Note: Descriptions are shown in the official language in which they were submitted.
CA 02646971 2008-09-19
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Paclitaxel combination
This invention relates to a novel combination of compounds and processes for
its
preparation, methods of treating diseases, particularly cancer, comprising
administering
said combination, and methods of making said pharmaceutical composition for
the
treatment or prevention of disorders, particularly cancer.
Cancer is a disease resulting from an abnormal growth of tissue. Certain
cancers
have the potential to invade into local tissues and also metastasize to
distant organs. This
disease can develop in a wide variety of different organs, tissues and cell
types. Therefore,
the term "cancer" refers to a collection of over a thousand different
diseases.
Over 4.4 million people worldwide were diagnosed with breast, colon, ovarian,
lung, or prostate cancer in 2002 and over 2.5 million people died of these
devastating
diseases (Globocan 2002 Report). In the United States alone, over 1.25 million
new cases
and over 500,000 deaths from cancer were expected in 2005. The majority of
these new
cases will be cancers of the colon (-100,000), lung (-170,000), breast (-
210,000) and
prostate (-230,000). Both the incidence and prevalence of cancer is predicted
to increase
by approximately 15% over the next ten years, reflecting an average growth
rate of 1.4%
(American Cancer Society, Cancer Facts and Figures 2005).
Cancer treatments are of two major types, either curative or palliative. The
main
curative therapies for cancer are surgery and radiation. These options are
generally
successful only if the cancer is found at an early localized stage. Once the
disease has
progressed to locally advanced cancer or metastatic cancer, these therapies
are less
effective and the goal of therapy aims at symptom palliation and maintaining
good quality
of life. The most prevalent treatment protocols in either treatment mode
involve a
combination of surgery, radiation therapy and/or chemotherapy.
Cytotoxic drugs (also known as cytoreductive agents) are used in the treatment
of
cancer, either as a curative treatment or with the aim of prolonging life or
palliating
symptoms. Cytotoxics may be combined with radiotherapy and/or surgery, as neo-
adjuvant treatment (initial chemotherapy aimed at shrinking the tumor, thereby
rendering
local therapy such as surgery and radiation more effective) or as adjuvant
chemotherapy
(used in conjunction or after surgery and/or localized therapy). Combinations
of different
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drugs are frequently more effective than single drugs: they may provide an
advantage in
certain tumors of enhanced response, reduced development of drug resistance
and/or
increased survival. It is for these reasons that the use of combined cytotoxic
regimens in
the treatment of many cancers is very common.
Cytotoxic agents in current use employ different mechanisms to block
proliferation
and induce cell death. One group of such agents are the microtubule modulators
that
interfere with the polymerization or depolymerization of microtubules. Taxolo
(Paclitaxel), chemical name benzenepropanoic acid, beta=(benzoylamino)-alpha-
hydroxy-,
6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-
dodecahydro-
4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-lH-
cyclodeca(3,4)benz(1,2-
b)oxet-9-ylester,(2aR-(2aalpha,4beta,4abeta,6beta,9alpha(alphaR*,betaS *),
11alpha,l2alpha,l2aalpha,i2balpha))-, or tax-ll-en-9-one,5beta,20-epoxy-
1,2a1pha,4,7beta,10beta,13alpha-hexahydroXy-, 4,10-diacetate 2-benzoate, 13 -
ester with
(2R,3S)-N-benzoyl-3-phenylisoserine, Chemical Abstracts Service Registry
Number
[33069-62-4], Orange Book application No. 020262 by Bristol Myers Squibb,
hereinafter
referred to as Paclitaxel, is such a microtubule modulator.
In one embodiment, the present invention provides a combination of 6-(6-
aminopyridin-3-yl)-N 4- {4-[(2-methylpyridin-4-yl)oxy]phenyl} pyrimidine-2,4-
diamine:
H3C
N r ~ NH
N ~
(
H2N N~ N
N H2
with Paclitaxel.
Unless otherwise stated, the following definitions apply for the technical
expressions used
throughout this specification and claims:
Combination refers to the result or product of combining two or more
iridividual units,
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ingredients or compounds. In a first meaning, combination refers to a spatial
relationship.
