooled samples:
% of total non-parent plus parent in the pooled samples=(total
radioactivity of the peak/total radioactivity of the parent and
non-parent peaks).times.100
[0184] The percent of total radioactivity in the pooled sample for
each peak was converted to the percent of parent in the pooled
samples:
% of parent in the pooled samples=(total radioactivity of the
peak/total radioactivity of the parent peak).times.100
[0185] The limit of quantification for a radioactivity detector was
defined as the ratio of signal to noise (3 to 1) on the
radio-chromatogram. The low limits of quantification were 10 and
500 dpm for the TopCount and n-RAM radio flow-through detector,
respectively.
Results and Discussion
[0186] Radioactive Recovery
[0187] The initial extraction recovery was determined using plasma
samples from a subject at 4 hours and 72 hours post-dose with three
volumes of MeOH:ACN (20:80) extracting three times. The mean
extraction recoveries of radioactivity from 4 and 72 hour samples
were 98.43% and 94.99%, respectively. After drying down and
reconstitution into MeOH:ACN solution, the reconstitution
recoveries were 92.73% and 85.90%, respectively. The overall
recoveries were 91.27% and 81.60%, respectively.
[0188] Urine centrifugation recoveries determined using 0-8, 24-48,
72-96, and 120-144 hour post-dose samples from the subject ranged
between 102% and 104%. Urine reconstitution recovery after
lyophilization was 94.7% using pooled samples from a subject.
[0189] For pooled fecal samples from 0-48 hours post dose, the
extraction, reconstitution, and overall recoveries were 98.48%,
88.80%, and 87.37%, respectively. For pooled fecal samples of
120-168 hours post dose, the extraction, reconstitution, and
overall recoveries were 85.85%, 87.69%, and 75.24%,
respectively.
[0190] The radioactivity recovery from HPLC column for urine sample
was 97.05%.
[0191] No correction factor was applied to the plasma, urine, and
fecal radio-quantitation to account for the recovery.
[0192] Metabolite Profiling
[0193] In a subject, cabozantinib, compound 9 (cabozantinib
sulfate), and compound 19 (cabozantinib N-oxide) were the major
peaks on the radio-chromatograms. Compound 2 (demethylated and
sulfated fluoroaniline cleavage product) was the major metabolite
in plasma samples after 72 hours post-dose. Metabolite compound 7
(fluoroaniline cleavage product) accounted for one of the minor
peaks. Metabolite compounds 7, 3 (demethyl cabozantinib glucuronide
B), 9, and 10 (methyl ester of fluoroaniline cleavage product)
co-eluted using HPLC Method 1.
[0194] Representative human urine metabolite profiles, the
radio-chromatograms (using HPLC Method 1) of human urine samples
from 0-72 hours, 144-192 hours, and 288-336 hours post-dose were
collected from a subject. Metabolite compound 6 was the major
metabolite in 0-72 hours, 144-192 hours, and 288-336 hours post
dose pooled urine samples. In addition to compound 6, metabolite
compounds 1, 4, 5, 7, and 19 were observed in the pooled sample of
0-72 hours post dose. Metabolite compounds 1, 4, 5, and 7 were
observed in the pooled sample of 144-192 hours post dose.
Metabolite compounds 1 and 5 were detected in the pooled sample of
288-336 hours post dose. Parent compound cabozantinib was not
observed in urine samples.
[0195] Representative human fecal metabolite profiles, the
radio-chromatograms (using HPLC Method 1) of human fecal samples
from 0-72 hours, 144-192 hours, and 288-336 hours post-dose from a.
Parent cabozantinib and metabolites compound 4, 7, 11, and 15
(including compound 16) were observed in the pooled sample of 0-72
hours post dose. Metabolite compounds 4, 7, 11, 15, 16, and 18 were
observed in the pooled sample of 144-192 hours post dose.
Metabolite compounds 4 and 11 were observed in the pooled sample of
288-336 hours post dose.
[0196] Metabolite Identification Using HPLC/MS Analysis
[0197] HPLC/MS analysis of authentic standards using HPLC Method 1
showed that the retention times of cabozantinib, fluoroaniline
cleavage product (compound 7), sulfate (compound 9), and N-oxide
(compound 19) were 20.3, 14.4, 16.5, and 23.1 minutes,
respectively.
