Specification
DESCRIPTION
ALPHA-TEA SALT FORMS: COMPOSITIONS AND USES FOR TREATING DISEASE
This application claims the benefit of US 61/874,823 filed on September 6, 2013, the entirety
of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
I. Field of the Invention
The present disclosure relates generally to salts of a-tocopheryloxyaeetic acid (a-TEA),
polymorphic forms thereof, and pharmaceutical compositions thereof. In other aspects, the present
disclosure also relates methods for preparation and use of these substances.
II. Description of Related Art
Vitamin E analogs such as a-toeophcryloxyacetic acid (a-TEA) have been previously
reported (US 6, 7,223, US 6,703,384 B2, US 6,770, 672 B ί , and US 7,3 2,232 B2). The derivative
a-TEA has been shown to suppress the growth of tumors through a mechanism which includes
mitochondrial depolarization and the generation of reactive oxygen species which trigger cell death
(Yu, et. al, 20 ; Neuzil, et. al, 2 07; Kline, et. al, 2007). Studies have shown that the compound
inhibits the growth of a variety of different cancer cell lines without significant negative effects on
normal tissues (Lawson, et al, 20 3; Anderson, e t al, 2 04; Hahn, e t al, 2009; Hahn, et al, 2006).
In view of these promising biological properties, and in consideration that biological activity
profiles and physical properties can be expected vary across different salt forms of a compound, it
remains desirable to obtain novel salts forms with advantageous properties, including one that mayease
process scale-up and formulation issues and/or possess biological activity or pharmacokinetic
profiles that is well suited for the treatment or prevention of a given disease or indication n view of
the high degree of unmet medical need represented by the diseases and indications discussed herein, t
is therefore desirable to synthesize new solid forms, including salt forms, of a-TEA.
SUMMARY OF THE INVENTION
aspect, the present invention provides compounds of the formula:
wherein X is:
or a hydrate thereof. In some embodiments, X is:
or a hydrate thereof. In some embodiments, X is:
or a hydrate, thereof. In some embodiments, X + is
a hydrate, thereof.
In some embodiments, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising two peaks at about 11.84 °2Qand 19.74 °2Qor
a hydrate, thereof. In some embodiments, the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising a peak at about 20.73 °2Q. In some embodiments, the polymorphic
form has an X-ray powder diffraction pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, or all of peaks at about 6.24, 9.35, 14.92, 15.5 1, 17.03, 18.49, 2 1.70, 22.81 ,
23.5 1, 24.85, 27.69, 28.3 1, 30.93, 32.30, 33.2 1, 35.02, and 39.30 °2Q. In some embodiments, the
polymorphic form has an X-ray powder diffraction pattern (CuKa) that is substantially as shown in
FIG. 4. In some embodiments, the polymorphic form has a differential scanning calorimetry (DSC)
isotherm comprising two endotherms from about 125 °C to about 140 °C and from about 170 °C to
about 195 °C. In some embodiments, the polymorphic form has a differential scanning calorimetry
(DSC) isotherm comprising two endotherms centered at about 131 °C and at around 184 °C,
respectively. In some embodiments, the polymorphic form has a differential scanning calorimetry
isotherm that is substantially as shown in FIG. 5. In some embodiments, the polymorphic form has a
thermogravimetric analysis (TGA) indicating a total volatile content of about 0.3 wt% over the
temperature range of about 25-1 94 °C. In some embodiments, the polymorphic form has a dynamic
vapor sorption (DVS) kinetic data and isotherm indicating a hemihydrate formation at about 40%
relative humidity. In some embodiments, the polymorphic form is anhydrous. In other embodiments,
the polymorphic form is a hydrate. In some embodiments, the polymorphic form is a hemihydrate.
In yet another embodiment, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 9.69 °2Qor a hydrate, thereof.
In some embodiments, the X-ray powder diffraction pattern (CuKa) further comprises a peak at about
20.10 °2Q. In some embodiments, the X-ray powder diffraction pattern (CuKa) further comprises a
peak at about 19.78 °2Q. In some embodiments, the X-ray powder diffraction pattern (CuKa) further
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all of peaks at about 5.90, 7.77, 14.08,
17.58, 17.91, 18.31, 21.26, 22.17, 23.16, 24.77, 28.57, 29.71, 31.10, 31.69, 34.45, 34.99, and
36.24 °2Q. In some embodiments, the X-ray powder diffraction pattern (CuKa) that is substantially as
shown in FIG. 6. In some embodiments, the polymorphic form has a differential scanning calorimetry
(DSC) isotherm comprising two endotherms from about 53 °C to about 60 °C and from about 180 °C
to about 235 °C. In some embodiments, the polymorphic form has a differential scanning calorimetry
(DSC) isotherm comrpising two endotherms centered at about 55.8 °C and at about 205 °C,
respectively. In some embodiments, the polymorphic form has a differential scanning calorimetry
isotherm that is substantially as shown in FIG. 7. In some embodiments, the polymorphic form has a
thermogravimetric curve from thermogravimetric analysis (TGA) indicating a total volatile content of
about 0.4 wt% over the temperature range of about 25-168.8 °C. In some embodiments, the
polymorphic form has a dynamic vapor sorption (DVS) kinetic data and isotherm indicating a hydrate
formation above about 75% relative humidity. In some embodiments, the polymorphic form is
anhydrous. In other embodiments, the polymorphic form is a hydrate.
In another embodiment, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 20.14 °2Q or a hydrate,
thereof. In some embodiments, the X-ray powder diffraction pattern (CuKa) further comprises peaks
at about 13.63, 14.40, 19.31, and 22.34 °2Q. In some embodiments, the X-ray powder diffraction
pattern (CuKa) further comprises two peaks at about 18.25 and 18.49 °2Q. In some embodiments, the
X-ray powder diffraction pattern (CuKa) further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of
peaks at about 9.67, 15.60, 21.47, 23.57, 27.07, 28.39, 30.49, 32.21, 33.30, 34.29, 34.96, 35.89, and
36.39 °2Q. In some embodiments, the polymorphic form has an X-ray powder diffraction pattern
(CuKa) that is substantially as shown in FIG. 10. In some embodiments, the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising two endotherms from about 97.5 °C to
about 120 °C and from about 185 °C to about 225 °C. In some embodiments, the polymorphic form
has a differential scanning calorimetry (DSC) isotherm comprising two endotherms at about 106 °C
and at about 192 °C, respectively. In some embodiments, the polymorphic form has a differential
scanning calorimetry isotherm that is substantially as shown in FIG. 11. In some embodiments, the
polymorphic form has a thermogravimetric curve from thermogravimetric analysis (TGA) indicating a
total volatile content of about 0.6 wt% over the temperature range of about 25-144 °C. In some
embodiments, the polymorphic form has a dynamic vapor sorption (DVS) kinetic data and isotherm
indicating a hemihydrate formation at about 55% relative humidity. In some embodiments, the
polymorphic form is anhydrous. In other embodiments, the polymorphic form is a hydrate. In some
embodiments, the polymorphic form is a hemihydrate.
In yet another embodiment, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 19.4 °2Qor a hydrate, thereof.
In some embodiments, the X-ray powder diffraction pattern (CuKa) further comprises peaks at about
10.4, 14.1, and 17.8 °2Q. In some embodiments, the X-ray powder diffraction pattern (CuKa) further
comprises two peaks at about 20.8 °2Q. In some embodiments, the polymorphic form has an X-ray
powder diffraction pattern (CuKa) that is substantially as shown in FIG. 2. In some embodiments, the
polymorphic form has a differential scanning calorimetry (DSC) isotherm comprising five endotherms
from about 85 °C to about 97 °C, from about 97 °C to to about 105 °C, from about 110 °C to about
125 °C, from about 145 °C to about 155 °C and from about 160 °C to about 210 °C. In some
embodiments, the polymorphic form has a differential scanning calorimetry (DSC) isotherm
comprising five endotherms at about 90.1 °C, at about 100.7 °C, at about 117.9 °C, at about 149.7 °C,
and at about 181.6 °C, respectively. In some embodiments, the polymorphic form has a differential
scanning calorimetry isotherm that is substantially as shown in FIG. 3. In some embodiments, the
polymorphic form has a thermogravimetric curve from thermogravimetric analysis (TGA) indicating a
total volatile content of about 0.4 wt% over the temperature range of about 25-95.5 °C.