These units, ingredients or compounds can remain unchanged in the combination,
or they
can to a lesser or greater extent react or interact. Also, they can be
physically brought and
blended together to form one mixture, or they can be brought together into a
container
without blending. Combination also refers to the result or product of
combining said
ingredients, if they are further processed or treated after having been
combined. For
example, an example of a combination is cookie dough, which consists of
several
ingredients being mixed together. If the cookie dough in above example is
baked and
covered with frosting, it still remains a combination. In another example,
combination
refers to the product of combining two or more pharmaceutical ingredients,
such as
phannaceutically active compounds, and also refers to this product if it is
further
processed and packaged, such as two pharmaceutically active compounds being
brought
together to form a mixture, the mixture then being pressed into a tablet, the
tablet then
subsequently being coated and packaged. The tablet still remains a
combination.
In a second meaning, combination refers to a temporal relationship, i.e. two
or more acts
are performed simultaneously or in temporal proximity. Acts are performed
simultaneously, when they are done at the same point in time or in overlapping
time
periods. For example, a tv set simultaneusly transmits sound and picture and
thus transmits
a combination of the two.
Two or more acts can also be perfomed in temporal proximity, i.e. after one
another, to
form a combination. What counts as temporal proximity is dependent on the
totality of
circumstances; one indicator being that the acts are being performed to
achieve a certain
purpose; another that the acts are being performed shortly after one another
on a timescale
relevant for the observed system.
For example, a sequence of chess moves is often referred to as a combination,
while the
moves are being done one after the other with the opponent making his
intermittent
moves. They are done with the purpose of eventually defeating the opponent.
The actual time elapsed between the acts performed in a combination can thus
be
irrelevant.
In another example of a combination as acts being perfomed in temporal
proximity,
combination refers to a drug regimen, wherein a patient is administered two or
more drugs
to treat a disease. Depending on the disease or disorder, the exact
circumstances of
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administering the drug can vary, but would still represent a combination. For
example,
when treating an acute bacterial infection, a physician might consider to
administer two
antibacterial drugs to the patient, where administering these drugs within an
interval of
several minutes to hours or even a few days would be considered a combination.
For a
chronic disease, administration of two drugs within an interval of days or
weeks could be
considered a combination, as long as it is done by the physician as parts of a
treatment,
that is with the intention of treating the respective disease.
To combine refers to the act of bringing two or more individual units,
ingredients or
compounds into close relationship. They can be brought together in a spatial
sense, e.g.
combined into one mixture, also referred to as intermixing or blending. For
example, to
prepare a cookie dough several ingredients are being mixed together. This can
happen by
filling them all into a container and mixing them, or by placing one
ingredient in a
container and subsequently, while mixing, adding the remaining ingredients.
In the example described above, one can bring two or more pharmaceutical
ingredients
together, form a mixture, press the mixture into a tablet, then subsequently
coat and
package the tablet. By bringing the ingredients together one combines them
within the
meaning of the present invention.
In a second meaning, 'to combine' refers to a temporal relationship, i.e. two
or more acts
are performed simultaneously or in temporal proxirnityz as discussed above
under
Combination.
Throughout this document, for the sake of simplicity, the use of singular
language is given
preference over plural language, but is generally meant to include the plural
language if not
otherwise stated. E.g., the expression "A method of treating a disease in a
patient,
comprising administering to a patient an effective amount of a compound of
claim 1" is
meant to include the simultaneous treatment of more than one disease as well
as the
administration of more than one compound of claim 1.
In another embodiment, the present invention provides a process for preparing
a
combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel, comprising combining 6-
(6-
aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-
diamine and
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Paclitaxel. For example, this process can be performed using techniques known
to the
person skilled in the pharmaceutical art.
In another embodiment, the present invention provides a pharmaceutical
composition comprising a combination of 6-(6-aminopyridin-3-yl)-N4- {4-[(2-
methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel; and a
process for
preparing a pliarmaceutical composition comprising a combination of 6-(6-
aminopyridin-
3-yl)-N4-{4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with
Paclitaxel,
comprising combining 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine and Paclitaxel. and converting them into
a suitable
pharmaceutical composition. For example, this process can be performed using
techniques known to the person skilled in the pharmaceutical art.