[0198] Plasma, urine, and fecal samples were next analyzed by
HOPLC/MS, and the compounds were identified based on their
protonated molecular ions and fragmentation patterns.
[0199] Metabolite Identification of Cabozantinib and its
Metabolites in Human Plasma
[0200] The mass spectrum of the peak at approximately 19.1 minutes
in the XIC showed the protonated molecular ions at m/z 502. Its
product ion spectra showed major fragments at m/z 391, 323, and
297, which is consistent with those of cabozantinib standard. The
MS data is summarized in Table 1 and 2.
TABLE-US-00005 TABLE 1 HPLC Radiochromatogram Retention Times of
Metabolites in Samples from Single Oral Dose of [.sup.14C]
Cabozantinib Compound HPLC Method Retention Time (min) Standards 7
1 14.13 9 1 16.45 I 1 20.26 19 1 23.06 Plasma 1 1 4.13 2a/2b 1 9.33
4 1 11.87 5 1 12.80 6 1 13.47 7 1 14.13 9 1 14.67 I 1 18.67 19 1
23.47 Urine 1 1 4.13 4 1 11.87 5 1 12.80 6 1 13.47 7 1 14.13 19 1
23.47 Feces 4 1 12.67 7 1 13.47 11 1 16.07 15 1 17.87 I 1 19.60 18
1 21.03 Hamilton Pooled Sample Plasma 9 3 17.36 7 3 19.32 8 3 19.32
(shoulder) 19 3 25.20 I 3 37.52
TABLE-US-00006 TABLE 2 MS Data for Metabolites Using HPLC Compound
HPLC Method HPLC Retention Time MS (m/z) I 1 19.10 502 19 1 21.85
518 9 1 15.29 518 (loss of SO.sub.3 from m/z molecular ion m/z at
598) 7 1 13.36 409 2a 1 10.70 473, 395 (2a) (loss of SO.sub.3 from
m/z molecular ion m/z at 473) 3 2 15.87 488 8 2 19.43 488 10 2
33.56 423 5 1 13.00 489 6 1 13.39 393 15 1 17.60 488 16 1 17.60 518
13 1 16.45 518 12 1 16.45 518 17 1 18.43 518
PK Parameters of Cabozanintinib and Metabolites
[0201] PK parameters for cabozantinib and various metabolites were
measured in eight human subjects who were administered cabozantinib
140 mg/day. The metabolites that were observed included
##STR00044##
[0202] Parameters measured included maximum observed concentration
(C.sub.max) measured as ng/mL; Area under the concentration-time
curve (AUC) measured as hng/mL at time 0 to 24 hours
(AUC.sub.0-24), at time 0 to 72 hours (AUC.sub.0-72), at time 0 to
the time of last measurable concentration (AUC.sub.0-t). AUC Ratios
were also calculated, including the ratio of AUC (cabozantinib) to
the AUC of the sum of cabozantinib plus measured cabozantinib
metabolites. Results are summarized in the following Table 3.
TABLE-US-00007 TABLE 3 Selected PK Parameters for Cabozantinib and
Measured Metabolites Cabozantinib ##STR00045## C.sub.max, 1250 .+-.
238 (19) 52.9 .+-. 17.3 (33) ng/mL T.sub.max,h.sup.a 1.49 (1.00,
3.00) 18.99 (5.0, 24.10) AUC.sub.0-24, 14300 .+-. 2600 1080 .+-.
341 (32) hng/mL (18) AUC.sub.0-72, 35000 .+-. 6770 3120 .+-. 976
(31) hng/mL (19) AUC.sub.0-t, 67200 .+-. 6880 6540 .+-. 1680 (26)
hng/mL (10) Ratio .sup.b, NA 9.93 .+-. 3.20 (32) % Ratio .sup.c, 32
.+-. 6.06 (19) 3.63 .+-. 1.76 (48) % AUC.sub.0-inf, 68000 .+-. 6910
6770 .+-. 1700 (25) hng/mL (10) k.sub.el, 1/h 0.00712 .+-. 0.001
0.00807 .+-. 0.00218 (27) 76 (25) t.sub.1/2, h 102 .+-. 23.3 (23)
91.8 .+-. 25.4 (28) ##STR00046## C.sub.max, 118 .+-. 33.7 (28)
ng/mL T.sub.max,h.sup.a 13.50 (2.00, 24.30) AUC.sub.0-24, 2030 .+-.