In another embodiment, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising two peaks at about 18.40 and 19.77 °2Qor
hydrate, thereof. In some embodiments, the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising peaks at about 4.28 and 10.75 °2Q. In some embodiments, the
polymorphic form has an X-ray powder diffraction pattern (CuKa) further comprising two peaks at
about 19.00 °2Q. In some embodiments, the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of peaks
at about 6.45, 12.90, 16.17, 16.73, 17.31, 17.72, 20.96, 21.72, 28.08, 28.71, 29.70, 31.05, 31.63,
32.25, 34.60, 35.18, 36.60, and 39.02 °2Q. In some embodiments, the polymorphic form has an X-ray
powder diffraction pattern (CuKa) that is substantially as shown in FIG. 8. In some embodiments, the
polymorphic form has a differential scanning calorimetry (DSC) isotherm comprising three
endotherms from about 50 °C to about 60 °C, from about 75 °C to about 85 °C, and from about 150
°C to about 250 °C, respectively. In some embodiments, the polymorphic form has a differential
scanning calorimetry (DSC) isotherm comprising three endotherms at about 50 °C, at about 80 °C,
and at about 150 °C, respectively. In some embodiments, the polymorphic form has a differential
scanning calorimetry isotherm that is substantially as shown in FIG. 9. In some embodiments, the
polymorphic form has a thermogravimetric curve from thermogravimetric analysis (TGA) indicating a
total volatile content of about 2.5 wt% over the temperature range of about 25-1 88.2 °C.
In another embodiment, the present disclosure provides a compound of the formula:
or a hydrate, thereof. In some embodiments, the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 21.73 °2Q or a hydrate,
thereof. In some embodiments, the polymorphic form has an X-ray powder diffraction pattern
(CuKa) further comprising peaks at about 9.34, 14.90, 16.06, 17.33, 18.51, and 22.94 °2Q. In some
embodiments, the polymorphic form has an X-ray powder diffraction pattern (CuKa) further
comprising two peaks centered at about 20.22 °2Q. In some embodiments, the polymorphic form has
an X-ray powder diffraction pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of
peaks at about 10.05, 11.19, 14.13, 24.04, 27.65, 28.14, 29.73, 30.30, 31.06, 32.02, 33.45, and
34.49 °2Q. In some embodiments, the polymorphic form has an X-ray powder diffraction pattern
(CuKa) that is substantially as shown in FIG. 12. In some embodiments, the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising three endotherms from about 45 °C to
about 55 °C, from about 65 °C to about 80 °C, and from about 80 °C to about 95 °C. In some
embodiments, the polymorphic form has a differential scanning calorimetry (DSC) isotherm
comprising three endotherms at about 47 °C, at about 73 °C, and at about 87 °C, respectively. In
some embodiments, the polymorphic form has a differential scanning calorimetry isotherm that is
substantially as shown in FIG. 13. In some embodiments, the polymorphic form has a
thermogravimetric curve from thermogravimetric analysis (TGA) indicating a total volatile content of
about 7.7 wt% over the temperature range of about 25-207 °C.
In another aspect, the present disclosure provides a pharmaceutical composition comprising a
compound, salt, or polymorphic form of the present disclosure and an excipient. In some
embodiments, the pharmaceutical composition is formulated for administration: orally,
intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally,
intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally,
intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically,
intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally,
parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally,
transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous
infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion. In
some embodiments, the pharmaceutical composition is formulated for oral, intraarterial, intravenous,
intraperitoneal, topical, or inhalational administration.
In yet another aspect, the present disclosure provides a method of reacting a compound of the
formula:
to obtain a polymorphic form comprising combining an about equal molar amount of a base and the
compound in an alcoholic solvent and mixing for a time period from about 1 to about 24 hours. In
some embodiments, the base is NaOH, KOH,
In some embodiments, the base is:
In some embodiments, the alcoholic solvent is of the formula: - 1 wherein R is alkyl(c<6). In
some embodiments, the alcoholic solvent is methanol, 1-butanol, or isopropyl alcohol. In some
embodiments, the alcoholic solvent is isopropyl alcohol. In some embodiments, the method further
comprises allowing the solution to slowly evaporate at room temperature. In some embodiments, the
method further comprises allowing the evaporation to occur under a constant flow of nitrogen gas. In
some embodiments, the constant flow of nitrogen has a pressure of about 2 psi.
In still another aspect, the present invention provides a method of treating or preventing a
hyperproliferative disease, comprising administering to the patient a therapeutically effective amount
of the pharmaceutical composition, salt, polymorphic form, or compound of the present disclosure. In
some embodiments, the hyperproliferative disease is a cancer. In some embodiments, the cancer is
breast cancer, lung cancer, colon cancer, an endometrial tumor, cervical cancer, ovarian cancer,
mammary cancer, prostate cancer, liver cancer, melanoma, lymphoma, or myeloma. In some
embodiments, the cancer is breast cancer. In some embodiments, the method further comprises
administering to the patient a second therapy. In some embodiments, the second therapy is an
immunotherapy or surgery. In some embodiments, the second therapy is an immunotherapy. In some
embodiments, the immunotherapy comprises administering a pharmaceutically effective amount of
trastuzumab, adoptively transferred T lymphocytes, or a therapeutic antibody. In some embodiments,
the therapeutic antibody selectively binds HER2/neu. In some embodiments, the immunotherapy is
trastuzumab. In some embodiments, the immunotherapy comprises administering adoptively
transferred T lymphocytes to the patient. In some embodiments, the adoptively transferred T
lymphocytes are engineered to express a chimeric antigen receptor (CAR) to the patient. In some
embodiments, the immunotherapy comprises administering an immune modulating antibody to the
patient. In some embodiments, the immune modulating antibody is an anti-PD-1, anti-4-l-BB, anti-
GITR, anti-TIM3, anti-LAG3, anti-TIGIT, anti-CTLA-4 or an anti-LIGHT antibody. In other
embodiments, the second therapy is radiotherapy or a chemotherapeutic agent. In other embodiments,
the second therapy is surgery. In other embodiments, the second therapy is a chemotherapeutic agent.
In some embodiments, the chemotherapeutic agent is anthracyclines, taxanes, methotrexate,
mitoxantrone, estramustine, doxorubicin, etoposide, vinblastine, carboplatin, vinorelbine, 5-
fluorouracil, cisplatin, topotecan, ifosfamide, cyclophosphamide, epirubicin, gemcitabine, vinorelbine,
irinotecan, etoposide, vinblastine, pemetrexed, melphalan, capecitabine, oxaliplatin, BRAF inhibitors,
and TGF-beta inhibitors.
In another a of the formula:
wherein: X is a protonated base; comprising reacting a compound of the formula:
with the base sufficient to form a salt; or a hydrate thereof. In some embodiments, the base is: sodium
hydroxide, potassium hydroxide, magnesium hydroxide, meglumine, diethylamine, ethanolamine,
diethanolamine, L-arginine, L-lysine, choline, tris-hydroxymethylamine, N,N-dimethylethanolamine,
2-diethylethanolamine, calcium hydroxide, dibenzylethylenediamine, or procaine; or a hydrate
thereof. In some embodiments, the base is ethanolamine, L-arginine, L-lysine, choline, trishydoxymethylamine,
or N,N-dimethylethanolamine. In some embodiments, the base is L-arginine, Llysine,
or tris-hydroxymethylamine.
