Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL FORMULATIONS CONTAINING 17-ALLYLAMINO-
17-DEMETHOXYGELDANAMYCIN
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
[0001] This invention relates to pharmaceutical formulations containing 17-
allylamino-17-
demethoxygeldanamycin ("17-AAG") and methods for their preparation and use.
2. DESCRIPTION OF RELATED ART
[0002] Geldanamycin belongs to the ansamycin family of natural products, whose
members
are characterized by a benzenoid nucleus (typically a benzoquinone or
hydroquinone nucleus)
connected at two naeta positions to form a macrolactam. Besides geldanamycin,
the
ansamycins include the macbecins, the herbimycins, the TAN-420s, and
reblastatin.
[0003] Geldanamycin and its derivatives are the most extensively studied of
the ansamycins.
Although geldanamycin was originally identified as a result of screening for
antibiotic
activity, current interest in it is based primarily on its cytotoxicity
towards tumor cells and,
therefore, its potential as an anticancer agent. It is an inhibitor of heat
shock protein-90
("Hsp90"), which is involved in the folding, activation and assembly of a wide
range of
proteins ("client proteins"), including key proteins involved in signal
transduction, cell cycle
control and transcriptional regulation. The binding of geldanamycin to Hsp90
disrupts
Hsp90-client protein interactions, preventing the client proteins from folding
correctly and
rendering them susceptible to proteasome-mediated destruction. Among the Hsp90
client
proteins are many mutated or overexpressed proteins implicated in cancer: p53,
Bcr-Abl
lcinase, Raf-I kinase, Akt kinase, Npm-Alk kinase p185ErB2 transmembrane
kinase, Cdk4,
Cdk6, Weel (a cell cycle-dependent kinase), Her2/Neu (ErbB2), and hypoxia
inducible
factor-la (HIF-la). However, the hepatotoxicity and poor bioavailability of
geldanamycin
have led to its discontinuation as a clinical candidate.
[0004] Nevertheless, interest persists in the development of geldanamycin
derivatives or
analogs having geldanamycin-like bioactivity, but with a more pharmaceutically
acceptable
spectrum of properties. Position 17 of geldanamycin has been an attractive
focal point,
chemically speaking, for the synthesis of geldanamycin derivatives because the
methoxy
group there is readily displaced by a nucleophile, providing a convenient
entry into 17-
substituted-l7-demethoxygeldanamycins. Further, structure-activity
relationship (SAR)
studies have shown that chemically and sterically diverse 17-substituents can
be introduced
without destroying antitumor activity. See, e.g., Sasaki et al., US 4,261,989
(1981); Schnur
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et al., US 5,932,566 (1999); Schnur et al., J. Med. Chem., 38, 3806-3812
(1995); Schnur et
al., J Med. Cliem. 38, 3813-3820 (1995); and Santi et at., US 2003/0114450 Al
(2003); the
disclosures of which are incorporated by reference. The SAR inferences are
supported by the
X-ray crystal co-structure of the complex between Hsp90 and a geldanamycin
derivative (17-
DMAG, v. infra), showing that the 17-substituent projects out from the binding
pocket and
into the solvent (Jez et al., Claemistry & Biology, 10, 361-368 (2003)). Thus,
position 17 is
an attractive one for the introduction of property-modulating substituents,
such as a
solubilizing group. The best-lcnown 17-substituted geldanamycin derivative is
17-AAG, first
disclosed in Sasalci et at., cited supra, and currently undergoing clinical
trials. Another
noteworthy 17-substituted geldanamycin compound is 17-(2-
dimethylaminoethyl)amino-17-
demethoxygeldanamycin ((" 17-DMAG") (Snader et al., WO 02/079167 Al (2002),
incorporated by reference), also undergoing clinical trials.
[0005] A limitation in the preparation of pharmaceutical formulations
containing
geldanamycin compounds such as geldanamycin itself and 17-AAG, especially for
parenteral
administration, is their very poor water solubility, only about 0.1 mg/mL at
neutral pH for 17-
AAG. (17-DMAG, having an alkyl amino group, is more soluble.) Addressing this
issue,
Tabibi et al., WO 00/37050 (2000) disclosed a formulation for a water
insoluble drug such as
17-AAG comprising (a) the drug, (b) a water-miscible organic solvent for the
drug, (c) a
surfactant, and (d) water. The water miscible solvent can be dimethylsulfoxide
(DMSO),
dimethylformamide, ethanol, glycerin, propylene glycol, or polyethylene
glycol. The
surfactant preferably is a phospholipid (especially egg phospholipid).
[0006] Another disclosure of interest is Ulm et al., WO 03/086381 (2003),
which discloses a
method for preparing pharmaceutical formulations for ansamycins by (a)
providing the
ansamycin dissolved in ethanol; (b) niixing the product of step (a) with a
medium chain
triglyceride to form a first mixture; (c) substantially removing the ethanol
from the first
mixture; (d) combining the product of step (c) with an emulsifying agent and a
stabilizer to
form a second mixture; and (e) emulsifying the second mixture. The emulsified
second
mixture optionally can be lyophilized and then re-hydrated. In a specific
combination, the
medium chain triglyceride comprises caprylic and/or caproic acid, the
emulsifying agent
comprises phosphotidylcholine, and stabilizer comprises sucrose. The
aforementioned
documents are incorporated herein by reference.
BRIEF SUMMARY OF THE INVENTION
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[0007] The present invention provides a pharmaceutical formulation for 17-AAG,
a method
for administering 17-AAG to a patient in need thereof, and a method for
preparing a
pharmaceutical formulation comprising 17-AAG.
[0008] In one aspect, the present invention provides a formulation including
17-AAG that is
suitable for intravenous administration. The formulation comprises 17-AAG in a
concentration of between about 1.0 mg/mL and about 5.0 mg/mL dissolved in a
vehicle
comprising (i) a first component that is an aprotic, polar solvent in an
amount between about
0.1 and about 10 volume %; and, (ii) a second component that is an aqueous
mixture
comprising between about 5.0 and about 55 volume % long chain triglycerides,
in an amount
between about 90.0 and 99.9 volume %.
[0009] In another aspect, the present invention provides a method for
administering 17-AAG
to a patient in need thereof, comprising the steps of:
(a) providing a pharmaceutical formulation comprising 17-AAG in a
concentration
of between about 1.0 mg/mL and about 5.0 ing/mL dissolved in a vehicle
comprising (i) a first component that is an aprotic, polar solvent in an
amount
between about 0.1 and about 10 volume %; and, (ii) a second component that is
an aqueous mixture comprising between about 5.0 and about 55 volume % long
chain triglycerides, in an amount between about 90.0 and 99.9 volume %.
(b) including the pharmaceutical formulation into an apparatus adapted to
deliver it
intravenously to a patient through an intravenous route; and,
(c) administering the formulation intravenously to a patient.
[0010] In yet another aspect, the present invention provides a method for
preparing a
pharmaceutical formulation comprising 17-AAG, comprising the steps of:
(a) providing an amount of 17-AAG;
(b) combining the 17-AAG with a first component, which is an aprotic, polar
solvent, to provide a solution of the 17-AAG in the aprotic, polar solvent;
and,
(c) combining the solution with a second component, wherein the second
component comprises an aqueous mixture comprising between about 5.0 and
about 55.0 volume % long chain triglycerides in an amount between about 90.0
and 99.9 volume % of the final formulation;
thereby forming the pharmaceutical formulation.
DETAILED DESCRIPTION OF THE INVENTION
Definitiotts
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[0011 ] Aprotic, polar solvents are solvents that do not contain an 0-H group
but still possess
a fairly large dipole. Examples of aprotic, polar solvents include, without
limitation,
dimethylsulfoxide (i.e., DMSO), N,N-dimethylacetamide (i.e., DMA), N,N-
dimethylformamide (i.e., DMF), and N-methylpyrrolidone (i.e., NMP).