In yet another embodiment, the present invention provides a pharmaceutical
composition comprising a combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-
methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel and a
pharmaceutically acceptable carrier; and a process for preparing a
pharmaceutical
composition comprising a combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-
methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel and a
pharmaceutically acceptable carrier, comprising combining 6-(6-aminopyridin-3-
yl)-N4-
{4-[(2-methylpS,ridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel
and a
pharmaceutically acceptable carrier.
In yet another embodiment, the present invention provides a pharmaceutical
composition comprising a combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-
methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel and a
pharmaceutically acceptable carrier, wherein said pharmaceutical composition
is suitable
for parenteral administration.
Parenteral administration can be carried out by avoiding an absorption step
(e.g. by
intravenous, intraarterial, intracardial, intraspinal or intralumbar
administration) or by
including absorption (e.g. by intramuscular, subcutaneous, intracutaneous or
intraperitoneal administration). Suitable parenteral administration forms are
for example
injection and infusion formulations in the form of solutions, suspensions,
emulsions,
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lyophilisates and sterile powders. Such parenteral pharmaceutical compositions
are
described in Part 8, Chapter 84 of Remington's Pharmaceutical Sciences, 18"'
ed. 1990,
Mack Publishing Group, Enolo.
In one embodiment, the invention relates to intravenous (i.v.) application of
the
active compounds, e.g. as bolus injection (that is as single dose, e.g. per
syringe), infusion
over a short period of time (e.g. for up to one hour) or infusion over a long
period of time
(e.g. for more than one hour). The 'application can also be done by
intermittent dosing. The
applied volume can vary dependent on the conditions and usually is 0.5 to 30,
or 1 to 20
ml for bolus injection, 25 to 500, or 50 to 250 ml for infusion over a short
period of time
and 50 to 1000, or 100 to 500 ml for infusion over a long period of time.
Thus, in yet
another embodiment, the present invention provides a pharmaceutical
composition
suitable for intraveneous administration over a short or long period of time.
The application forms should be sterile and free of pyrogens. They can be
based on
aqueous solvents or mixtures of aqueous and organic solvents. Examples are
ethanol,
polyethyleneglycol (PEG) 300 or 400, aqueous solutions containing
cyclodextrins or
emulsifiers, such as lecithin, Pluronic F68 , Solutol HS15 or Cremophor .
Aqueous
solutions are preferred.
For intravenous application the solutions are generally isotonic and -
euhydric, for example
with a pH of 3 to 11, 6 to 8 or about 7.4.
Glass or-plastic containers can be employed as packaging for i.v.-solutions,
e.g. rubber
seal vials. They can contain liquid volumes of I to 1000, or 5 to 50 ml. The
solution can
directly be withdrawn from the vial to be applied to the patient. For this
purpose, it can be
advantageous to provide the active compound in solid form (e.g. as
lyophilisate) and
dissolve by adding the solvent to the vial directly before administration.
Solutions for infusion can advantageously be packaged in containers made from
glass or plastic, for example bottles or collapsible containers such as bags.
They can
contain liquid volumes of I to 1000, or 50 to 500 ml.
In yet another embodiment, the present invention provides a pharmaceutical
composition comprising 6-(6-aminopyridin-3-yl)-1V4-{4-[(2-methylpyridin-4-
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yl)oxy]phenyl}pyrimidine-2,4-diamine, Paclitaxel, purified Cremophor EL and
dehydrated ethyl alcohol.
In yet another embodiment, the present invention provides a packaged
pharmaceutical composition, comprising a container comprising a combination of
6-(6-
aminopyridin-3 -yl)-N - {4-[(2-methylpyridi n-4-yl) oxy]phenyl } pyrimi dine-
2,4-diamine
with Paclitaxel and a pharmaceutically acceptable carrier and instructions for
using the
pharmaceutical composition to treat a disease in a-patient; and.a packaged
pharmaceutical
-composition, wherein the container is a bottle or collabsible bag.
In yet another embodiment, the present invention provides a kit-of-parts,
comprising a plurality of containers comprising 6-(6-aminopyridin-3-yl)-N4 -{4-
[(2-
methylpyridin-4-yl)oxy]phenyl}pyrirnidine-2,4-diamine and Paclitaxel or both,
optionally
further comprising instructions for using the kit-of-parts to treat a disease
in a patient.