Other objects, features and advantages of the present disclosure will become apparent from
the following detailed description. It should be understood, however, that the detailed description and
the specific examples, while indicating specific embodiments of the invention, are given by way of
illustration only, since various changes and modifications within the spirit and scope of the invention
will become apparent to those skilled in the art from this detailed description. Note that simply
because a particular compound is ascribed to one particular generic formula does not mean that it
cannot also belong to another generic formula.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further
demonstrate certain aspects of the present disclosure. The invention may be better understood by
reference to one of these drawings in combination with the detailed description of specific
embodiments presented herein.
FIG. 1 - The CuKa powder x-ray diffractogram of the a-TEA free acid. The diffractogram shows the
dominate peaks of the free acid to be about 17.78 °2Q.
FIG. 2 - A CuKa powder x-ray diffractogram of the ethanolamine salt candidates from panels 1-3
compared to the spectra of the a-TEA free acid. The spectra for the free acid is shown at the bottom,
the ethanolamine salt from panel 2 is shown second from the bottom, the ethanolamine salt from panel
1 is shown in the second from the top spectra, and the ethanolamine salt from panel 3 is shown at the
top.
FIG. 3 - A differential scanning calorimetry (DSC) isotherm of the ethanolamine salt candidate of the
a-TEA free acid showing five endotherms with the three largest endotherms at about 90, 118, and 182
°C.
FIG. 4 - A CuKa powder x-ray diffractogram of a representative arginine salt candidate from panels
1-3. The spectra shows a dominate peak at about 19.74 °2Q.
FIG. 5 - A differential scanning calorimetry (DSC) isotherm of the arginine salt candidate of the a-
TEA free acid showing two endotherms with the endotherms at about 131 and 184 °C.
FIG. 6 - A CuKa powder x-ray diffractogram of a representative lysine salt candidate. The spectra
shows a dominate peak at about 9.69 °2Q.
FIG. 7 - A differential scanning calorimetry (DSC) isotherm of the lysine salt candidate of the a-TEA
free acid showing two endotherms with the endotherms at about 56 and 205 °C.
FIG. 8 - A CuKa powder x-ray diffractogram of a representative choline salt candidate from panels
1-3. The spectra shows a pair of dominate peaks at about 18.4 and 19.77 °2Q.
FIG. 9 - A differential scanning calorimetry (DSC) isotherm of the choline salt candidate of the a-
TEA free acid showing three endotherms with the endotherms at about 50, 80 and 150 °C.
FIG. 10 - A CuKa powder x-ray diffractogram of a representative tris salt candidate. The spectra
shows a dominate peak at about 20.14 °2Q.
FIG. 11 - A differential scanning calorimetry (DSC) isotherm of the tris salt candidate of the a-TEA
free acid showing two endotherms with the endotherms at about 106 and 192 °C.
FIG. 12 - A CuKa powder x-ray diffractogram of a representative N,N-dimethyl-ethanolamine salt
candidate from panels 1-3. The spectra shows a dominate peak at about 2 1.73 °2Q.
FIG. 13 - A differential scanning calorimetry (DSC) isotherm of the N,N-dimethyl-ethanolamine salt
candidate of the a-TEA free acid showing three endotherms with the endotherms at about 47, 73, and
87 °C.
FIG. 14 - A graph showing HPLC traces of different a-TEA free acid samples that have been
subjected to different stresses to test the stability of the compound in that form.
FIG. 15 - A graph showing HPLC traces of different a-TEA lysine salt samples that have been
subjected to different stresses to test the stability of the compound in that form.
FIG. 16 - A graph showing HPLC traces of different a-TEA arginine salt samples that have been
subjected to different stresses to test the stability of the compound in that form.
FIG. 17 - A graph showing HPLC traces of different a-TEA tris salt samples that have been
subjected to different stresses to test the stability of the compound in that form.
FIG. 18 - A graph of the average tumor size of BALB/c mice with a 4T1 mammary tumor in
mammary fat pad for control group and mice feed a diet which contained the a-TEA lysine salt.
F G, 19 - A graph of the Kaplan-Meier analysis of survival of the BALB/c mice on a diet of a-TEA
lysine sa compared to mice fed a nutrient analogous control diet.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present disclosure provides in one aspect, salts of the compound:
and polymorphic forms of the compound, thereof. In another non-limiting example, the present
disclosure relates to pharmaceutical compositions of the polymorphic forms and the method of
preparation of these compounds and the method of use of these compounds to treat diseases.
I. Definitions
When used in the context of a chemical group: "hydrogen" means -H; "hydroxy" means
-OH; "oxo" means =0; "carboxylic acid", "carboxy", or "carboxyl" means -C(=0)OH (also written
as -COOH or -C0 2H); "carboxy late" means -C(=0)0 (also written as -COCT or -C0 2 ); "halo"
means independently -F, -CI, -Br or -I. Any undefined valency on an atom of a structure shown in
this application implicitly represents a hydrogen atom bonded to the atom.
In the context of chemical formulas, the symbol "-" means a single bond, "=" means a double
bond; and "º" means triple bond. The symbol " " - " means a single bond where the group attached
to the thick end of the wedge is "out of the page." The symbol " ""HI " means a single bond where the
group attached to the thick end of the wedge is "into the page". The symbol " L " means a single
bond where the conformation (e.g., either or S or the geometry is undefined (e.g., either E or ).
For the groups and classes below, the following parenthetical subscripts further define the
group/class as follows: "(Cn)" defines the exact number (n) of carbon atoms in the group/class.
"(C£n)" defines the maximum number (n) of carbon atoms that can be in the group/class, with the
minimum number as small as possible for the group in question, e.g., "alkyl (C<8)" For example,
"alkyl (c
of the weight of the unit, or between about 25%o to about 60%>, or between about 1% to about 10%, for
example, and any range derivable therein.
Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of
multiple doses can be determined by one of ordinary skill in the art employing no more than routine
experimentation. As an example, subjects may be administered two doses daily at approximately 12
hour intervals. In some embodiments, the agent is administered once a day.
The salts of a-TEA may be administered on a routine schedule. As used herein a routine
schedule refers to a predetermined designated period of time. The routine schedule may encompass
periods of time which are identical or which differ in length, as long as the schedule is predetermined.
For instance, the routine schedule may involve administration twice a day, every day, every two days,
every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or
any set number of days or weeks there-between. Alternatively, the predetermined routine schedule
may involve administration on a twice daily basis for the first week, followed by a daily basis for
several months, etc. In other embodiments, the disclosure provides that the agent(s) can be taken
orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the
agent can be taken every morning and/or every evening, regardless of when the subject has eaten or
will eat. In other embodiments, the disclosure is taken as a dietary supplement. In some
embodiments, the a-TEA salts are taken before the onset of the tumor as a prophylaxis measure. In
other embodiments, the a-TEA salts are taken as a treatment option for use as an antiproliferative
agent.
V. Combination Therapy
In addition to being used as a monotherapy, the salts of a-TEA described in the present
invention may also find use in combination therapies. Effective combination therapy may be
achieved with a single composition or pharmacological formulation that includes both agents, or with
two distinct compositions or formulations, administered at the same time, wherein one composition
includes a salt of a-TEA, and the other includes the second agent(s). The other therapeutic modality
may be administered before, concurrently with, or following administration of the salts or
polymorphic forms of a-TEA. The therapy using the salts or polymorphic forms of a-TEA may
precede or follow administration of the other agent(s) by intervals ranging from minutes to weeks. In
embodiments where the other agent and the salts or polymorphic forms of a-TEA are administered
separately, one would generally ensure that a significant period of time did not expire between the
time of each delivery, such that each agent would still be able to exert an advantageously combined
effect. In such instances, it is contemplated that one would typically administer the salts or
polymorphic forms of a-TEA and the other therapeutic agent within about 12-24 hours of each other
and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours
being most preferred. In some situations, it may be desirable to extend the time period for treatment
significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8)
lapse between the respective administrations.