682 (34) hng/mL AUC.sub.0-72, 5610 .+-. 1940 (35) hng/mL
AUC.sub.0-t, 10100 .+-. 3210 (32) hng/mL Ratio .sup.b, 15.0 .+-.
3.80 (25) % Ratio .sup.c, 5.25 .+-. 2.15 (41) % AUC.sub.0-inf,
10300 .+-. 3170 (31) hng/mL k.sub.el, 1/h 0.00846 .+-. 0.00256 (30)
76 (25) t.sub.1/2, h 89.2 .+-. 29.2 (33) ##STR00047## C.sub.max,
236 .+-. 66.7 (28) ng/mL T.sub.max,h.sup.a 24.00 (3.00, 48.00)
AUC.sub.0-24, 3970 .+-. 1350 (34) hng/mL AUC.sub.0-72, 12600 .+-.
4180 (33) hng/mL AUC.sub.0-t, 28900 .+-. 10700 (37) hng/mL Ratio
.sup.b, 42.9 .+-. 14.4 (33) % Ratio .sup.c, 13.4 .+-. 5.6(43) %
AUC.sub.0-inf, 29500 .+-. 10600 (36) hng/mL k.sub.el, 1/h 0.00859
.+-. 0.0022 (26) 76 (25) t.sub.1/2, h 86.0 .+-. 24.3 (28)
##STR00048## C.sub.max, 229 .+-. 91 (40) ng/mL T.sub.max,h.sup.a
168 (72-240) AUC.sub.0-24, 950 .+-. 376 (40) hng/mL AUC.sub.0-72,
-- hng/mL AUC.sub.0-t, 99362 .+-. 34386 (35) hng/mL Ratio .sup.b,
150 .+-. 51.3 (34) % Ratio .sup.c, 46 .+-. 11.2 (25) %
AUC.sub.0-inf, 177682 .+-. 62496(35).sup.d hng/mL k.sub.el, 1/h --
76 (25) t.sub.1/2, h 494 .+-. 99 (20) .sup.a: median (range);
.sup.b: ratio of AUC.sub.0-t (metabolite)/AUC.sub.0-t (parent);
.sup.c: ratio of AUC.sub.0-t (each analyte/AUC.sub.0-t
(parent.varies.measured metabolites); NA: Not Applicable; .sup.d
> 40% of AUCinf was extrapolated C.sub.max, maximum observed
concentration; T.sub.max, time of the maximum concentration;
AUC.sub.0-1, area under the concentration-time curve from time zero
to the time of the last measurable concentration; AUC.sub.0-24,
area under the concentration-time curve from time zero to 24 hours
post XL184 dose; AUC.sub.0-72, area under the concentration-time
curve from time zero to 72 hours post XL 184 dose; AUC.sub.0-inf,
area under the concentration-time curve from time zero to infinity;
k.sub.el, apparent terminal elimination rate constant; t.sub.1/2,
apparent terminal elimination half-life; CL/F, apparent total body
clearance; V/F, apparent total volume of distribution.
Kinase Activity of Cabozantinib Metabolites
[0203] Kinase Dilution
[0204] Kinase Activity was measured and profiled by EMD Millipore
according to the Kinase Profiler Service Assay Protocols Protocol
Guide Volume 57. The results are summarized below in Table 4.
Inhibition is indicated as IC.sub.50 with the following key:
A=IC.sub.50 less than 50 nM, B=IC.sub.50 greater than 50 nM, but
less than 500 nM, C=IC.sub.50 greater than 500 nM, but less than
5000 nM, and D=IC.sub.50 greater than 5,000 nM. Depending upon the
functionality about the quinazoline or quinoline, exemplary
compounds of the invention exhibit selectivity for any of c-Met,
KDR, c-Kit, flt-3, and flt-4. Abbreviations for enzymes listed in
Table 3 are defined as follows: c-Met refers to hepatocyte growth
factor receptor kinase; RET refers to RET proto-oncogene kinase;
KDR refers to kinase insert domain receptor tyrosine kinase; fit-1
alpha, flt-3, and flt-4, fins-like tyrosine kinases, representative
of the FLK family of receptor tyrosine kinases; and Aurora B MP
refers to Aurora B kinase. When a percentage is listed instead of
an IC.sub.50 value, it indicates percent inhibition at 1 .mu.M.