[0012] Intralipid, either 10% or 20%, includes soybean oil, phospholipids
(lecithin), glycerin
and water for injection. One liter of Intralipid 10% contains the following:
100 g of purified
soybean oil; 12 g of purified egg phospholipids, 22 g anhydrous glycerin, and
water to a
volume of 1 liter. The pH is adjusted with sodium hydroxide to approximately
8. One liter
of Intralipid 20% contains the following: 200 g of purified soybean oil; 12 g
of purified egg
phospholipids, 22 g anhydrous glycerol, and water to a volume of 1 liter. The
pH is adjusted
with sodium hydroxide to approximately 8. The Intralipids are typically stored
at a
controlled temperature below 25 C.
[0013] Liposyn II, either 10% or 20%, includes safflower oil, soybean oil,
phospholipids
(lecithin), glycerin and water for injection. Liposyn 1110% contains 5 weight
% safflower
oil, 5 weight % soybean oil, up to 1.2 weight % egg phosphatides added as an
emulsifier, and
2.5 weight % glycerin in water for injection. The pH may be adjusted with
sodium hydroxide
to between 6 and 9. Liposyn II 20% contains 10 weight % safflower oil, 10
weight %
soybean oil, 1.2 weight % egg phosphatides, and 2.5 weight % glycerin in water
for injection..
The pH may be adjusted with sodium hydroxide to between 6 and 9.
[0014] Lecithin is a mixture of the diglycerides of stearic, palmitic, and
oleic acids, linked to
the choline ester of phosphoric acid. Commercial lecithin is typically soybean
lecithin, which
contains 11.7% palmitic acid, 4.0% stearic acid, 8.6% palmitoleic acid, 9.8%
oleic acid,
55.0% linoleic acid, 4.0% linolenic acid, and 5.5% C20 to C22 acids. Egg
lecithins are one
suitable type of lecithin.
[0015] Linoleic acid is a fatty acid of the formula C1$H32O2.
[0016] Linolenic acid is a fatty acid of the formula C18H3002.
[0017] Long chain triglycerides are triglyceride compositions that include
fatty acids ranging
from 12 to 22 carbons in length as the predominant constituent, preferably 16
to 20 carbons
in length.
[0018] Medium chain triglycerides are triglyceride compositions that include
fatty acids
ranging from 7 to 11 carbons in length as the predominant constituent,
preferably 8 to 10
carbons in length.
[0019] Oleic acid is a fatty acid of the formula C18H3402.
[0020] Palmitic acid is a fatty acid of the formula C16H3202.
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[0021] Safflower oil is a mixture of triglycerides of palmitic acid (6.4%),
stearic acid (3.1%),
arachidic (0.2%), oleic acid (13.4%), linoleic acid (76.6-79.0%), and
linolenic acid (0.04-
0.13%).
[0022] Sesame oil comprises primarily triglycerides of oleic and linoleic
acids. However, it
appears to adversely affect the stability of 17-AAG and is not as desirable
for this reason.
[0023] Stearic acid is a fatty acid of the foimula C18H3G02.
[0024] Soybean oil is a mixture of triglycerides of oleic acid (26%), linoleic
acid (49%)
linolenic acid (11%), and saturated acids (14%).
[0025] Soybean oil/safflower oil in a 50/50 mixture includes triglycerides
composed of
approximately 65.8% linoleic acid, 17.7% oleic acid, 8.8% palmitic acid, 3.4%
stearic acid,
and 4.2% linolenic acid.
Description
[0026] The pharmaceutical formulation of the present invention includes 17-AAG
and is
suitable for intravenous administration. The forinulation comprises 17-AAG in
a
concentration of between about 1.0 mg/mL and about 5.0 mg/mL dissolved in a
vehicle
comprising (i) a first component that is an aprotic, polar solvent in an
amount between about
0.1 and about 10 volume %; and, (ii) a second component that is an aqueous
mixture
comprising between about 5.0 and about 55.0 volume % long chain triglycerides,
in an
amount between about 90.0 and 99.9 volume %.
[0027] The concentration of 17-AAG in the formulation is typically between
about 1.25
mg/mL and 4.0 mg/mL. Where the aprotic, polar solvent in the formulation is
DMSO, the
concentration of 17-AAG oftentimes ranges from about 2.0 mg/mL to about 3.0
mg/mL;
where the aprotic, polar solvent is DMA, the concentration oftentimes ranges
from about 1.50
mg/mL to about 3.0 mg/mL.
[0028] Aprotic, polar solvents are typically present in the formulation in an
amount between
about 0.5 and about 5.0 volume %. The concentration of such solvents is
maintained within
FDA acceptable limits due to toxicity at higher concentrations. For example,
the tolerated
dose for DMA is approximately 14.8 g/m2. Oftentimes, the concentration of
aprotic, polar
solvent ranges from about 1.0 to about 4.0 volume %.
[0029] The second component of the vehicle is typically an aqueous mixture
comprising
between about 5.0 and about 55.0 volume % long chain triglycerides. Examples
of suitable
second components include, without limitation, the following: Intralipid 10%;
Intralipid
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20%; Liposyn II 10%; and Liposyn II 20%. For these mixtures, the triglyceride
content
oftentimes ranges from about 7.5 to about 30 volume %.
[0030] Phospholipids, preferably egg phospholipids, are an optional third
component of the
vehicle. Where the second component is Intralipid or Liposyn II, the
phospholipid
concentration oftentimes ranges from about 0.8 to about 3.0 volume percent,
with 1.0 to 2.0
volume percent being preferred.
[0031 ] Examples of suitable 17-AAG of the present invention include, without
limitation, the
following:
[0032] Formulation 1-comprising
a) concentration of 17-AAG of between about 1 mg/niL and about 3 mg/mL;
b) first component is DMSO present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0033] Formulation 2-conzprising
a) concentration of 17-AAG of between about 2.0 mg/mL and about 3.0 mg/mL;
b) first component is DMSO present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0034] Fonnulation 3-conaprisiyzg
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 voluMe % long chain triglycerides.
[0035] Fot-naulation 4-comprising
a) concentration of 17-AAG of between about 2.25 mg/mI, to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0036] Formulation 5-comprising
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
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b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides.
[0037] Formulation 6-comprising
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid, oleic acid and palmitic acid.
[0038] Forniaulation 7-conapr-ising
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids).
[0039] Fonnulatlon 8-Cofnprising
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %;
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids); and,
d) third component-phospholipids-present in an amount between about 1.0
percent and 2.0 volume percent.
[0040] Fonnulation 9-cofnprising
a) concentration of 17-AAG of between about 2.25 mg/mL to about 2.75 mg/mL;
b) first component is DMSO present in an amount between about 2.0 to about 3.5
volume %;
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c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids);
d) third component-egg phospholipids-present in an amount between about 1.0
w and 2.0 volume percent; and,
e) glycerin in an amount between about 2.0 and 3.0 volume %.
[0041] Fonnulation 10-comprising
a) concentration of 17-AAG of between about 1.0 mg/mL and about 3.0 mg/mL;
b) first component is DMA present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0042] Formulation 11-coinprising
a) concentration of 17-AAG of between about 1.5 mg/mL and about 3 mg/mL;
b) first component is DMA present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0043] Fof-niulation 12-comprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 1.0 to about 4.0
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0044] Fof-inulation 13-eofnprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 7.5 and
30.0 volume % long chain triglycerides.
[0045] Fonazulation 14-comprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
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b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides.