It also is conceivable that more than one administration of a salt or polymorphic form of a-
TEA, or the other agent will be desired. In this regard, various combinations may be employed. By
way of illustration, where the salts or polymorphic forms of a-TEA is "A" and the other agent is "B",
the following permutations based on 3 and 4 total administrations are exemplary:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are likewise contemplated. Non-limiting examples of pharmacological
agents that may be used in the present invention include any pharmacological agent known to be of
benefit in the treatment of a cancer or hyperproliferative disorder or disease. In some embodiments,
combinations of the salts or polymorphic forms of a-TEA with a cancer targeting immunotherapy,
radiotherapy, chemotherapy, or surgery are contemplated. Also contemplated is a combination of a
salt or polymorphic form of a-TEA with more than one of the above mentioned methods including
more than one type of a specific therapy. In some embodiments, it is contemplated that the
immunotherapy is a monoclonal antibody which targets HER2/neu such trastuzumab (Herceptin®) or
a similar antibody. In other embodiments, the immunotherapy can be other cancer targeting
antibodies such as alemtuzumab (Campath®), bevacizumab (Avastin®), cetuximab (Eribitux®), and
panitumumab (Vectibix®) or conjugated antibodies such as ibritumomab tiuxetan (Zevalin®),
tositumomab (Bexxar®), brentuximab vedotin (Adcetris®), ado-trastuzumab emtansine (Kadcyla™),
or denileukin dititox (Ontak®) as well as immune cell targeting antibodies such as ipilimumab
(Yervoy®), tremelimumab, anti-PD-1, anti-4-l-BB, anti-GITR, anti-TIM3, anti-LAG-3, anti-TIGIT,
anti-CTLA-4, or anti-LIGHT. Furthermore, in some embodiments, the salts or polymorphic forms of
a-TEA are envisioned to be used in combination therapies with dendritic cell-based immunotherapies
such as Sipuleucel-T (Provenge®) or adoptive T-cell immunotherapies.
Furthermore, it is contemplated that the salts or polymorphic forms of a-TEA are used in
combination with a chemotherapeutic agent such as anthracyclines, taxanes, methotrexate,
mitoxantrone, estramustine, doxorubicin, etoposide, vinblastine, carboplatin, vinorelbine, 5-
fluorouracil, cisplatin, topotecan, ifosfamide, cyclophosphamide, epirubicin, gemcitabine, vinorelbine,
irinotecan, etoposide, vinblastine, pemetrexed, melphalan, capecitabine, oxaliplatin, BRAF inhibitors,
and TGF-beta inhibitors. In some embodiments, the combination therapy is designed to target a
cancer such as those listed above. In the preferred embodiments, the cancer the combination therapy
is designed to treat is an endometrial tumor, mammary cancer, lung cancer, ovarian cancer, prostate
cancer, breast cancer, cervical cancer or colon cancer.
VI. Examples
The following examples are included to demonstrate preferred embodiments of the invention.
It should be appreciated by those of skill in the art that the techniques disclosed in the examples which
follow represent techniques discovered by the inventor to function well in the practice of the
invention, and thus can be considered to constitute preferred modes for its practice. However, those
of skill in the art should, in light of the present disclosure, appreciate that many changes can be made
in the specific embodiments which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
A, Methods
MICROSCOPY
A Zeiss Universal microscope configured with a polarized visible light source and polarizable
analyzer was used to evaluate the optical properties of the samples. Specimens were typically
mounted on a microscope slide with a cover glass. Because of the "waxy" nature of the material slide
preparations were manually smeared with a spatula a d. appearance noted. Observations of
particle/crystal size and shape and birefringence were recorded.
2. HOT STAGE MICROSCOPY (HSM)
A Linkam hot stage accessory was used in tandem with the microscope. Specimens were
mounted on a microscope slide w th a cover glass. Samples were heated from room temperature
through melting using a Linkam TMS 94 temperature control and Linksys 32 data capture software
system. Observations of possible phase change, melting, decomposition, etc. were
recorded.
.?. PROTON NUCLEAR MAGNETIC RESONANCE (' NMR)
Samples were analyzed by 1 NMR to determine stoiebiometry (molar ratio of acid to base
counter ion). Spectra of salt candidates were compared to that of the free acid and changes in
chemical shifts indicating salt formation observed. Samples were prepared by dissolving in a
chloroform: methanol : water mixture with 0.05% (v/v) tetramethylsilane (TMS). Spectra were
collected at ambient temperature on a Bruker Avance 1 400 MHz FT-NMR spectrometer and Bruker
Topspin software (version 2.1). Prior to each sample analysis, the magnetic field surrounding the
sample was optimized by an automated shimming program.
4. DIFFERENTIAL SCANNING CALORIMETRY (BSC)
Differential scanning calorimetry (DSC) is a technique used to measure characteristic heat
flux of a test article as it is scanned through a temperature gradient under a controlled atmosphere.
Thermal phase transitions such as endothermie melting and exothermic decomposition were recorded.
DSC data were collected on a TA Instruments DSC. In general, samples in the mass range of 1 to 1 0
g were crimped in aluminum sample pans and scanned from 25 to approximately 300 °C at 10
°C/min using a nitrogen purge of 0 mL/min.
5. T E M G A ViMETRlC ANAL S S (TGA)
Themiogravimetxic analysis involves the detemrination of the mass of a specimen as a
function of temperature. TGA data were collected on a TA Instruments Q500 TGA. In general,
samples in he mass range of 2 to 0 mg were placed in an open, prc-tared platinum sample pan and
attached by fine wire to a microhalanee. The sample was suspended into a furnace which was heated
from 25 to about 250 °C at 0 °C/min using a nitrogen purge at 100 mL/min. Sample weight change
as a function of temperature was observed.
6. X~RA POWDER DIFFRACTION fXR
X- ay powder diffraction patterns were obtained using a Broker D8 Discovery diffractometer
equipped with an XYZ stage, laser video microscope for positioning, and a two dimensional HiStar
area Detector or a scintillation detector. A CuKa radiation 1.5406 angstrom source operating at 40 kV
and 40 mA was used to irradiate samples. The X-ray optics consists of a Gobe mirror coupled with a
pinhole collimator of 0,5 or 1.0 mm Theta-tbeta conti nuous scans were employed with a sampledetector
distance of approximately 30 cm, which gives an effective 2Q range of 4-40 °C. Samples
were mounted in ow background quartz plates.
7. SOLUBILITY
Milligram size (2 mg) quantities of each sample were placed into a vial. Buffered water (pH
4, 7, and 0) was added and the vials were stirred for a few minutes, followed by visual observation
for remaining solids. The solvent was incrementally added until the solids were dissolved, or a
maximum volume of solvent (10 ml.) was added and the experiment was terminated.
& TYG OSC PIC - DYNAMIC VAPOR SORPTION {DVS}
DVS is a gravimetric screening technique that measures how quickly and how much of a
solvent (water) is adsorbed by a sample. The relative humidity or vapor concentration surrounding the
sample is varied while the change in mass of the sample is measured. A vapor sorption isotherm
shows the equilibrium amount of vapor sorbed as a function of relativity humidity. The mass values at
each relative humidity step are used to generate the isotherm isotherms are divided in two
components: sorption for increasing humidity steps and desorpiion for decreasing humidity steps. A
plot of kinetic data is also supplied which shows the change in mass an humidity as a function of
time.
Samples were analyzed using a TA Q2000 automated dynamic vapor sorption analyzer. The
samples were dried at 40 C for 5 hours and then cooled to 25 °C with a dry nitrogen purge over them
until they no lo ger lost mass at % RH. Tire samples were then subjected to 0 to 95% RH, back to
0% RH at 25 °C in 5% RH steps
9. STABILITY
The scaled up salts and free acid were challenged by heat (solids stored at 25 and 60 C for 1
week), oxidation {solids stored in oxygen headspace at 25 C for 1 week), light (solids exposed > 1 "
1CH international Conference on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use UV confirmatory conditions), and solutions (HPLC diluentrnethanoi)
at 25 and 40 °C for 1 week.
These samples were analyzed, along with unstressed controls, by HPLC to characterize their
stability.
0. HPLC ANALYSIS
Salt candidates were analyzed by total area normalization (TAN). The samples were dissolved
in methanol at a concentration of 0.8 mg/mL.