Empty cells in the tables indicate lack of data only.
TABLE-US-00008 TABLE 4 Kinase Activity c-Met RET KDR Flt-1 Std Std
Std Alpha Compound (IC50) (IC50) (IC50) (IC50) ID MOLSTRUCTURE (nM)
(nM) (nM) (nM) Cabozantinib ##STR00049## A A A A 16 ##STR00050## A
A C 13 ##STR00051## A A C 2a ##STR00052## .gtoreq.50% @ 1.mu.M
.ltoreq.25% @ 1.mu.M .ltoreq.25% @ 1.mu.M .ltoreq.25% @ 1.mu.M 2b
##STR00053## Not Detected 9 ##STR00054## .gtoreq.75% @ 1.mu.M
.gtoreq.75% @ 1.mu.M .ltoreq.25% @ 1.mu.M .gtoreq.50% @ 1.mu.M 19
##STR00055## B B 7 ##STR00056## D D Aurora B Flt-3 Flt-4 MP 8pt Std
Std Std Compound (IC50) (IC50) (IC50) ID (nm) (nm) (nm)
Cabozantinib A A 16 A B 13 A B 2a .ltoreq.25% .gtoreq.25%
.ltoreq.25% @ 1.mu.M @ 1.mu.M @ 1.mu.M 2b 9 .gtoreq.50% .gtoreq.75%
.gtoreq.75% @ 1.mu.M @ 1.mu.M @ 1.mu.M 19 C C 7 C C
Metabolite Synthesis and Structural Data
[0205] 6-Desmethyl Acid
##STR00057##
[0206] In a vessel, cabozantinib (15.0 g; 53.3 mmol) and potassium
carbonate (29.5 g; 213.3 mmol; 4 equiv) were suspended in THF (210
mL; 14 vol) and water (90 mL; 6 vol) at 20.degree. C. In a separate
vessel, sodium 1-(methoxycarbonyl)cyclopropanecarboxylate (17.71 g;
106.6 mmol; 2 equiv.) was suspended in THF (90 mL; 6 vol). DMF (120
.mu.L; 3 mol %) was added and cooled to less than 15.degree. C.
Oxalyl chloride (9.34 mL; 106.6 mmol; 2 equiv.) was added over 90
minutes, and the reaction was aged 2 hours at 10-15.degree. C. The
acid chloride slurry was added to the cabozantinib suspension over
2 hours at 20-25.degree. C. and aged at least 3 hours, whereupon
HPLC analysis showed greater than 99% conversion to a mixture of
the mono- and biscarbonylated material. The reaction mixture was
filtered over Celite.RTM., washed with THF (30 mL; 2 vol), and the
layers were separated. 1 M NaOH (150 mL; 10 vol) was added to the
upper THF layer, and the mixture was heated at 40.degree. C. for 1
hour whereupon HPLC analysis showed greater than 99% saponified
product. The mixture was cooled to 25.degree. C., and the upper THF
layer was removed. The aqueous layer was acidified to pH 3-4 with 1
M HCl to precipitate the product and was aged for 1 hour. The
precipitate was filtered, washed with water (90 mL, 6 vol), and
dried under vacuum (greater than 20 psig) with nitrogen bleed at
50.degree. C. to give a grey to brown powder. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz) .delta. 10.8-11.0 (br s, 1H), 10.7 (s, 1H),
8.65 (d, J=6.9 Hz, 1H), 7.81 (d, J=9.3 Hz, 2H), 7.67 (s, 1H), 7.58
(s, 1H), 7.32 (d, J=9.3 Hz, 2H), 6.69 (d, J=6.9 Hz, 1H), 4.01 (s,
3H), 2.48-2.53 (m, 4H). MS (ESI-) m/z 393 [M-H].sup.-.