[0046] Fonzzulation 1 S-comprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid, oleic acid and palmitic acid.
[0047] Formulation 16-comprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %; and,
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids).
[0048] Fo>~mulation 17-compYising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %;
c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids); and,
d) third component-phospholipids-present in an amount between about 1.0 and
2.0 volume percent.
[0049] Fonnulation 18-comprising
a) concentration of 17-AAG of between about 1.65 mg/mL to about 2.50 mg/mL;
b) first component is DMA present in an amount between about 2.0 to about 3.5
volume %;
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c) second component is an aqueous mixture comprising between about 15.0 and
30.0 volume % long chain triglycerides, wherein the fatty acid component of
the triglycerides comprises linoleic acid (50% to 75% of fatty acids) , oleic
acid
(15% to 25% fatty acids) and palmitic acid (7% to 10% fatty acids);
d) third component-egg phospholipids-present in an amount between about 1.0
and 2.0 volume percent; and,
e) glycerin in an amount between about 2.0 and 3.0 volume %.
[0050] The formulations of the present invention exhibit stability for a
period of hours at a
variety of temperatures. For instance, the 17-AAG of Formulations 1, 2, 3, 4,
5, 6, 7, 8, and
9-at room temperature, 4 C, and -20 C-degrades less than 5% over a period of
7 hours,
preferably less than 2.5%, and more preferably less than 1%; the 17-AAG of
Formulations
10, 11, 12, 13, 14, 15, 16, 17, and 18 exhibits the same stability.
Furthermore, the 17-AAG
of Formulations 1 through 18-at room temperature, 4 C, and -20 C-degrades
less than
5% over a period of 14 hours, preferably less than 2.5%, and more preferably
less than 1%.
[0051 ] Compared to prior art formulations, the present formulation offers a
number of
advantages. It is easily prepared and stored, and it avoids the use of
excessive amounts of
DMSO, which can have poor patient acceptance due to its odor. The present
formulation
furthermore allows the delivery of a requisite amount of 17-AAG within an
acceptable
infusion time (e.g., 90 min.).
[0052] The present invention also provides a method for administering 17-AAG
to a patient
in need thereof, comprising the steps of:
(a) providing a pharmaceutical formulation comprising 17-AAG in a
concentration
of between about 1 mg/mL and about 5 mg/mL dissolved in a vehicle
comprising (i) a first component that is an aprotic, polar solvent in an
amount
between about 0.1 and about 10 volume %; and, (ii) a second component
comprising between about 5.0 and about 55.0 volume % long chain
triglycerides in an amount between 90.0 and 99.9 volume %;
(b) including the pharmaceutical formulation into an apparatus that can
deliver it to
a patient through an intravenous route; and,
(c) administering the formulation intravenously to a patient.
[0053] Examples of 17-AAG formulations administered to patients include,
without
limitation, Formulations 1 through 18 discussed above. Any suitable apparatus
may be used
to delivery the formulation intravenously, including an IV bag with attached
medical tubing.
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Finally, the rate of intravenous delivery can be readily determined by one of
ordinary skill in
the art using well-known methods.
[0054] The present invention further provides a method for preparing a
pharmaceutical
foimulation comprising 17-AAG, comprising the steps of:
(a) providing an amount of 17-AAG;
(b) combining the 17-AAG with a first component, which is an aprotic, polar
solvent to provide a solution;
(c) combining the solution with a second component, wherein the second
component comprises an aqueous mixture comprising between about 5.0 and
about 55.0 volume % long chain triglycerides in an amount between about 90.0
and 99.9 volume % of the final formulation;
thereby forming the pharmaceutical formulation.
[0055] The amount of 17-AAG provided is typically such that a concentration
between about
1.0 mg/mL and 5 mg/mL results in the formulation. Where the first component is
DMSO, an
amount is oftentimes provided that results in a formulation concentration from
about 2.0
mg/mL to about 3.0 mg/mL; where the first component is DMA, an amount is
oftentimes
provided that results in a formulation concentration from about 1.50 mg/mL to
about 3.0
mg/mL.
[0056] For the preparation, the 17-AAG is dissolved in the first component-an
aprotic,
polar solvent such as DMSO or DMA-to provide a solution. The solution is
coinbined with
components that typically include a substantial amount of water, along with
long chain
triglycerides and phospholipids. Examples of suitable components include,
without
limitation, Intralipid 10%; Intralipid 20%; Liposyn 1110%; and, Liposyn 1120%.
Should
some degree of precipitation occur upon combining the solution with the
vehicle, the
resulting formulation is usually filtered. Examples of formulations prepared
through this
method include, without limitation, Formulations 1 through 18 discussed above.
[0057] Geldanamycin is a well-known natural product, obtainable by culturing
the producing
organism, Streptofnyces hygroscopicus var. geldafzus NRRL 3602. 17-AAG is made
semi-
synthetically from geldanamycin, by reaction of geldanamycin with allylamine,
as described
in Sasaki et al., US 4,261,989 (1981), the disclosure of which is incorporated
herein by
reference.
[0058] 17-AAG administered via a pharmaceutical solution formulation of this
invention can
be used for treating diseases such as, but not limited to, hyperproliferative
diseases,
including: cancers of the head and neck which include tumors of the head,
neck, nasal cavity,
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paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx,
salivary
glands, and paragangliomas; cancers of the liver and biliary tree,
particularly hepatocellular
carcinoma; intestinal cancers, particularly colorectal cancer; treat ovarian
cancer; small cell
and non-small cell lung cancer; breast cancer sarcomas, such as fibrosarcoma,
malignant
fibrous histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma,
neuioflbrosarcoma,
osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma,
neoplasms of
the central nervous systems, particularly brain cancer; lymphomas such as
Hodglcin's
lymphoma, lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associated
lymphoid tissue lymphoma, mantle cell lymphoma, B-lineage large cell lymphoma,
Burkitt's
lymphoma, and T-cell anaplastic large cell lymphoma. Clinically, practice of
the methods
and use of compositions described herein will result in a reduction in the
size or number of
the cancerous growth and/or a reduction in associated symptoms (where
applicable).
Pathologically, practice of the method and use of compositions described
herein will produce
a pathologically relevant response, such as: inhibition of cancer cell
proliferation, reduction
in the size of the cancer or tumor, prevention of further metastasis, and
inhibition of tumor
angiogenesis. The method of treating such diseases comprises administering a
therapeutically effective amount of an inventive combination to a subject. The
method may
be repeated as necessary.
[0059] Non-cancer disorders that are characterized by cellular
hyperproliferation can also be
treated by 17-AAG administered in accordance with this invention. Illustrative
examples of
such disorders include but are not limited to: atrophic gastritis,
inflammatory hemolytic
anemia, graft rejection, inflammatory neutropenia, bullous pemphigoid, coeliac
disease,
demyelinating neuropathies, dermatomyositis, inflammatory bowel disease
(ulcerative colitis
and Crohn's disease), multiple sclerosis, myocarditis, myositis, nasal polyps,
chronic
sinusitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, surgical
adhesions,
stenosis or restenosis, scleritis, scleroderma, eczema (including atopic
dermatitis. irritant
dermatitis, allergic dermatitis), periodontal disease ( periodontitis),
polycystic kidney disease,
and type I diabetes.