HPLC Conditions
HPLC Column: Sunfire C , 3.5 mih, 4.6 x 150 m
Column Temp; 45 C
Autosanipler Flush 25:75 Acetonitriie: reagent alcohol
Flow Rate: 1.5 mL/m
injection Volume; m .
UV Detection: 2 5 n
Mobile Phase: A - 0.1% acetic acid in acetonitriie
B - 0.1% acetic acid in reaeen! alcohol
Gradient Pump Pro
Step Time (minutes) %A % B Curve
0.5 92.0 8.0 0.0
6.0 92.0 8.0 0.0
6.0 10.0 90.0 1.0
2.0 10.0 90.0 0.0
7.0 92.0 8.0 0.0
/. CHA RACTERIZATIONOF THE FREE ACID
The -TEA free acid, lot 54543-7-34, was analyzed by XRD, DSC, TGA, DVS and
microscopy. The sample is crystalline as shown by the XRD pattern in F G, 1. The DSC thermogram
of the free acid shows a melting endotherm onset at 54.5 °C. The TGA thermogram shows the free
acid to be free of vo!attlcs, with a weight loss of 0.2% at 207.5 °C.
DVS was performed on a sample of the free acid. The free acid exh its a total weight gain of
abo t 0.2 weight% at 95% RI-I. This data indicates that the free acid does not form hydrates or exhibit
deliquescence.
The free acid was examined using optical microscopy. The sample exhibited areas of both
birefringent and amorphous agglomerates. Manual manipulation of the sample under magnification
resulted in a smearing and a droplet like appearance. This behavior suggests thai the free acid has
some of the characteristics of a liquid crystal. Liquid crystals are a state of matter that have properties
between those of a conventional liquid and those of a solid. Liquid crystals are substances that are not
as rigidly ordered as a solid but have some degree of alignment. Molecules which tend to be
candidates for having liquid crystal phases are often long having a rigid central region and end groups
that are slightly flexible.
2. PRIMARY SALT SCREEN (SO MGSCALE) PREPARATIVE PROCEDURES
The predicted pK value of the -Ί Έ A free acid is 3.2.3. Based o n these predicated pK values,
a library of salt forming bases were chosen and is shown in Table .
Table : Salt Forming Bases
*Safety and pharmaceutical acceptability of the counter-ions: class J-unrestricted used based on
physiological ubiquitous ions or intermediate metabolites; class Ώ-ttniversally approved, but not
naturally occurring. Low toxicity and good talerability; class Mi-case by case limited approval, some
reactivity, somepharmacological activity.
Initially, a series of experiments consisted of 7 salts prepared on an approximately 0 mg
scale were carried out and are panel . A solution of free acid was prepared in methanol and
appropriate molar equivalents of base solution and/or solids were added. Samples were mixed several
hours and allowed to slowly evaporate at room temperature with about 2. psi nitrogen purge.
Characterization of the resultant salts was performed using X-ray diffraction (XRD). One
sample (ethanolamine) exhibited a crystalline pattern different than the free acid starting material
indicating possible salt formation. The remaining six samples produced amorphous materials. These
six samples were solvent ripened in an attempt to increase the crystallinity of the resultant saits.
Unfortunately, this technique had limited successful in increasing the crystallinity.
The crystalline salt candidate, ethanolamine, was further characterized by differen
scanning calorimetry (DSC). An overview of the results is summarized in Table 2.
Table 2; Panel 1
1glassy materials are amorphous (not crystalline) nonfree flowing solids which often do not exhibit a
sharp melting endolherm during DSC analysis.
The next group of expenmenls, panel 2, consists of 8 saits prepared on an approximately 50
g scale. A solution of free acid was prepared in i -butanol and appropriate molar equivalents of base
solution and or solids were added. Samples were mixed several hours and allowed to slowly evaporate
at room temperature with about 2 psi nitrogen purge.
Characterization was performed using X-ray Diffraction (XRD). One sample (ethanolamine)
exhibited a crystalline pattern similar to the pattern from Panel , indicating possible salt formation.
Two samples exhibited evidence of the base eo nte on, which would indicate that no salt was
formed. The remaining five samples produced amorphous materials. These five samples were solvent
ripened in an attempt to increase the crystallinity. Again, this technique was not successful in
increasing the crystallinity.
The results of Panel 2 are listed in Table 3.
Table 3: Panel 2
Finally, another set of experiments, Panel 3, consisting of 16 salts was prepared on
approximately 50 mg scale. A solution of free acid was prepared in isopropyl alcohol and appropriate
molar equivalents of base solution and or solids were added. Samples were mixed several hours and
allowed to slowly evaporate at room temperature with about 2 psi nitrogen purge.
Characterization was performed using XRD. Six samples (ethanolamine arginine, lysine,
choline, tris, and deanol) exhibited crystalline XRD patterns indicating possible salt formation. The
eight remaining samples produced amorphous materials. These eight samples were solvent ripened in
an attempt to increase the crystal linity. This technique was not successful in increasing the
cr tallinity. Two samples exhibited evidence of the base counter-ion, indicating that no salt
for alion.
The Panel 3 results are listed in Table 4.
Table 4: Panel 3
3. PRIMARYSALT SCREEN (SO MGSCALE) RESULTS AND DISCUSSION
Panels 1 through 3 produced six crystalline salt co po d . The characteristics of these
compounds are further described below.
a) E a o a ne
The ethanolamine salt of a-Ί A is a waxy crystalline solid. The unique X-ray diffraction
characteristics were observed in all three panels and are shown in FIG, 2. The DSC thermogram
exhibited multiple endothermic transitions with temperatures of about 88, 100, 115, 149 and 169 °C.
The DSC plot is shown in FIG. 3. The total volatile content by TGA over the temperature range 25-
95.5 C was 0.4 wt%.
The H M of this compound shows the stoich tometry of free acid to base to be in a 1:1
ratio. A change in chemical shifts from the free acid spectrum wa s observed suggesting the compound
is a salt.
b) -Ar g nm
The arginine salt of a-TEA was a partially crystalline solid that was less "waxy" than the free
acid. The unique X-ray diffraction pattern of the L-arginine sample is shown in FIG. 4. The DSC
thermogram exhibited multiple endothermic transitions at about 31 and 4 °C. The DSC plot is
shown in FIG. 5. The total volatile content by TGA over the temperature range 25-1 4 C was about
0.3 wt%.
The Ή NMR of this candidate shows the stoichiometry of free acid to base to be in a 1:1
ratio. A change in chemical shifts from the free acid spectrum was observed suggesting this candidate
is a salt.
The dynamic vapor sorption (DVS) isotherm indicates a possible hemihydrate formation
( 1.3% weight gain) at about 40% RH. The desorption cycle indicates that the hydration process is
reversible. The particle size of this material was very small as evidenced by photomicrograph. The
salt produced some liquid crystal-like behavior, but to a lesser degree than observed with the free
acid. The approximate visual solubility of the arginine salt in pH 4, 7, and 10 buffers was < 0.2
mg/niL.
) L-Lysme
The lysine salt of a-TEA is a crystalline solid that is less "waxy" than the free acid. The
unique X-ray diffraction pattern of this sample is shown in FIG. 6. The DSC thermogram exhibited
multiple endothermic transitions at about 55 and 200 °C. The DSC plot is shown in FIG. 7. The total
volatile content by TGA over the temperature range 25-168.8 °C was 0.4 wt%.
The MR of the lysine salt shows the stoichiometry of free acid to base to be in a :0.7
ratio. A change in chemical shifts from the free acid spectrum was observed suggesting the compound
is a salt.