6-Hydrogen Sulfate 6-Desmethyl Acid
##STR00058##
[0208] 6-Desmethyl acid (120 mg; 0.30 mmol), potassium hydroxide
(118 mg; 2.1 mmol; 7 equiv.), and sulfur trioxide trimethyl amine
complex (292 mg; 2.1 mmol; 7 equiv.) was dissolved in water (3 mL;
25 vol) and heated to 70.degree. C. for 2 hours whereupon analysis
by HPLC showed greater than 99% conversion. The reaction mixture
was then cooled in an ice bath and acidified dropwise with 1 N aq.
H.sub.2SO.sub.4 to approximately pH 1. The slurry was aged at
25.degree. C. for 1 hour, filtered, washed with water (3 mL; 25
vol), and deliquored. The wet cake was then washed with acetone (3
mL; 25 vol) and dried at 35.degree. C. under vacuum (greater than
20 psig) with nitrogen bleed for 24 hours to give a beige
powder.
[0209] Alternatively, 6-desmethyl acid (120 mg; 0.30 mmol) was
suspended in MeCN (50 vol, 6 mL), and triethylamine (1.27 mL, 9.12
mmol, 30 equiv.) was added and then cooled in an ice bath.
Chlorosulfonic acid (101 .mu.L, 1.52 mmol, 5 equiv.) was added
dropwise, and the reaction was then heated to 70.degree. C. for 1
hour whereupon analysis by HPLC showed greater than 98 percent
conversion. The reaction mixture was then cooled in an ice bath for
2 hours in which a precipitate was formed. The precipitate was
removed with filtration, rinsing with cold MeCN (50 vol). The MeCN
solution was then concentrated to approximately 20 vol
(approximately 2 mL) and quenched with 100 vol IN HCl and cooled in
an ice bath to give a fine precipitate that was filtered, washed
with 50 vol water and 50 vol acetone, and dried at 35.degree. C.
under vacuum (greater than 20 psig) with nitrogen bleed for 24
hours to give a beige powder. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz)
.delta. 10.8 (s, 1H), 8.83 (d, J=5.9 Hz, 1H), 8.5 (s, 1H), 7.85 (d,
J=8.5 Hz, 2H), 7.52 (s, 1H), 7.40 (d, 0.7=8.5 Hz, 2H), 6.84 (d,
J=5.9 Hz, 1H), 4.04 (s, 3H), 3.20-3.70 (br s, 1H), 1.39-1.48 (br s,
4H). MS (ESI-) m/z 473 [M-H].sup.-, 236.
Ortho-Hydroxy-Cabozantinib
##STR00059##
[0211] A flask was charged with the carboxylic acid (0.84 g; 2.1
mmol), THF (1.2 mL), and DMF (5 .mu.L), and cooled to 15.degree. C.
To this slurry was added oxalyl chloride (0.17 mL; 2.1 mmol)
dropwise over approximately 20 minutes. After 2 hours, the acid
chloride slurry was added to another vessel containing a stirred
suspension of the aniline (0.2 g, 1.6 mmol), potassium carbonate
(0.63 g, 4.6 mmol) in THF (2.8 mL), and water (1 mL) over
approximately 15 minutes. After 3 hours, HPLC analysis showed
complete conversion to the product. Stirring was stopped, the lower
aqueous layer was removed, and water (30 mL) was added to
precipitate the product. The product was then collected by
filtration and washed with 1:1 THF-water solution (2.times.10 mL)
to afford a pale grey solid. It was then further purified by flash
chromatography on silica gel using methanol/dichloromethane as the
mobile phase.
[0212] Alternatively, a suspension of the carboxylic acid (4.08 g;
10 mmol), aniline (1.52 g; 12 mmol), and triethylamine (2.7 mL; 20
mmol) in acetonitrile (100 mL) was treated with EDAC (2.30 g; 12
mmol) and HOBt (0.5 g; 3 mmol). The slurry was stirred overnight at
room temperature, and the reaction progress was monitored by HPLC.
At the end of the reaction, 150 mL of water was added, and the
precipitated product was collected by filtration, washed with
water, and then purified by flash chromatography. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz) .delta. 10.46 (br s, 1H), 10.29 (br s, 1H),
10.0 (br s, 1H), 8.47 (d, 1H), 7.92 (dd, 1H), 7.73 (dd, 2H), 7.51
(s, 1H), 7.40 (s, 1H), 7.28 (dd, 2H), 6.68 (dd, 1H), 6.62 (dt, 1H),
6.45 (d, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 1.60-1.55 (m, 4H).