In yet another embodiment, the present invention provides a process for
preparing
a combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel, comprising combining 6-
(6-
aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-
diamine
with Paclitaxel, purified Cremophor EL and dehydrated ethyl alcohol.
In another embodiment, the present invention provides a method for treating a
disorder, comprising administering to a patient a pharmaceutically effective
amount of a
combination of 6-(6-arninopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel; or a method for treating
a disorder,
comprising administering to a patient a pharmaceutically effective amount of a
pharmaceutical composition comprising a combination of 6-(6-aminopyridin-3-yl)-
N'4-{4-
[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel and a
pharmaceutically acceptable carrier; or said method, wherein the is suitable
for
intraveneous administration; or wherein the intraveneous administration is
intraveneous
administration over a short or long period of time; or said pharmaceutical
composition
suitable for intraveneous administration; or a method for treating a disorder,
comprising
administering to a patient a pharmaceutically effective amount of a
pharmaceutical
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composition comprising a combination of 6-(6-aminopyridin-3-yi)-N4-{4-[(2-
methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine, Paclitaxel, purified
Cremophor
EL and dehydrated ethyl alcohol.
In yet another embodiment, the present invention provides said method for
treating
a disorder, wherein said disorder is cancer. In yet another embodiment, the
present
invention provides a method for treating cancer, wherein said cancer is small
cell lung
cancer.
In yet another embodiment, the present invention provides a method for
treating a
disorder, comprising administering to a patient a pharmaceutically effective
amount of a
combination of 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel, wherein 6-(6-
aminopyridin-3-yl)-
N¾-{4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine and Paclitaxel
are
administered simultaneously or in temporal proximity, such as within 24 hours.
In yet another embodiment, the present invention provides a method for
treating a
disorder, comprising administering to a patient a pharmaceutically effective
amount of a
combination of 6-(6-aminopyridin-3-yl)-N4- {4-[(2-rnethylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel, wherein 6-(6-
aminopyridin-3-yl)-
N4-{4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine together with
6mg
Paclitaxel, 527 mg of purified Cremophor EL (polyoxyethylated castor oil) and
49.7%
(v/v) dehydrated alcohol per ml are diluted in 0.9% Sodium Chloride Injection,
USP; 5%
Dextrose Injection, USP; 5% Dextrose and 0.9% Sodium Chloride Injection, USP,
or 5%
Dextrose in Ringer's Injection and administered into a vein (intravenous
infusion,
optionally further by administering certain premedications to reduce or
prevent the chance
of having a severe allergic reaction.
In yet another embodiment, the present invention provides a method for
treating a
disorder, comprising administering to a patient a pharmaceutically effective
amount of a
combination of 6-(6-aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine with Paclitaxel, wherein less than the
maximum
tolerated dose is ' administered or patient weight loss is minimized.
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Examnles
Paclitaxel is commercially available or can be prepared similarly to known
processes. 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-yl)oxy]phenyl}
pyrimidine-
2,4-diamine is a dry powder with a color ranging from white to ivory or light
yellow and
can be prepared as follows:
Step 1: Preparation of i4-((2-Methyl)yridin-4-yl)oxyl phenyl} amine
0
N
N H 2
4-aminophenol (44.9 g, 410 mmol) was added to the 1L 3-neck flask and
dissolved with
N,N-dirnethylacetamide (600 mL). The stirred mixture was then cooled to 9 C
and
potassium t-butoxide (46.18 g, 410 mmol) was added portionwise; the solution
tumed
green and solidified before potassium t-butoxide addition was completed.
Stirring was
reestablished and a solution containing the 4-chloro-2-picoline (50 g, 390
mmol) in N,N-
dimethylacetamide (400 mL) was slowly added and the mixture was heated at 90
C for 17
h. The mixture was then allowed to cool to 45 C, filtered and concentrated to
near
dryness in vacuo to leave brown residue. The residue was slowly added to
vigorously
stirred water (1 L) and the suspension was stirred for lhr. The solids were
then collected
by suction filtration and washed with small amount of isopropanol, ether and
dried to
afford {4-[(2-Methylpyridin-4-yl) oxy] phenyl} amine (49.9 g, 64%) as light
tan solid. 'H
NMR (DMSO-d6) S 8.21 (d, 1H), 6.78 (d, 214), 6.57-6.63 (m, 4H), 5.10 (s, 2H),
2.35 (s,
3H); MS ES 201 (M+H){, calcd 201, RT = 1.04 min.