The DVS adsorption isotherm shows onset of weight gain at about 75% RH, with a rapid total
weight gain of about 12% at 95% RH. The desorption cycle shows hysteresis, in which upon
desorption (decreasing humidity) the sample does not lose the sorbecl water. The residual 2 weight %
corresponds to a putative hydrate of the lysine salt. The stoichiometry of the hydrated lysine salt is
not defined. The kinetic plot indicates the weight change on desorption does not come to equilibrium
indicating that hydrate formation may be reversible given enough time. The particle size of this
material appears was very small as evidenced by photomicrograph. The salt exhibits some liquid
crystal-like behavior, but to a lesser degree than observed with the free acid. The approximate visual
solubility of the lysine salt in pH 4, 7, and buffers was < .2 mg/mL.
d) Choline
The choline salt of -TEA is a partially crystalline "waxy" solid. The unique X-ray diffraction
pattern of the sample was observed is shown in FIG. 8. The DSC thermogram exhibited multiple
endothermic transitions at about 50, 80, and 150 °C. The DSC plot is shown in FIG. 9. The total
volatile content by "I'GA over the temperature range 25- 88.2 C was 2.5 wt%.
The NMR of this candidate shows the stoichiometry of free acid to base to be in a :0.8
ratio. A change n chemical shifts from the free acid spectrum was observed suggesting this candidate
a salt.
e) Tris-hydroxyinethyl me (Tris)
The tris candidate was a partially crystalline " semi-waxy" solid. The unique X-ray diffraction
pattern of the sample was observed is shown in FIG. 10. The DSC thermogram exhibited multiple
endothermic transitions at about 10 1 and 75 °C. The DSC plot is shown in FIG, 11. The total
volatile content by I GA over the temperature range 25- 144 C wa s 0.6 wt%.
The H NMR of this candidate shows the stoichiometry of free acid to base to be in a 1:1+
ratio. A change n chemical shifts from the free acid spectrum was observed suggesting this candidate
a salt.
The DVS isotherm indicates a possible hemihydrate formation ( .5% weight gain) starting at
about 55% RH. The desorption cycle shows hysteresis indicating that the water is well bound. The
particle size of this material was very small based upon photomicrograph images. There is some
liquid crystal-like behavior, but to a lesser degree than observed with the free acid. The approximate
visual solubility of the tris salt i pH 4, 7, and 10 buffers was < 0.2 mg/mL.
1) iY - i ei e ¾sn i m an l)
T re deanol candidate is a crystalline "waxy" solid. The unique X-ray diffraction pattern of the
sample was observed is shown in FIG. 12, The DSC thermogram exhibited multiple endoihermic
transitions at about 47, 73, and 87 °C. The DSC plot is shown in FIG. 13. The total volatile content by
TGA over the temperature range 25-207 °C was 7.7 wt%.
The NMR of this candidate shows the stoichiometry of free acid to base to be in a :0.7
ratio. A change in chemical shifts from the free acid spectrum was observed suggesting this candidate
is a salt.
4. SALTSCREEN SCALE UP RESULTS AND DISCUSSION
Based on results of the primary salt screening panels, the lysine, arginine and tris nionosaits
were selected for further investigation. This analysis served two purposes; firstly to provide a
sufficient quantity of material for further testing, and secondly, to gather information on the
processibility and physical handling characteristics of the salt candidates. Samples were analyzed by
XRD, DSC, Hot. Stage Microscopy (HSM), HPLC for purity, static humidity, stoichiometry, and
stability.
a) Lysine (Sample 901)
The scaled-up lysine salt had more desirable handling properties compared to the free acid,
including being less "tacky" than the free acid. Furthermore, the salt was a crystalline powder and
easier to formulate into a capsule than the free acid.
The XR.D pa tem of the scaled-up lysine salt candidate was consistent with the pattern of the
small scale sample. The DSC profile of this sample is very similar to the small scale sample, both
exhibiting endoihermic transitions at about 55 and 200°C. Hot stage microscopy (HSM) was carried
out on this salt candidate in order to aid in explaining the multiple endotberms observed by DSC.
Visual onset of melting was observed at about 200 °C with complete melting by about 5 °C. No
obvious transition was observed at about 55 °C.
The HPLC total area normalization (TAN) purity of the salt wa determined to be 98 area
given a relative purity of the compound.
Formation of hydrates can be a very slow kinetic process. A second approach to identify
hydrate formation is performing static humidity experiments and monitor gravirnetrically for weight
change. Samples of lysine salt were placed in 75 and 84% RH environments for 33 and 9 days
respectively. After 33 days at 75% RH the lysine sample gained 0.6 weight %. At 84% RH, after 9
days, the lysine salt exhibited a weight gain of 1.8%. The X-ray diffraction pattern of th is 84% RH
sample showed no change from the prehumidified sample. Given the DVS behavior of the small scale
sample, these studies suggest an isostruetural hemihydrate exists.
The Ή NMR analysis showed this candidate to have a monomolar ratio of free acid to base.
The chemical shift of the methylene group in the polar part of the molecule suggests that the
compound appears to be a salt.
b) Arginme {Sample 903)
The scaled up argmine salt exhibited better handling relative to the free acid.
The XRD pattern of the scaled p arginine salt matches the pattern of the small scale sample
pattern. The DSC profile of this sample matches small scale exhibiting 2 endoihermic transitions at
about 136, and 7 °C respectively. Hot stage microscopy (HSM) on this salt candidate showed
visual melting at about 180 C and above. No changes in the sampie were visually observed in the 30
to 0 °C temperature range. The eodothermic transition observed in the DSC profile in this
temperature region may be due to a thermally induced liquid crystal phase change. Some variable
temperature XRD studies may be useful in studying this transition further.
T re HPLC-Total Area Normalization (TAN purity) of the salt was found to be 98 area%
given a relative purity of the compound.
Samples of arginine salt were placed in 75 and 84% RH environments for 33 and 9 days
respectively. After 33 days at 75% RH the arginine sample gained 0.7 weight%. At 84% RH, after 9
days, the argmine salt exhibited a weight gain of 2.3%. The X-ray diffraction pattern of this 84% RH
sample showed no change from the pre-humidified sampie, suggesting that if a hydrate is forming, t
has the same XRD pattern.
The -I NMR analysis showed this salt to have a mole ratio of free acid to base of one. The
chemical shi ft of the methylene group in the polar part of the molecule suggests that a salt l as been
formed.
c) Tris (Sample 902)
The scaled up tris salt appears to have more normal handling than the free acid but is slightly
waxier than both the lysine and arginine salts.
The XR.D pattern of the sealed up tris salt matches small scale sample pattern. The DSC
profile is very similar to the small scale sample. The tris scaled up sait exhibits an endolherm with an
onset on 106 °C. Visual melting was observed using HSM in the 95 to 105 °C temperature range,
which corresponds to the endotherm observed in the DSC.
The HPLC-Total Are Normalization (TAN purity) of the sait was found to be 97.5 area
given a relative purity of the compound.
A sample of tris salt was placed in 75% RH environment for 33 days. The sample exhibited a
weight gain of 4.0%. This fact is consistent with the DVS data collected earlier. These data suggest
that this salt forms a hydrate upon exposure to sufficient moisture.
The Ή NMR analysis showed this salt to have a ole ratio of free acid to base of about one.
The chemical shift of the methylene group in the polar part of the molecule suggests that the
compound formed is likely a salt.
A stacked plot of the NMR spectra of the free acid, lysine, arginine, and tris salts shows the
movement of the methylene group supporting the conclusion of the formation of salts with the
compound.
d) STABILITY OF SALTS
The three salts (lysine, arginine, and iris) were challenged using heat (solids stored at 25 and
60 C for 1 week), oxidation (solids stored in oxygen headspaee at 25 C for 1 week), light (UV
source under ICH confirmatory conditions^ 200 Whr/' ), and solutions (in HPLC diluent) at 25 and
40 °C for 1 week. Stressed samples were analyzed using HPLC to determine their impurity profiles.
A summary of the stability data generated using HPLC analysis is shown below in Table 5.
Table 5: Summary of HPLC Stability Bates o Stressed Salts
PC Total Area o i i - Area % P
Conditions Free A d Lysine Tris
Sample 734 Sample 901 Sample 903 Sample 902
Photo Stability Dark Control 97 7 98.7 97.9 98.2
Photo Stability Exposed 98.2 98.8 98.5 98.7
The stability results shown i Table 5 represent the averages of two injections of duplicate
sample preparations. While the data can only suggest the short term stability, they do provide some
insight into the stability of the sal forms which could be extrapolated to the salts long term stability.