.sup.13C NMR (DMSO-d.sub.6, 100 MHz) .delta. 169.82, 167.67,
159.91, 157.51, 152.58, 149.97, 149.35, 149.09, 148.98, 148.86,
146.49, 135.72, 123.00, 122.97, 122.91, 122.43, 121.30, 115.17,
107.86, 105.10, 104.87, 103.16, 102.43, 102.19, 99.08, 55.74,
55.71, 55.66, 30.02, 16.51.
[0213] MS (APCI+) m/z 518.3 [M+H].sup.+, 500.3.
Cabozantinib-Hydroxysulfate
##STR00060##
[0215] A suspension of the hydroxy-cabozantinib (0.95 g; 1.9 mmol)
in THF (20 mL) was added triethylamine (5 mL; 36 mmol), and cooled
to below 5.degree. C. Chlorosulfonic acid (1 mL; 15 mmol) was added
dropwise such that the temperature remained below 10.degree. C.,
over approximately 15 minutes. After stirring overnight at room
temperature, HPLC analysis showed approximately 5 percent of
starting material remaining. The reaction mixture was treated with
aqueous 1 N HCl (25 mL). The precipitated product was collected by
filtration, washed with water (4.times.25 mL), and dried under
vacuum to yield an off-white solid (937 mg; 82 percent crude
yield). Analysis by AN-HPLC showed that the product was 90.8% pure,
the major impurity being the starting material. The product was
purified to greater than 99 percent (AN-HPLC) by preparative HPLC
on a C18 column, using aqueous ammonium acetate/acetonitrile mobile
phase system. .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) .delta. 10.39 (s,
1H), 9.69 (s, 1H), 8.81 (d, 1H), 7.95 (dd, 1H), 7.85 (d, 2H), 7.77
(s, 1H), 7.51 (s, 1H), 7.11 (s, 1H), 7.08 (dd, 1H), 6.93 (dd, 1H),
6.45 (d, 1H), 4.05 (s, 3H), 4.04 (s, 3H), 1.53 (s, 4H). MS (ESI-)
m/z 596.0 [M-H].sup.-.
Meta-Hydroxy-Cabozantinib
##STR00061##
[0217] A flask was charged with the carboxylic acid (0.84 g; 2.1
mmol), THF (1.2 mL), and DMF (5 .mu.L), and cooled to 15.degree. C.
To this slurry was added oxalyl chloride (0.17 mL; 2.1 mmol)
dropwise over approximately 20 minutes. After 2 hours, the acid
chloride slurry was added to another vessel containing a stirred
suspension of the aniline (0.2 g, 1.6 mmol), potassium carbonate
(0.63 g, 4.6 mmol) in THF (2.8 mL), and water (1 mL) over
approximately 15 minutes. After 90 minutes, HPLC analysis showed
complete conversion to the product. Stirring was stopped, and the
lower aqueous layer was removed and extracted with ethyl acetate
(15 mL). The organic layers were combined, dried over anhydrous
MgSO.sub.4, filtered, and concentrated to yield a brown solid. The
solid was then further purified by flash chromatography on silica
gel using ethyl acetate/heptane as the mobile phase. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz) .delta. 10.15 (br s, 1H), 9.96 (br s, 1H),