Step 2: Preparation of 6-chloro-N4-(4-1f2-methypyridin-4-
ylloxylphenyl)pyrimidine-2,4-
diamine
0
N
NH
N
~
H2N~N CI
{4-[(2-Methylpyridin-4-yl) oxy] phenyl} amine (47.5 g, 237 mmol) and 2-amino-
4,6-
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dichloropyrimidine (40.8 g, 249 mmol) were suspended in water (900 mL) and 2-
propanol
(300 mL). 2M aqueous hydrochloric acid solution (23.7 mL) was then added and
the
mixture was then heated at 95 C for 17 h. The mixture was then allowed to
come to rt and
solids were collected by suction filtration and washed with small amount of
isopropanol.
The solids were then resuspended in DMF and heated at 90 C. Triethylamine (20
mL)
was then added and the mixture was stir for additional 10 min at 90 C. Water
was then
added in excess until cloudiness persisted at temperature. This was cooled to
about 5 C
and precipitate formed were collected by suction filtration, washed with water
and dried in
vacuum oven at 40 C to afford desired product (50 g, 64%) as light tan solid.
' H NMR
(DMSO-d6) S 9.40 (s, 1H), 8.27 (d, 1H), 7.76 (d, 2H), 7.06 (d, 2H), 6.75 (brs,
2H), 6.72
(d, IH), 6.66 (s, 1H), 5.98 (s, 1H); MS ES 328 (M+H)+, calcd 328, RT = 1.45
min.
Step 3: Preparation of. 6-(6-aminopyridin-3-yl)-N4-{4-f(2-methylpyridin-4-
yl)oxylnhenyl lpyrimidine-2,4-diamine
To a mixture of 6-chloro-N4-(4-{[2-methylpyridin-4-yl]oxy}phenyl)pyrimidine-
2,4-
diamine (2.0 g, 6.1 mmol) and 2-Amino-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-
yl)pyridine (1.6 g, 7.3 mmol) in DMF (30 mL) was added aqueous Na2CO3 (2 M,
9.0 mL)
and 1,1'-bis(diphenylphosphino)ferrocenepalladium (II) chloride (223 mg, 0.3
mmol).
The resulting mixture was degassed for 10 min before it was heated at 80 C
overnight.
The resulting mixture was cooled to rt before it was concentrated under vacuo
and
dissolved in EtOAc. The resulting mixture was washed with water and brine and
the
organic layer was dried over NaZSO4. Removal of the solvent under vacuo gave
the crude
material, which was purified with 40 M biotage eluting with 100% EtOAc first
and then
with 95% CH2CI2 and 5% 2 N ammonia in MeOH to provide the title compound as an
off-
white solid (1340 mg, 57%): 'H NMR (DMSO-d6): S 9.20 (s, 1H), 8.51 (dd, 1H),
8.27 (d,
IH), 7.81-7.88 (m, 3H), 7.03-7.06 (m, 2H), 6.72 (d, IH), 6.68 (dd, 1H), 6.47
(dd, 1H),
6.33 (s, IH), 6.32 (s, 2H), 6.23 (s, 2H), 2.39 (s, 3H) ppm; MS ES 386 (M+H)+,
RT = 1.06
min.
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A. Physiological activity
The utility of the compounds of the present invention can be illustrated, for
example, by their activity in vitro in the in vitro tumor cellular
proliferation assay
described below. The .link between activity in tumor cell proliferation assays
in vitro and
anti-tumor activity in the clinical setting has been very well established in
the art. For
example, the therapeutic utility of taxol (Silvestrini et al. Stem Cells 1993,
11(6), 528-35),
taxotere (Bissery et al. Anti Cancer Drugs 1995, 6(3), 339), and topoisomerase
inhibitors
(Edelman et al. Cancer Chemother. Pharmacol. 1996, 37(5), 385-93) were
demonstrated
with the use of in vitro tumor proliferation assays.