FIGS, 14-17 show the chromatogram overlay plots for samples analyzed during the stability portion
of the study. The HPL stability data show that the salts exhibited little to no degradation under the
stress conditions used n the study. Ali three salt candidates show improved solution stability in
methanol compared to the free acid.
5. SUMMARYOF SALT SCREENING STUDIES
A salt screening study wa.s performed to identify suitable salt candidates of a-TEA. During
the course of the study, different potential bases were examined. Of these bases, 6 bases formed
crystalline salts. The most promising of the group salts of lysine, arginine, and iris were isolated and
studied n detail . These three salts were scaled-up to 2 grams and eval uated using multiple analytical
techniques.
Based on the data obtained during ti s study, all three of these salts appear to be viable
candidates. These salts exhibit improved physical properties over the free acid. A l of the salts are
crystalline and have less "waxy" physical properties relative to the free acid. The lysine and arginine
salts exhibit the most improvement in this characteristic. The visual melting temperature of ali three
salts is higher than the free acid. Ali three salts exhibit equal stability under all conditions tested with
improved stability over the free acid in solution. The visual solubility of each salt was comparable to
the free acid in buffered water (< 0.2 mg/mL).
The salt samples ali exhibited the potential to form putative hydrates ail which appear to be
reversible. The arginine and tris salts exhibited D S water uptake near critical humidities 40% and
55% H respectively. The lysine salt shows DVS water uptake above about 75% RH.
Table Summar o Final Salt Characteristics
C. Anil-Tumor Efficacy of o TEA Saiis m Animal Models
Studies of the anti-tumor efficacy of the a-TEA salts were arn ed out using a well
characterized but difficult to treat 4TI mouse model of metastatic mammary cancer with the a-TEA
lysine salt (a-TEA-Lys). The lysine salt was incorporated into standard mouse chow at an amount of
3 g kg chow {0.3% w/w) by Harlan Teklad, Madison W . Half of the mice were placed on an a-TEALys
diet after the tumors were established (9 days post tumor injection.) and the rest placed on an
analogous nutrient diet without a-TEA-Lys which served as the control group. The average tumor
size of mice that ate a diet containing a-TEA-Lys showed a significant decrease in average tumor size
compared to mice feed a control diet without the a-TEA salt (FIG. 18). Furthermore, mice
consuming a diet which contains the a-TEA salt showed an increased survival compared to mice who
consumed a control diet of standard chow (FIG. 19). These data suggest that converting a-TEA into a
salt appear to have little change on the efficacy of the compound with significant tumor growth
suppression, prolongation of overall survival, and complete tumor regression in about 25% of the
mice treated.
* * * * * * * * * * * * * * * *
All of the compounds, polymorphs, formulations, and methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the present disclosure. While the
compounds, polymorphs, formulations, and methods of this invention have been described in terms of
preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to
the compounds, polymorphs, formulations, and methods, as well as in the steps or in the sequence of
steps of the method described herein without departing from the concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described herein while the same or similar
results would be achieved. All such similar substitutes and modifications apparent to those skilled in
the art are deemed to be within the spirit, scope and concept of the invention as defined by the
appended claims.
REFERENCES
The following references to the extent that they provide exemplary procedural or other details
supplementary to those set forth herein, are specifically incorporated herein by reference.
US 6,41 7,223
US 6,703,384
US 6,770,6 72
US 7,3 12,232
Anderson, etal, Cancer Res, 64:4263-4269, 2004.
Hahn, et al, Cancer Res, 66:9374-9378, 2006.
Hahn, et al, Mol Cancer Ther, 8:1570-1578, 2009.
Hahn, et al, BMC Cancer, 11:471, 2011.
Kline, et al, Vitam Horm, 76:435-461, 2007.
Lawson, et al, Cancer Chemother Pharmacol, 54:421-431, 2004.
March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 2007.
Neuzil, et al, Mol Aspects Med, 28:607-645, 2007.
Yu, et al, Mol Carcinog, 49:964-973, 2010.
CLAIMS
What Is Claimed Is:
1. A comp
wherein
or a hydrate thereof.
2. The compound of claim 1, wherein X is:
or a hydrate thereof.
The compound of either claim 1 or claim 2, wherein the X
or a hydrate, thereof.
The compound according to any one of claims 1-3, wherein the X
or a hydrate, thereof.
5. The compound according to any one of claims 1-4, wherein the formula is further defined as:
or a hydrate, thereof.
6. The compound of claim 5, wherein the compound is present as a polymorphic form having an
X-ray powder diffraction pattern (CuKa) comprising two peaks at about 11.84 °2Qand 19.74
°2Qor a hydrate, thereof.
7. The compound of claim 6, wherein the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising a peak at about 20.73 °2Q.
8. The compound of either claim 6 or 7, wherein the polymorphic form has an X-ray powder
diffraction pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, or all of the peaks at about 6.24, 9.35, 14.92, 15.51, 17.03, 18.49, 21.70, 22.81, 23.51,
24.85, 27.69, 28.31, 30.93, 32.30, 33.21, 35.02, and 39.30 °2Q.
9. The compound according to any one of claims 6-8, wherein the polymorphic form has an Xray
powder diffraction pattern (CuKa) substantially as shown in FIG. 4.
10. The compound according to any one of claims 6-9, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising two endotherms from about 125
°C to about 140 °C and from about 170 °C to about 195 °C.
11. The compound of claim 10, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comprising two endotherms centered at about 131 °C and at
around 184 °C, respectively.
12. The compound of either claim 10 or claim 11, wherein the polymorphic form has a
differential scanning calorimetry isotherm substantially as shown in FIG. 5.
13. The compound according to any one of claims 6-12, wherein the polymorphic form is
anhydrous.
14. The compound according to any one of claims 6-12, wherein the polymorphic form is a
hydrate.
15. The compound of claim 14, wherein the polymorphic form is a hemihydrate.
16. The compound according to any one of claims 1-4, wherein the formula is further defined as:
or a hydrate, thereof.
17. The compound of claim 16, wherein the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 9.69 °2Qor a hydrate,
thereof.
18. The compound of claim 17, wherein the X-ray powder diffraction pattern (CuKa) further
comprises a peak at about 20.10 °2Q.
19. The compound of either claim 17 or 18, wherein the X-ray powder diffraction pattern (CuKa)
further comprises a peak at about 19.78 °2Q.
20. The compound according to any one of claims 17-19, wherein the X-ray powder diffraction
pattern (CuKa) further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all of the
peaks at about 5.90, 7.77, 14.08, 17.58, 17.91, 18.31, 21.26, 22.17, 23.16, 24.77, 28.57, 29.71,
31.10, 31.69, 34.45, 34.99, and 36.24 °2Q.
21. The compound according to any one of claims 17-20, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) that is substantially as shown in FIG. 6.
22. The compound according to any one of claims 17-21, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising two endotherms from about 53
°C to about 60 °C and from about 180 °C to about 235 °C.
23. The compound of claim 22, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comrpising two endotherms centered at about 55.8 °C and at
about 205 °C, respectively.
24. The compound of either claim 22 or claim 23, wherein the polymorphic form has a
differential scanning calorimetry isotherm that is substantially as shown in FIG. 7.
25. The compound according to any one of claims 17-24, wherein the polymorphic form is
anhydrous.
26. The compound according to any one of claims 17-24, wherein the polymorphic form is a
hydrate.
27. The compound according to any one of claims 1-4, wherein the formula is further defined as:
or a hydrate, thereof.
28. The compound of claim 27, wherein the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 20.14 °2Q or a
hydrate, thereof.
29. The compound of claim 28, wherein the X-ray powder diffraction pattern (CuKa) further
comprises peaks at about 13.63, 14.40, 19.31, and 22.34 °2Q.
30. The compound of either claim 28 or 29, wherein the X-ray powder diffraction pattern (CuKa)
further comprises two peaks at about 18.25 and 18.49 °2Q.