[0060] Other examples include vasculitis (e.g., Giant cell arteritis (temporal
arteritis,
Takayasu's arteritis), polyarteritis nodosa, allergic angiitis and
granulomatosis (Churg-
Strauss disease), polyangitis overlap syndrome, hypersensitivity vasculitis
(Henoch-
Schonlein purpura), serum sickness, drug-induced vasculitis, infectious
vasculitis, neoplastic
vasculitis, vasculitis associated with connective tissue disorders, vasculitis
associated with
congenital deficiencies of the complement system, Wegener's granulomatosis,
Kawasaki's
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disease, vasculitis of the central nervous system, Buerger's disease and
systemic sclerosis);
gastrointestinal tract diseases (e.g., pancreatitis, Crohn's disease,
ulcerative colitis, ulcerative
proctitis, primary sclerosing cholangitis, benign strictures of any cause
including ideopathic
(e.g., stiictures of bile ducts, esophagus, duodenum, small bowel or colon);
respiratory tract
diseases (e.g., asthma, hypersensitivity pneumonitis, asbestosis, silicosis
and other forms of
pneumoconiosis, chronic bronchitis and chronic obstructive airway disease);
nasolacrimal
duct diseases (e.g., strictures of all causes including ideopathic); and
eustachean tube diseases
(e.g., strictures of all causes including ideopathic).
[0061] 17-AAG can be administered in combination with other anti-cancer or
cytotoxic
agents, including alkylating agents, angiogenesis inhibitors, antimetabolites,
DNA cleavers,
DNA crosslinlcers, DNA intercalators, DNA minor groove binders, heat shock
protein 90
inhibitors, histone deacetylase inhibitors, microtubule stabilizers,
nucleoside (purine or
pyrimidine) analogs, proteasome inhibitors, topoisomerase (I or II)
inhibitors, tyrosine kinase
inhibitors. Specific anti-cancer or cytotoxic agents include (3-lapachone, 17-
DMAG,
bicalutamide, bleomycin, bortezomib, bisulfan, calicheamycin, camptothecin,
capecitabine,
callistatin A, CC-1065, cisplatin, cryptophycins, daunorubicin,
discodermolide, docetaxel,
doxorubicin, duocarmycin, dynemycin A, epothilones, etoposide, floxuridine,
fludarabine,
fluoruracil, gefitinib, geldanamycin, gemcitabine, hydroxyurea, imatinib,
interferons,
interleukins, irinotecan, leptomycin B, methotrexate, mitomycin C,
oxaliplatin, paclitaxel,
spongistatins, suberoylanilide hydroxamic acid (SAHA), thiotepa, topotecan,
trichostatin A,
vinblastin, vincristine, and vindesine.
[0062] The co-administered anti-cancer or cytotoxic agent can be a protein
kinase inhibitor,
including: quinazolines, particularly 4-anilinoquinazolines such as Iressa
(AstraZeneca; N-
(3- chloro-4-fluorophenyl)-7-methoxy-6-[3(4- quinazolinamine) and Tarceva
(Roche/Genentech; N-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy)-4-
quinazolinamine
monohydrochloride); phenylamino-pyrimidines such as Gleevec (Novartis; 4-[(4-
methyl-l-
piperazinyl)methyl]-N-[4-methyl-3-[ [4-(3-pyridinyl)-2-
pyrimidinyl]amino]phenyl]benzamide); pyrrolo- and pyrazolopyrimidines such as
BIBX
1382 (Boehringer Ingelheim; N8-(3-chloro-4-fluorophenyl)-N-2-(1-rnethyl-4-
piperidinyl)-
pyrimido[5,4-d] pyrimidine-2,8-diamine); indoles and oxindoles such as
Semaxinib
(Pharmacia; 3-[(3,5-dimethyl-1 H-pyrrol-2-yl)methylene]-1,3-dihydro-2H- Indol-
2-one);
benzylidene malononitriles; flavones such as flavopiridol (Aventis; 2=(2-
chlorophenyl)-5 ,7-
dihydroxy-8-[(3S,4R)-3-hydroxy-l-methyl- 4-piperidinyl]-4H-1 -benzopyran-4-
one);
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staurosporines such as CEP-701 (Cephalon); antibodies such as Herceptin
(Genentech); and,
ribozyrnes such as Angiozyme (Ribozyme Pharmaceuticals).
[0063] Using a pharmaceutical solution formulation of this invention, 17-AAG
may be
administered in a dose ranging from about 4 mg/m2 to about 4000 mg/m2
depending on the
frequency of administration. A preferred dosage regimen for 17-AAG is about
450 mg/m2
weelcly (Banerji et al., Proc. Ana, Soc. Clin. Oncol. , 22, 199 (2003,
abstract 797), "A
Pharmacokinetically (PK)-pharmacodynamically (PD) Guided Phase I Trial of the
Heat
Shock Protein 90 (HSP90) Inhibitor 17-Allyl-17-demethoxygeldanamycin
(17AAG)").
Alternatively, a dose of about 308 mg/m2 weekly can be administered. See Goetz
et al., Eur.
J. Cancer, 38 (Supp. 7), S54-S55 (2002), "A phase I trial of 17-Allyl-Amino-
Geldanamycin
(17-AAG) in patients with advanced cancer."
[0064] The practice of this invention can be further understood by reference
to the following
examples, which are provided by way of illustration and not of limitation.
General Experimental Section
[0065] Instruments and reagents used for the experiments described below were
obtained
from the indicated vendor, where applicable: water bath (VWR Polyscience Model
1167);
balance (Mettler-Toledo AT261 Delta Range); centrifuge (Sorval Super T21);
HPLC (Agilent
1100 Series); reverse-phase column (Agilent ZORBAX Eclipse C8 Column); guard
column
(Agilent ZORBAX Eclipse XDB-C8 Column); osmometer (Wescor VaproTM Vapor
Pressure Osmometer); dimethylacetamide (Sigma); diinethyl sulfoxide (J.T.
Baker);
Safflower Oil (commercial); soybean oil (commercial); sesame oil (commercial);
and
Miglyol 810 (Sasol North America).
Example 1
Solubility of 17-AAG in Various Oils
[0066] 1 mg of 17-AAG reference standard was initially dissolved in 1 mL of
100%
methanol. The sample was diluted to 0.4 mg/mL concentration with methanol and
used as a
reference standard. 17-AAG was dissolved in various organic solvents or oils.
The
formulations were lightly vortexed to ensure homogeneity and subsequently
filtered through
0.2 gm PVDF syringe filters. The 17-AAG stock formulations were diluted in
their
respective solvents to determine the initial concentration and purity as well
as the recovery of
17-AAG by RP-HPLC analysis.
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[0067] 20 mg of 17-AAG was taken in different vials and 0.2 mL of various oils
was added
as listed in Table 1. The samples were vortexed to dissolve 17-AAG. Upon
preparation, the
samples showed varying degrees of precipitation and were subsequently filtered
through 0.2
m PVDF filters and analyzed by RP-HPLC.
Table 1: Visual appearance, solubility and purity of 17-AAG/oil formulations.
17-AAG Formulations Visual Appearance (0 Solubility (mg/mL) % Purity
hr)
Safflower Oil Precipitate 1.01 97%
L Soybean Oil Precipitate 0.86 98%
Sesame Oil Precipitate 1.05 98%
Mig1yo1810 Precipitate
1.88 96%
E
Example 2
Evaluation of DMA in Combinati.on wit/i Oils on 17-AAG Solubility
[0068] 17-AAG stocks were prepared in 100% DMA (50 mg/mL or 120 mg/mL). The
formulations listed in Tables 2 to 5 were prepared by two different methods.
Either the 17-
AAG/solvent stock was slowly added to excess oil (Method I), or the oil was
added to an
aliquot of the 17-AAG/solvent stock (Method II). For example, solvent to oil
(Method I) was
prepared as follows: A 20 L or 50 mg/mL 17-AAG/DMA stock was added to vial
containing 500 L of the Safflower oil under stirring conditions. Oil to
solvent (Method II)
was prepared by adding 500 L of oil dropwise to a vial containing 20 gL of 50
mg/mL 17-
AAGlDMA stock over a period of 5 minutes. The sample was stirred on a magnetic
stir plate
for a period of 30 minutes. Upon preparation, the DMA samples showed varying
degrees of
precipitation. The formulations were subsequently filtered using 0.2 m PVDF
filters and
analyzed by RP-HPLC. There was no change in appearance observed in any of the
formulations tested after 24 hours of incubation at room temperature.