The in vitro effect of the compounds according to the invention can be
demonstrated in the following Cellular Proliferation Assay:
Tumor cells are seeded on Day 0 at subconfluent densities in RPMI growth
medium with 10% FBS and incubated at 37 C in a humidified incubator
containing 5%
COa. On Day 1, cultures are treated with various concentrations of 6-(6-
aminopyridin-3-
yl)-N4-{4-[(2-methylpyridin-4-yl)oxy]phenyl}pyrimidine-2,4-diamine and
Paclitaxel (0 -
10 M, 0.1% final DMSO (dimethylsulfoxide) concentration) for 72 hours. Cell
viability
is assessed using Alarnar Blue staining per the manufacturer's protocol (Trek
Diagnostic
Systems, Inc., Cleveland, Ohio) or measuring the ATP content using the
Ce1lTiter-Glo
Assay (Promega, Wisonsin, USA).
The in vivo effect of the compounds according to the invention can be
demonstrated in the NCI-H460 NSCLC xenoQraft model:
Animals
Ncr nulnu female mice (Taconic Farms, Germantown, NY, USA) were used for all
in vivo studies. The mice were housed and maintained within the Comparative
Medicine
Department at Bayer Corporation, West Haven, CT in accordance with Bayer
IACUC,
State, and Federal guidelines for the humane treatment and care of laboratory
animals.
Mice received food and water ad libitum.
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Compounds
6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-yl)oxyJphenyl}pyrimidine-
2,4-diamine (here called Compound 1) was stored in the dark until used.
Clinical grade Paclitaxel (Bristol Myers Squibb, USA) came supplied as a dry
powder. It was stored at room temperature as indicated on the package insert.
Vehicles
Cremophor EL /Ethanol (50:50) (Sigma Cremophor EL Cat.# C-5135; 500g, 95%
Ethyl Alcohol), was prepared as a stock solution, wrapped with aluminum foil,
and stored
at room temperature. Paclitaxel was formulated at 4-fold (4X) of the highest
dose in this
Cremophor EL/Ethanol (50:50) solution. This 4X stock solution was prepared
fresh daily.
Final dosing solutions were prepared by dilution to IX with endotoxin screened
distilled
H20 (GIBCO, Cat.# 15230-147) and mixed by vortexing immediately prior to
dosing.
Solutol HS15 /Ethanol (50:50) (BAXF Solutol HS15, 95% ethyl alcohol) was
prepared as a stock solution, wrapped with aluminum foil, and stored at room
temperature.
Compound 1 was formulated at 5-fold (5X) of the highest dose in this
Solutol/Ethanol
(50:50) solution. This 5X stock solution was prepared fresh daily. Final
dosing solutions
were prepared by dilution to 1X with 0.9% saline and mixed by vortexing
immediately
prior to dosing.
Tumor Lines
The NCI-H460 human non-small-cell lung carcinoma line was obtained from the
American Type Tissue Culture Collection Repository. NCI-H460 cells were
maintained
and passaged in vitro using DMEM (GIBCO cat. # 11995-065: 500 mis)
supplemented
with 10% heat inactivated fetal bovine serum (JRH Biosciences cat.# 12106-
500M), 2mM.
L-glutamine (GIBCO cat. # 25030-81), 10mM HEPES buffer (GIBCO cat # 15630-080)
and penicillin-streptomycin (GIBCO cat. # 15140-122: 5 mis/ 50 mis DMEM).
Cells were
maintained at 37 C and 5% CO2 in a Fisher Scientific 610 CO2 incubator.
Tumor Xenograft Experiments
Studies with the NCI-H460 cells were initiated by harvesting cells from an in
vitro
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culture by adding Trypsin-EDTA (GIBCO cat#25200-056 ) for 2 minutes followed
by
centrifugation of the cells into a pellet and resuspension in HBSS (GLBCO cat#
14025-
092) to a final cell count of 5 x 107 viable cells/ml. A volume of 0.1ml of
the cell
suspension was injected s.c. in the right flank of each mouse. All treatment
was initiated
when all mice in the experiment had established tumors ranging in size from
100 to 150
mg. The general health of mice was monitored and mortality was recorded daily.
Tumor
dimensions and body weights were recorded two to three times a week starting
with the
first day of treatment. Animals were euthanized according to Bayer IACUC
guidelines.
The maximum tolerated dose (MTD) is defined as the highest dose that produces
less than
20% lethality and/or 20% net body weight loss.