9.89 (br s, 1H), 8.46 (d, 1H), 7.76 (d, 1H), 7.50 (s, 1H), 7.41 (d,
2H), 7.39 (s, 1H), 7.22 (d, 2H), 7.07-6.98 (m, 2H), 6.42 (d, 1H),
3.94 (s, 3H), 3.93 (s, 3H), 1.46 (br s, 4H). .sup.13C NMR
(DMSO-d.sub.6, 100 MHz) .delta. 168.27, 167.95, 160.02, 152.56,
149.48, 149.33, 148.86, 148.56, 146.46, 146.21, 144.52, 144.39,
136.45, 135.33, 135.31, 122.23, 121.22, 115.63, 115.44, 115.15,
111.29, 111.23, 110.26, 107.85, 103.04, 99.08, 55.73, 55.71, 31.66,
15.40. MS (APCI+) m/z 518.3 [M+H].sup.+, 502.3.
Cabozantinib N-Oxide
##STR00062##
[0219] A flask was charged with
N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N'-(4-fluorophenyl)cyc-
lopropane-1,1-dicarboxamide (3.21 g; 6.4 mmol), acetic acid (32.1
mL), and sodium perborate tetrahydrate (1.98 g, 12.8 mmol) and
heated to 65.degree. C. and stirred overnight. After 24 hours, HPLC
analysis showed about 38:62 starting material: product. More
oxidant (1.98 g; 12.8 mmol) was added, and heating continued
overnight. Solvents were removed under vacuum, and the residue was
purified by flash chromatography using dichloromethane-methanol
gradient (dichloromethane to 10% methanol-dichloromethane) to
obtain 0.95 g of the product as a white solid. .sup.1H-NMR
(DMSO-d.sub.6, 400 MHz) .delta. 10.20 (br s, 1H), 10.08 (br s, 1H),
8.28 (d, 1H), 7.90 (s, 1H), 7.74 (d, 2H), 7.64 (dd, 2H), 7.48 (s,
1H), 7.23 (d, 2H), 7.15 (t, 2H), 6.45 (d, 1H), 3.97 (s, 3H), 3.94
(s, 3H), 1.47 (br s, 4H). .sup.13C NMR (DMSO-d.sub.6, 100 MHz)
.delta. 172.11, 168.18, 168.13, 159.49, 157.09, 153.34, 150.72,
150.57, 149.98, 137.41, 136.32, 135.24, 135.21, 134.06, 122.44,
122.36, 122.19, 120.65, 117.23, 11.17, 114.95, 104.37, 100.34,
99.12, 56.09, 56.03, 31.59, 15.42. MS (APCI+) m/z 518.3
[M+H].sup.+.
1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-phenylcarbamoyl]-cyclopropane
carboxylic Acid
##STR00063##
[0221] To the cyclopropyl di-carboxylic acid (449 mg, 3.45 mmol) in
THF (3.5 mL) was added TEA (485 .mu.L, 3.45 mmol). The resulting
solution was stirred at room temperature under a nitrogen
atmosphere for 40 minutes before adding thionyl chloride (250
.mu.L, 3.44 mmol). The reaction was monitored by LCMS for the
formation of mono acid chloride (quenched the sample with MeOH and
looked for corresponding mono methyl ester). After 3 hours stirring
at room temperature, 4-(6,7-dimethoxy-quinolin-4-yloxy)-phenylamine
(1.02 g, 3.44 mmol) was added as a solid, followed by more THF (1.5
mL). The reaction continued to stir at room temperature for 16
hours. The resulting thick slurry was diluted with EtOAc (10 mL)
and extracted with IN NaOH. The biphasic slurry was filtered, and
the aqueous phase was acidified with concentrated HCl to pH or
approximately 6 and filtered. Both solids were combined and washed
with EtOAc, then dried under vacuum. The desired product,
1-[4-(6,7-dimethoxy-quinolin-4-yloxy)-phenylcarbamoyl]-cycloprop-
anecarboxylic acid, was obtained (962 mg, 68.7 percent yield, 97
percent pure) as a white solid. .sup.1H NMR (D.sub.2O/NaOH): 7.97
(d, 1H), 7.18 (d, 2H), 6.76 (m, 4H), 6.08 (d, 1H), 3.73 (s, 3H),
3.56 (s, 3H), 1.15 (d, 4H).
[0222] The foregoing disclosure has been described in some detail
by way of illustration and example for purposes of clarity and
understanding. The invention has been described with reference to
various specific and preferred embodiments and techniques. However,
it should be understood that many variations and modifications can
be made while remaining within the spirit and scope of the
invention. It will be obvious to one of skill in the art that
changes and modifications can be practiced within the scope of the
appended claims. Therefore, it is to be understood that the above
description is intended to be illustrative and not restrictive. The
scope of the invention should, therefore, be determined not with
reference to the above description, but should instead be
determined with reference to the following appended claims, along
with the full scope of equivalents to which such claims are
entitled. Unless otherwise stated all patent references cited
herein are incorporated by reference.
* * * * *