[0069] Table 2 provides data regarding an evaluation of 17-AAG solubility
using DMA in
combination with various oils. The combinations were prepared in accordance
with Method I
where a 50 mg/mL stock solution of 17-AAG/DMA was used. Percent recovery was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(2 mg/mL).
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Table 2: Evaluation of DMA in combination with oils on 17-AAG solubility. RP-
HPLC
analysis (17-AAG/DMA stock: 50 mg/mL), Method I.
17-AAG Visual Appearance Solubility % Recovery % Purity
Formulations (30 min) (mg/mL)
DMA/Safflower Oil Slight ppt. 0.30 15% 97%
particulates
DMA/Soybean Oil Slight ppt. 0.29 15% 99%
particulates
DMA/Sesame Oil Slight ppt. 0.35 18% 99%
particulates
[0070] Table 3 provides data regarding an evaluation of 17-AAG solubility
using DMA in
combination with various oils. The combinations were prepared in accordance
with Method
II, where a 50 mg/mL stock solution of 17-AAG/DMA was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(2 mg/mL).
Table 3: Evaluation of DMA in combination with oils on 17-AAG solubility. RP-
HPLC
analysis (17-AAG/DMA stock: 50 mg/mL), Method TI.
17-AAG Visual Appearance Solubility (mg/mL) % Recovery % Purity
Formulations (30 min)
Safflower Oil/DMA Slight ppt. 0.44 22% 99%
particulates
Soybean Oil/DMA Slight ppt. 0.33 17% 99%
particulates
Sesame Oil/DMA Slight ppt. Not detected -- --
particulates
[0071 ] Table 4 provides data regarding an evaluation of 17-AAG solubility
using DMA in
combination with various oils. The combinations were prepared in accordance
with Method
I, where a 120 mg/mL stock solution of 17-AAG/DMA was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(4.8 mg/mL).
Table 4: Evaluation of combination of DMA with oils on 17-AAG solubility (17-
AAG/DMA stock: 120 mg/mL), Method I.
17-AAG Visual Appearance
Solubility % Recovery % Purity
j IT T 1~
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Formulations (30 min) (mg/mL)
DMA/Safflower Oil Slight ppt. 0.25 5% 97%
particulates
DMA/Soybean Oil Slight ppt. 0.21 4% 99%
particulates
DMA/Sesame Oil Slight ppt. 0.31 6% 99%
particulates
[0072] Table 5 provides data regarding an evaluation of 17-AAG solubility
using DMA in
combination with various oils. The combinations were prepared in accordance
with Method
II where a 120 mg/mL stock solution of 17-AAG/DMA was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(4.8 mg/mL).
Table 5: Evaluation of DMA in combination with oils on 17-AAG solubility (17-
AAG/DMA stock: 120 mg/mL), Method II.
17-AAG Visual Appearance Solubility (mg/mL) % Recovery % Purity
Formulations (30 min)
Safflower Oit/DMA Slight ppt. 0.35 7% 98%
particulates
Soybean Oil/DMA Slight ppt. 0.29 6% 99%
particulates
Sesame Oil/DMA Slight ppt. Not detected -- --
particulates
Example 3
Evaluation of DMSO in Coinbination witli Oils on 17-AAG Solubility
[0073] 17-AAG stocks were prepared in 100% DMSO (50 mg/mL or 200 mg/mL). The
formulations listed below in Tables 6 to 9 were prepared by two different
methods. Either
the 17-AAG solvent stock was slowly added to excess oil (Method I), or the oil
was added to
an aliquot of the 17-AAG/solvent stock (Method II). For example, solvent to
oil (Method I)
was prepared as follows: A 20 L of 50 mg/mL 17-AAG/DMSO stock was added to a
vial
containing 500 liL of the Safflower oil under stirring conditions. Oil to
solvent (Method II)
was prepared by adding 500 L of oils dropwise to a vial containing 20 L of
50 mg/mL 17-
AAG/DMSO stock over a period of 5 minutes. The sample was stirred on a
magnetic stir
17
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plate for a period of 30 minutes. Upon preparation, the formulations formed
tiny oil droplets
initially and precipitated after 10 minutes. The formulations were
subsequently filtered using
0.2 m PVDF filters and analyzed by RP-HPLC. The DMSO containing formulations
showed oil droplet formation even after filtering; thus supematant without the
oil droplets
was loaded on the RP-HPLC. There was no change in the appearance observed in
any of the
formulations tested after 24 hours of incubation at room temperature.
[0074] Table 6 provides data regarding an evaluation of 17-AAG solubility
using DMSO in
combination with various oils. The combinations were prepared in accordance
with Method
I, where a 50 mg/mL stock solution of 17-AAG/DMSO was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(2 mg/mL).
Table 6: Evaluation of DMSO in combination with oils on 17-AAG solubility (17-
AAG/DMSO stock: 50 mg/mL), Method I.
17-AAG Visual Appearance Solubility % Recovery % Purity
Formulations (30 min) (mg/mL)
DMSO/Safflower Slight ppt. 0.66 33% 99%
Oil particulates
DMSO/Soybean Oil Slight ppt. 0.45 22% 99%
particulates
DMSO/Sesame Oil Slight ppt. Not Detected -- --
particulates
[0075] Table 7 provides data regarding an evaluation of 17-AAG solubility
using DMSO in
combination with various oils. The combinations were prepared in accordance
with Method
II, where a 50 mg/mL stock solution of 17-AAG/DMSO was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(2 mg/mL).
Table 7: Evaluation of DMSO in combination with oils on 17-AAG solubility (17-
AAG/DMSO stock: 50 mg/mL), Method II.
17-AAG Visual Appearance Solubility (mg/mL) % Recovery % Purity
Formulations (30 min)
Safflower Oil/ Slight ppt. 0.60 30% 99%
DMSO particulates
Soybean Oil/ Slight ppt. 0.35 18% 99%
DMSO particulates
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FSesame Oil/ DMSO Slight ppt. Not Detected -- --
particulates
[0076] Table 8 provides data regarding an evaluation of 17-AAG solubility
using DMSO in
combination with various oils. The combinations were prepared in accordance
with Method
1, where a 200 mg/mL stock solution of 17-AAG/DMSO was used. Percent recovery
was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(8 mg/mL).
Table 8: Evaluation of DMSO in combination with oils on 17-AAG solubiiity (17-
AAG/DMSO stock: 200 mg/mL), Method I.
17-AAG Visual Appearance Solubility % Recovery % Purity
Formulations (30 min) (mg/mL)
DMSO/Safflower Solid Precipitate 1.82 23% 99%
Oil
DMSO/Soybean Oil Solid Precipitate 0.85 99%
DMSO/Sesame Oil Solid Precipitate 0.61 8% 99%
[0077] Table 9 provides data regarding an evaluation of 17-AAG solubility
using DMSO in
combination with various oils. The combinations were prepared in accordance
with Method
IT, where a 200 ing/mL stock solution of 17-AAG/DMSO was used. Percent
recovery was
calculated by dividing the solubility of 17-AAG by the targeted concentration
(8 mg/mL).
Table 9: Evaluation of DMSO in combination with oils on 17-AAG solubility (17-
AAG/DMSO stock: 200 mg/mL), Method II.