Compound I was administered i.v. on a q4d x 3 schedule and paclitaxel was
administered i.v on a q2d x 5 schedule. On days when both compounds were
administered, compound I was dosed first and paclitaxel was administered 4 h
later. Both
agents were dosed up to their respective MTD (maximum tolerated dose). Tumor
weights
(mg) were calculated using the equation (I x w2)/2, where I and w refer to the
larger and
smaller dimensions collected at each measurement. There were 10 mice per
group.
Results
An evaluation endpoint of three tumor mass doublings was used to evaluate the
anti-tumor efficacy of the combination of these agents at their MTDs to the
efficacy of the
corresponding single agents as represented in Table 1. The tumor growth delay
produced
by each individual therapy was approximately 8 days (median time to reach the
evaluation
endpoint in the control groups was 7 days and 15 days for both compound 1 and
paclitaxel. The tumor growth delay produced by the combination therapy was
approximately 14 days (the time to reach the evaluation endpoint in the
combination
therapy group was 21 days). There was no lethality on any treatment. The
average tumor
weight at each day of measurement (mean standard error of the mean, SEM) for
each
group is summarized in Table 1.
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Mean Tumor Weight (mg SEM on each day post-implant)
Day Day 10 Day 12 Day 14 Day 17 Day 20 Day Day
6 24 28
Control 135 428 t 794 t 1234 t 1768 t
6 23 50 79 110
Vehicles 126 t 409 t 762 1193 t 1873 t
39 67 115 162
compound 1 (40 124 t 239 t 295 t 351 464 t 802 t 1466
mg/kg) + vehicle 5 37 50 72 80 144 253
vehicle + Paclitaxel 124 263 326 366 528 962
(15 mg/kg) 4 44 53 87 131 289
compound 1 (40 129 t 176 t 172 t 147 t 132 t 221 t 526 t 1229
mg/kg) + PacEitaxel 5 22 24 27 24 55 155 t 286
(15 mg/kg)
Table 1: Effect of compound 1 and paclitaxel on growth of H460 xenografts in
NCr
mice
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= B. Operative examples relatintZ to pharmaceutical compositions
The compounds according to the invention can be converted into pharmaceutical
preparations as follows:
Solution for intravenous adrninistration 1:
Composition: I mg 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine, 1 mg Paclitaxel, 15 g polyethylenglykol
400 and
250 g water optionally with up to 15 % Cremophor EL, and optionally up to 15%
ethyl
alcohol, and optionally up to 2 equivalents of a pharmaceutically suitable
acid such as
citric acid or hydrochloric acid.
Preparation:
1 mg 6-(6-aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-
diamine, I mg Paclitaxel, and the polyethylenglyko1400 are dissolved in the
water with
stirring. The solution is sterile filtered (pore size 0.22 m) and filled into
heat sterilized
infusion bottles under aseptical conditions. The infusion bottles are being
sealed with
rubber seals.
Solution for intravenous administration 2:
Composition: I mg 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine, 1 mg Paclitaxel, saline solution,
optionally with
up to 15 % by weight of Cremophor EL, and optionally up to 15% by weight of
ethyl
alcohol, and optionally up to 2 equivalents of a pharmaceutically suitable
acid such as
citric acid or hydrochloric acid.
Preparation:
1 mg 6-(6-aminopyridin-3-yl)-N4- {4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-
diamine and 1 mg Paclitaxel are dissolved in the saline solution with
stirring. Optionally
Cremophor EL, ethyl alcohol or acid are added. The solution is sterile
filtered (pore size
0.22 m) and filled into heat sterilized infusion bottles under aseptical
conditions. The
infusion bottles are being sealed with rubber seals.
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Solution for intravenous administration 3:
Composition: 1 mg 6-(6-aminopyridin-3-yl)-N4-{4-[(2-methylpyridin-4-
yl)oxy]phenyl}pyrimidine-2,4-diamine, 6 mg Paclitaxel, 527 mg of purified
Cremophor
EL, 49.7% (v/v) dehydrated ethyl alcohol USP per mL solution, dilution with a
in 0.9%
Sodium Chloride Injection, USP; 5% Dextrose Injection, USP; 5% Dextrose and
0.9%
Sodium Chloride Injection, USP, or 5% Dextrose in Ringer's 7njection.
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