17-AAG Visual Appearance Solubility (mg/mL) % Recovery % Purity
Formulations (30 min)
Safflower OiU Solid Precipitate 2.57 32% 99%
DMSO
Soybean OiUDMSO Solid Precipitate 0.92 12% 99%
Sesame OiUDMSO Solid Precipitate Not Detected -- --
Example 4
Evaluation of Combination of Solvents and Oils oiz 17-AAG Solubility
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[0078] 17-AAG stocks were prepared in either 100% DMA (50 mg/rnL or 120 mg/mL)
or
DMSO (50 mg/mL or 200 mg/mL). The formulations were prepared as follows: A 10
or 20
gL aliquot of l 7-AAG/solvent stock was added to a vial containing 500 L of
the oil and
gently swirled for 20 seconds to ensure complete homogeneity. The samples were
subsequently incubated at room temperature and monitored over the course of 2
hours.
[0079] Table 10 presents the visual appearance and solubility profiles of 17-
AAG
formulations containing a combination of DMA and oils prepared with no
mechanical stress.
Upon preparation, the DMA samples showed either little or no evidence of
precipitation.
Table 10: Evaluation of Combination of Solvents and Oils on 17-AAG Solubility
17-AAG Stock Targeted Visual Appearance (2 Solubility % Recovery
Formulations Concentration Concentration hrs) mg/mL
used mg/mL
(mg/ML)
DMA/Safflower 50 1.0 Clear with few particles 0.88 88%
Oil
DMA/Soybean Oil 50 1.0 Clear with few particles 0.93 93%
DMA/Safflower 50 2.0 Clear with few particles 1.28 64%
Oil
DMA/Soybean Oil 50 2.0 Clear with few particles 0.61 31%
DMA/Safflower 120 2.4 Settling of particles 0.64 27%
Oil
DMA/Soybean Oil 120 2.4 Settling of particles 0.51 21%
DMA/Safflower 120 4.8 Settling of particles 0.61 13%
Oil
DMA/Soybean Oil 120 4.8 Settling of particles 0.26 3%
[0080] Table 11 presents the visual appearance and solubility profiles of 17-
AAG
formulations containing combinations of DMSO and oils prepared with no
mechanical
shearing. Upon preparation, the DMSO samples showed tiny oil droplets. After 2
hours of
incubation at room temperature, there was a large amount of particles settled
on the bottom of
the vial containing DMSO/oil combinations in comparison to the DMA/oil
formulations,
irrespective of the stock concentrations used.
Table 11: Evaluation of Combination of Solvents and Oils on 17-AAG Solubility
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17-AAG Formulations Stock Targeted Visual Appearance Solubility % Recovery
Concentration Concentration (2 hrs) mg/mL
used (mg/mL) mg/mL
DMSO/Safflower Oil 50 1.0 Settling of particles 0.70 70%
DMSO/Soybean Oil 50 1.0 Settling of particles 0.67 67%
DMSO/Safflower Oil 50 2.0 Settling of particles 0.41 41%
DMSO/Soybean Oil 50 2.0 Settling of particles 0.24 24%
DMSO/Safflower Oil 200 2.4 Settling of particles 1.38 34%
DMSO/Soybean Oil 200 2.4 Settling of particles 0.74 29%
DMSO/Safflower Oil 200 4.8 Settling of particles 1.37 17%
DMSO/Soybean Oil 200 4.8 Settling of particles 1.00 12%
Exaniple 5
17-AAG Solubility and Stability in 10% Intralipid
[0081 ] Different stock concentrations of 17-AAG were prepared either in 100%
DMA or
DMSO as follows: 17-AAG/DMA-50, 55, 60, and 65 mg/mL; 17-AAG/DMSO-50, 55,
60, and 65 mg/mL.
[0082] The formulations listed below in Tables 12 to 15 were prepared by
mixing different
aliquots of each 17-AAG/solvent stock in 1 mL of 10% Intralipid. For example,
a 50/25 of
17-AAG/DMA/10% Intralipid combination was prepared as follows: 25 L of 50
mg/ML
17-AAG/ DMA stock was slowly added to vial containing 1 mL of the 10%
Intralipid. The
sample was mixed by inverting the vials continuously for 20 seconds. All of
the formulations
were incubated at room temperature for a period of 24 hours to monitor the
stability of 17-
AAG in the lipid emulsions.
[0083] Table 12 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 50 mg/mL 17-AAG stock and formulated with 10%
Intralipid.
Table 12: Stability of 17-AAG in 10% Intralipid by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
50/25 1.22 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
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50/30 1.46 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
50/50 2.38 Settling of Settling of Settling of Settling of
particles particles particles particles
[0084] Table 13 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 55 mg/mL 17-AAG stock and foi7nulated with 10%
Intralipid.
Table 13: Stability of 17-AAG in 10% Intralipid by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stoclz/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
55/25 1.34 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
55/30 1.60 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
[0085] Table 14 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 60 mg/mL 17-AAG stock and formulated with 10%
Intralipid.
Table 14: Stability of 17-AAG in 10% Intralipid by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
60/25 1.46 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
60/30 1.75 Slight ppt. Slight ppt. Clear emulsion Clear emulsion
particulates particulates
[0086] Table 15 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 65 mg/mL 17-AAG stock and formulated with 10%
Intralipid.
Table 15: Stability of 17-AAG in 10% Intralipid by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
65/25 1.59 Slight ppt. Clear emulsion Slight ppt. Clear emulsion
particulates particulates
65/30 1.89 Slight ppt. Slight ppt. Slight ppt. Slight ppt.
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particulates particulates particulates r particulates
Example 6
17-AAG Solubility atzd Stability in 10% Liposyiz 2
[0087] Different stock concentrations of 17-AAG were prepared either in 100%
DMA or
DMSO as follows: 17-AAG/DMA-55, 60, and 65 mg/mL; 17-AAG/DMSO-55, 60 and
65 mg/mL. The formulations listed below in Tables 16 to 18 were prepared by
mixing
different aliquots of each 17-AAG/solvent stock in 1 mL of 10% Liposyn II. For
example, a
55/25 of 17-AAG/DMA/10% Liposyn II combination was prepared as follows: 25 L
of 55
mg/mL 17-AAG/DMA stock was slowly added to a vial containing 1 mL of the 10%
Liposyn
II. The sample was mixed by inverting the vials continuously for 20 seconds.
All of the
fonnulations were incubated at room temperature for a period of 24 hours to
monitor the
stability of 17-AAG in the lipid emulsions.
[0088] Table 16 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 55 mg/mL 17-AAG stock and formulated with 10%
Liposyn II.
Table 16: Stability of 17-AAG in 10% Liposyn II by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Liposyn (8 h) Liposyn (24 h) Liposyn (8 h) Liposyn (24 h)
added
55/25 1.34 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
55/30 1.60 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
55/40 2.12 Clear emulsion Slight ppt. Clear emulsion Clear emulsion
particulates
[0089] Table 17 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 60 mg/mL 17-AAG stock and formulated with 10%
Liposyn II.
Table 17: Stability of 17-AAG in 10% Liposyn II by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Liposyn (8 h) Liposyn (24 h) Liposyn (8 h) Liposyn (24 h)
added
60/25 1.46 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
60/30 J 1.75 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
23
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60/40 2.30 Clear emulsion Slight ppt. Clear emulsion Clear emulsion
particulates
[0090] Table 18 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 65 mg/mL 17-AAG stock and formulated with 10%
Liposyn II.
Table 18: Stability of 17-AAG in 10% Liposyn II by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
StocW .L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
65/25 1.59 Slight ppt. Clear emulsion Slight ppt. Clear emulsion
particulates particulates
65/30 1.89 Slight ppt. Slight ppt. Slight ppt. Slight ppt.
particulates particulates particulates particulates
Example 7
17-AAG Solubility and Stability in 10% or 20% Ib2tralipid
[0091 ] Different stock concentrations of 17-AAG were prepared either in 100%
DMA or
DMSO as follows: 17-AAG/DMA-70, 80, 90, 95 and 100 mg/mL; 17-AAG/DMSO-70,
80 90, 95 and 100 mg/mL. The formulations listed below in Tables 19 to 20 were
prepared
by mixing different aliquots of each 17-AAG/solvent stock in 1 mL of 10 or 20%
Intralipid.
For example, a 70/25 of 17-AAG/DMA/Intralipid combination was prepared as
follows: 25
gL of 70 mg/mL 17-AAG/DMA stock was slowly added to a vial containing 1 mL of
Intralipid. The sample was mixed by inverting the vials continuously for 40
seconds. All of
the formulations were incubated at room temperature for a period of 24 hours
to monitor the
stability of 17-AAG in the lipid emulsions.
[0092] Table 19 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 70 mg/mL 17-AAG stock and formulated with 10%
Intralipid.
Table 19: Stability of 17-AAG in 10% Intrali id by visual a earance following
P PP dilution in 10% Intralipid.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
70/20 1.37 Clear emulsion Clear emulsion Slight ppt. Slight ppt.
24
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particulates particulates
70/25 1.71 Slight ppt. Slight ppt. Slight ppt. Slight ppt.
particulates particulates particulates particulates
[0093] Table 20 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 70-80 ing/mL 17-AAG stock and formulated with 20%
Intralipid.
Table 20: Stability of 17-AAG in 20% Intralipid by visual appearance.
17-AAG Targeted conc. DMA/20% DMA/20% DMSO/20% DMSO/20%
Stock/ L (mg/mL) Intralipid (8 h) Intralipid (24 Intralipid (8 h) Intralipid
(24
added h) h)
70/25 1.71 Slight ppt. Clear emulsion Clear emulsion Clear emulsion
particulates
70/30 2.04 Slight ppt. Slight ppt. Clear emulsion Slight ppt.
particulates particulates particulates
80/25 1.95 Slight ppt. Slight ppt. Slight ppt. Slight ppt.
particulates particulates particulates particulates
Example 8
17-AAG Solubility and Stability in 10% or 20% Liposyn II
[0094] Different stock concentrations of 17-AAG were prepared either in 100%
DMA or
DMSO as follows: 17-AAG/DMA-70, 80, 90, 95 and 100 mg/mL; 17-AAG/DMSO-70,
80, 90, 95 and 100 mg/mL. The formulations listed below in Tables 21 to 23
were prepared
by mixing different aliquots of each 17-AAG/solvent stock in 1 mL of 10 or 20%
Liposyn H.
For example, a 70/25 of 17-AAG/DMA/Liposyn II combination was prepared as
follows: 25
L of 70 mg/mL 17-AAG/DMA stock was slowly added to vial containing 1 niL of
Liposyn
II. The sample was mixed by inverting the vials continuously for 40 seconds.
All of the
formulations were incubated at room temperature for a period of 24 hours to
monitor the
stability of 17-AAG in the lipid emulsions.
[0095] Table 21 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 70 or 80 mg/mL 17-AAG stocks and 10% Liposyn II.
Table 21: Stability of 17-AAG in 10% Liposyn II by visual appearance.
17-AAG Targeted conc. DMA/10% DMA/10% DMSO/10% DMSO/10%
Stock/ L (mg/mL) Liposyn (8 h) Liposyn (24 h) Liposyn (8 h) Liposyn (24 h)
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added
70/20 1.37 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
70/25 1.71 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
80/25 1.95 Slight ppt. Slight ppt. Slight ppt. Slight ppt.
particulates particulates particulates particulates
[0096] Table 22 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 70-80 mg/mL 17-AAG stocks and 20% Liposyn II.
Table 22: Stability of 17-AAG in 20% Liposyn II by visual appearance following
dilution in 20% Liposyn II.
17-AAG Targeted conc. DMA/20% DMA/20% DMSO/20% DMSO/20%
Stoclc/ L (mg/mL) Liposyn (8 h) Liposyn (24 h) Liposyn (8 h) Liposyn (24 h)
added
70/20 1.71 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
70/25 2.04 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
80/25 2.33 Clear emulsion Clear emulsion Clear emulsion Clear emulsion
[0097] Table 23 below presents the visual observation for 17-AAG/DMA or DMSO
emulsions prepared from a 90-100 mg/niL 17-AAG stock and 20% Liposyn II.
Table 23: Stability of 17-AAG in 20% Liposyn II by visual appearance.
17-AAG Targeted conc. DMA/20% DMA/20% DMSO/20% DMSO/20%
Stock/ L (mg/mL) Liposyn (8 h) Liposyn (24 h) Liposyn (8 h) Liposyn (24 h)
added
90/25 2.19 Slight ppt. Clear emulsion Clear emulsion Clear emulsion
particulates
90/30 2.62 Slight ppt. Clear emulsion Clear emulsion Clear emulsion
particulates
95/25 2.32 Clear emulsion Slight ppt. Clear emulsion Clear emulsion
particulates
95/30 2.76 -- -- Slight ppt. Clear emulsion
particulates
100/25 2.44 Slight ppt. Clear emulsion Clear emulsion Clear emulsion
particulates
26
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100/30 2.91 Slight ppt. Clear emulsion Slight ppt. Clear emulsion
particulates particulates
Example 9
Stability of 17-AAG Lipid Efnulsiorzs
[0098] Emulsion particle size was measured after adding 17-AAG to the fat
emulsions using
a light microscope. 10 L aliquots of 17-AAG emulsions were loaded onto a RP-
HPLC.
Osmolality was determined by removing 10 L aliquots of the 17-AAG emulsions
and
measuring the quantity using a Wescor VaproTM Vapor Pressure Osmometer.
[0099] Table 24 below presents the microscopic observations of lipid emulsions
before and
after the addition of 17-AAG. Using a light microscope, the oil droplet of the
emulsion was
categorized into 3 different sizes: small (< 200 iun); medium (200-400 nm) or
large droplets
(> 400 nm).
Table 24: Stability of 17-AAG in lipid emulsions by microscopic observation.
DMSO/17-AAG Targeted Before adding 17-AAG After adding 17-AAG
Stock (7/25) concentration (to & t24)
(mg/mL)
DMSO/10% 1.71 Heterogeneous, small droplets with More or less uniform sized
Intralipid few medium and large-sized droplets particles with very few
medium-sized droplets
DMSO/20% 1.71 Heterogeneous, small droplets with Homogeneous small sized oil
Intralipid very few medium sized droplets droplets
DMSO/10% 1.71 Heterogeneous, small droplets with Homogeneous small sized oil
Liposyn very few medium and large-sized droplets
droplets
DMSO/20% 1.71 Heterogeneous, small droplets with Homogeneous small sized oil
Liposyn very few medium-sized droplets droplets
[00100] Table 25 below presents the stability of 17-AAG/DMA or DMSO
emulsions prepared from a 55, 70 or 100 mg/mL 17-AAG stocks and 20% Liposyn R.
Table 25: Stability of 17-AAG in 20% Liposyn II by visual appearance following
dilution in 20% Liposyn II.
17-AAG Target DMA/20% Liposyn II DMSO/20% Liposyn II
Stock/ L concentration
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added (mg/mL) 8 hrs 24 hrs 8 hrs 24 hrs
%Rec %Purity %Rec %Purity %Rec %Purity %Rec %Purity
55130 1.60 99.2 98.4 97.7 98.3 99.2 98.7 98.2 98.3
70/30 2.04 99.3 98.4 97.8 98.6 99.3 98.7 98.2 98.6
100/25 2.44 -- -- -- -- 98.9 99.3 98.7 98.2
L- _ -- L -
[00101] Table 26 below presents the osmolality of the commercially available
lipid emulsions with no drug.
Table 26: Osmolality of commercially available lipid emulsions with no drug.
Lipid Emulsion Osmolality on the Container Measured Osmolality
(mOsmol/liter) (mOsinoUkg)
20% Liposyn II 258 377
10% Liposyn II 276 323
20% Intralipid 350 343
Table 27: Osmolality of 17-AAG/DMSO emulsions.
17-AAG Stock/pL added Lipid Emulsion Final 17-AAG Conc. Osmolality
(mg/mL) (mOsmol/kg)
90/30 20% Liposyn II 2.62 882
95/25 20% Liposyn II 2.32 741
100/25 20% Liposyn II 2.44 746
70/25 10% Liposyn II 1.71 688
70/25 20% Intralipid 1.71 760
Table 28: Osmolality of 17-AAG/DMA emulsions.
17-AAG Stock/ L added Lipid Emulsion Final 17-AAG Conc. Osmolality
(mg/rnL) (mOsmol/kg)
80/30 20% Liposyn II 2.33 543
70/30 20% Liposyn lI 2.04 551
28
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70/25 20% Liposyn II 1.71 521
70/25 10% Liposyn II 1.71 431
Example 10
17-A.A.G Emulsioras
j00102] Tables 29 and 30 below present various 17-AAG
formulations/emulsions with the final dosage/ volume of active and inactive
ingredients to be
infused.
Table 29: Final 17-AAG and DMSO concentrations in various 17-AAG formulations/
emulsions in fat emulsions.
Fat 17-AAG Final 17- Volume of Final Emulsion Total DMSO
Emulsions formulations AAG conc. 17-AAG DMSO vol. infused dosage g/m2
(mg/niL) stock (mL) conc. (%) (mL) (DMSO, g)
20% 90/30 2.62 6.66 2.91 229 7.33 4.30
Liposyn
20% 95/25 2.32 6.32 2.44 259 6.95 4.09
Liposyn
20% 100/25 2.44 6.0 2.44 246 6.60 3.88
Liposyn
10% 70/25 1.71 8.57 2.44 351 9.43 5.55
Liposyn
20% 70/25 1.71 8.57 2.44 351 9.43 5.55
Intralipid
Table 30: Final 17-AAG and DMA concentrations in various 17-AAG formulations/
emulsions in fat emulsions
Fat 17-AAG Final 17- Volume of Final Emulsion Total DMA
Emulsions formulations AAG conc. 17-AAG DMA vol. infused dosage glm2
(mglmL) stock cone. (%) (mL) (DMA, g)
(ML)
20% 80/30 2.33 7.50 2.91 258 7.03 4.14
Liposyn
20% 70/30 2.04 8.57 2.91 294 8.03 4.72
Liposyn ----IE 20% 70/25 1.71 8.57 E 2.44 351 8.03 1174T]
29
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Liposyn
10% 70/25 1.71 8.57 2.44 351 8.03 4.72
Liposyn
Example 11
Short-Ter}na Stability of 17-AAG Forrriulations
[00103] A 100 mg/mL stock of 17-AAG was prepared either in 100% DMA or
DMSO. Aliquots of 17-AAG/solvent formulations were placed in glass vials,
covered with
aluminum foil and incubated at -20 C, 4 C or room temperature (i.e., RT) for
a period up to
28 days. Samples were withdrawn at each time point (day 7, 14, 21 or 28), and
an aliquot
from each sample was subsequently diluted in and analyzed by RP-HPLC to
determine the
percent recovery and purity of the stock solutions. The stability of 17-
AAG/DMA or DMSO
test samples was also examined by diluting the stock samples in 20% Liposyn
II. The 17-
AAG emulsions were incubated at room temperature over a period of 24 hours to
determine
percent recovery via RP-HPLC.
[00104] Table 31 below presents the 28 day stability data of 17-AAG stock in
either DMA or DMSO at 100 mg/mL 17-AAG concentration incubated at 4 C, room
temperature, or -20 C. Percent recovery and purity were analyzed after time =
0, 7 hours, 14
hours, 21 hours, and 28 hours. The percent recovery was calculated by dividing
the AUP of
the t7 through t28 formulations by the AUP of to formulations.
Table 31: Short-term stability on 17-AAG formulations by RP-HPLC analysis.
17-AAG tp t7 t14 t21 t2g
Formulations
%Rec %Pur %Rec %Pur %Rec %Pur %Rec %Pur %Rec %Pur
DMA/4 C N/A 96.1% 98 97.8 101 96.9 100 96.4 94 92.0
DMA/RT N/A 96.1% 98 96.1 100 95.8 102 94.9 92 91.2
DMA/-20 C N1A 96.1% 97 95.7 101 96.5 100 96.0 103 98.2
DMS014 C N/A 99.5% 102 99.3 101 98.8 100 96.6 99 98.4
DMSO/RT N/A 99.5% 99 98.7 100 97.7 97 94.8 90 92.5
DMSO/-20 C N/A 99.5% 100 99.0 106 98.9 98 98.7 104 98.5
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[00105] Table 32 below presents 24 hour stability data of 17-AAG stock in
either DMA or DMSO at 100 mg/mL 17-AAG concentration incubated at 4 C, room
temperature, or -20 C.
Table 32: 17-AAG Lipid Emulsion Stability after 24 hours of incubation at RT
in 20%
Liposyn II.
17-AAG Lipid 17-AAG stock/ Target concentration Days-to, t7, t14, t21, t28
emulsions L added (mg/mL)
0 hour 8 hours 24 hours
DMA/4 C 100/25 2.44 Slight ppt. Clear Clear
emulsion emulsion
DMA/RT 100/25 2.44 Slight ppt. Clear Clear
emulsion emulsion
DMA/-20 C 100/25 2.44 Slight ppt. Clear Clear
emulsion emulsion
DMSO/4 C 100/25 2.44 Clear Clear Clear
emulsion emulsion emulsion
DMSO/RT 100/25 2.44 Clear Clear Clear
emulsion emulsion emulsion
DMSO/-20 C 100/25 2.44 Clear Clear Clear
emulsion emulsion emulsion
[00106] Table 33 below presents the percent recovery of t21 or t28 17-
AAG/DMA or DMSO lipid emulsions in 20% Liposyn after 24 hours of incubation at
room
temperature.
Table 33: 17-AAG Lipid Emulsion Stability after 24 hours of incubation at RT
in 20%
Liposyn II.
17-AAG Lipid emulsions Target concentration t21 t28
(mg/mL)
% Recovery % Purity % % Purity
Recovery
DMA/4 C 2.44 98 95.9 99 95.2
DMA/RT 2.44 90 93.6 94 91.8
DMA/-20 C 2.44 99 96.4 99 96.0
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DMSO/4 C 2.44 97 96.7 95 96.3
DMSO/RT 2.44 91 94.4 88 94.3
DMSO/-20 C 2.44 99 96.6 96 F7711
[00107] The foregoing detailed description of the invention includes passages
that are chiefly or exclusively concerned with particular parts or aspects of
the invention. It
is to be understood that this is for clarity and convenience, that a
particular feature may be
relevant in more than just the passage in which it is disclosed, and that the
disclosure herein
includes all the appropriate combinations of information found in the
different passages.
Similarly, although the various descriptions herein relate to specific
embodiments of the
invention, it is to be understood that where a specific feature is disclosed
in the context of a
particular embodiment, such feature can also be used, to the extent
appropriate, in the context
of another embodiment, in combination with another feature, or in the
invention in general.
32