31. The compound according to any one of claims 28-30, wherein the X-ray powder diffraction
pattern (CuKa) further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of peaks at about
9.67, 15.60, 21.47, 23.57, 27.07, 28.39, 30.49, 32.21, 33.30, 34.29, 34.96, 35.89, and
36.39 °2Q.
32. The compound according to any one of claims 28-31, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) substantially as shown in FIG. 10.
33. The compound according to any one of claims 28-32, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising two endotherms from about
97.5 °C to about 120 °C and from about 185 °C to about 225 °C.
34. The compound of claim 33, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comprising two endotherms at about 106 °C and at about 192 °C,
respectively.
35. The compound of either claim 33 or claim 34, wherein the polymorphic form has a
differential scanning calorimetry isotherm substantially as shown in FIG. 11.
36. The compound according to any one of claims 28-35, wherein the polymorphic form is
anhydrous.
37. The compound according to any one of claims 28-35, wherein the polymorphic form is a
hydrate.
38. The compound of claim 37, wherein the polymorphic form is a hemihydrate.
39. The compound according to any one of claims 1-3, wherein the formula is further defined as:
or a hydrate, thereof.
40. The compound of claim 39, wherein the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 19.4 °2Qor a hydrate,
thereof.
41. The compound of claim 40, wherein the X-ray powder diffraction pattern (CuKa) further
comprises peaks at about 10.4, 14.1, and 17.8 °2Q.
42. The compound of either claim 40 or claim 41, wherein the X-ray powder diffraction pattern
(CuKa) further comprises two peaks at about 20.8 °2Q.
43. The compound according to any one of claims 40-42, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) substantially as shown in FIG. 2.
44. The compound according to any one of claim 40-43, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising five endotherms from about 85
°C to about 97 °C, from about 97 °C to about 105 °C, from about 110 °C to about 125 °C,
from about 145 °C to about 155 °C and from about 160 °C to about 210 °C.
45. The compound of claim 44, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comprising five endotherms at about 90.1 °C, at about 100.7 °C,
at about 117.9 °C, at about 149.7 °C, and at about 181.6 °C, respectively.
46. The compound of either claim 44 or claim 45, wherein the polymorphic form has a
differential scanning calorimetry isotherm substantially as shown in FIG. 3.
47. The compound according to any one of claims 1-3, wherein the formula is further defined as:
or a hydrate, thereof.
48. The compound of claim 47, wherein the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising two peaks at about 18.40 and 19.77
°2Qor hydrate, thereof.
49. The compound of claim 48, wherein the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising peaks at about 4.28 and 10.75 °2Q.
50. The compound of either claim 48 or claim 49, wherein the polymorphic form has an X-ray
powder diffraction pattern (CuKa) further comprising two peaks at about 19.00 °2Q.
51. The compound according to any one of claims 48-50, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, or all of the peaks at about 6.45, 12.90, 16.17, 16.73, 17.31, 17.72, 20.96,
21.72, 28.08, 28.71, 29.70, 31.05, 31.63, 32.25, 34.60, 35.18, 36.60, and 39.02 °2Q.
52. The compound according to any one of claims 48-51, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) substantially as shown in FIG. 8.
53. The compound according to any one of claims 48-52, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising three endotherms from about 50
°C to about 60 °C, from about 75 °C to about 85 °C, and from about 150 °C to about 250 °C,
respectively.
54. The compound of claim 53, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comprising three endotherms at about 50 °C, at about 80 °C, and
at about 150 °C, respectively.
55. The compound of either claim 53 or claim 54, wherein the polymorphic form has a
differential scanning calorimetry isotherm substantially as shown in FIG. 9.
56. The compound according to any one of claims 1-3, wherein the formula is further defined as:
or a hydrate, thereof.
57. The compound of claim 56, wherein the compound is present as a polymorphic form having
an X-ray powder diffraction pattern (CuKa) comprising a peak at about 21.73 °2Q or a
hydrate, thereof.
58. The compound of claim 57, wherein the polymorphic form has an X-ray powder diffraction
pattern (CuKa) further comprising peaks at about 9.34, 14.90, 16.06, 17.33, 18.51, and
22.94 °2Q.
59. The compound of either claim 57 or claim 58, wherein the polymorphic form has an X-ray
powder diffraction pattern (CuKa) further comprising two peaks centered at about 20.22 °2Q.
60. The compound according to any one of claims 57-59, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or
all of peaks at about 10.05, 11.19, 14.13, 24.04, 27.65, 28.14, 29.73, 30.30, 31.06, 32.02,
33.45, and 34.49 °2Q.
61. The compound according to any one of claims 57-60, wherein the polymorphic form has an
X-ray powder diffraction pattern (CuKa) substantially as shown in FIG. 12.
62. The compound according to any one of claims 57-61, wherein the polymorphic form has a
differential scanning calorimetry (DSC) isotherm comprising three endotherms from about 45
°C to about 55 °C, from about 65 °C to about 80 °C, and from about 80 °C to about 95 °C.
63. The compound of claim 62, wherein the polymorphic form has a differential scanning
calorimetry (DSC) isotherm comprising three endotherms at about 47 °C, at about 73 °C, and
at about 87 °C, respectively.
64. The compound of either claim 62 or claim 63, wherein the polymorphic form has a
differential scanning calorimetry isotherm that is substantially as shown in FIG. 13.
65. A method of making a salt of the formula:
wherein: X + is a protonated base; comprising reacting a compound of the formula:
with the base sufficient to form a salt; or a hydrate thereof.
66. The method of claim 65, wherein the base is: sodium hydroxide, potassium hydroxide,
magnesium hydroxide, meglumine, diethylamine, ethanolamine, diethanolamine, L-arginine,
L-lysine, choline, tris-hydroxymethylamine, N,N-dimethylethanolamine, 2-
diethylethanolamine, calcium hydroxide, dibenzylethylenediamine, or procaine.
67. The method of either claim 65 or claim 66, wherein the base is ethanolamine, L-arginine, Llysine,
choline, tris-hydoxymethylamine, or N,N-dimethylethanolamine.
68. The method according to any one of claims 65-67, wherein the base is L-arginine, L-lysine, or
tris-hydroxymethy lamine .
69. A pharmaceutical composition comprising a compound, a salt, or a polymorphic form
according to any one of claims 1-68 and an excipient.
70. The pharmaceutical composition of claim 69, wherein the pharmaceutical composition is
formulated for administration: orally, intraadiposally, intraarterially, intraarticularly,
intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly,
intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally,
intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously,
intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally,
subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally,
vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous
infusion, via infusion, via inhalation, via injection, via local delivery, or via localized
perfusion.
71. The pharmaceutical composition of claim 70, wherein the pharmaceutical composition is
formulated for oral, intraarterial, intravenous, intraperitoneal, topical, or inhalational
administration.
72. A method of reacting a compound of the formula:
to obtain a polymorphic form comprising combining an about equal molar amount of a base
and the compound in an alcoholic solvent and mixing for a time period from about 1 to about
24 hours.
The method of claim 72, wherein the base is NaOH, KOH,
74. The method of either claim 72 or claim 73, wherein the base is
75. The method according to any one of claims 72-74, wherein the alcoholic solvent is of the
formula: O- w erem s alkyl(C<6).
76. The method according to any one of claims 72-75, wherein the alcoholic solvent is methanol,
1-butanol, or isopropyl alcohol.
77. The method according to any one of claims 72-76, wherein the alcoholic solvent is isopropyl
alcohol.
78. The method according to any one of claims 72-77, further comprising allowing the solution to
slowly evaporate at room temperature.
79. The method according to any one of claims 72-78, further comprising allowing the
evaporation to occur under a constant flow of nitrogen gas.
80. The method of claim 79, wherein the constant flow of nitrogen has a pressure of about 2 psi.
81. A method of treating or preventing a hyperproliferative disease, comprising administering to
the patient a therapeutically effective amount of the compound, salt, polymorphic form, or
pharmaceutical composition according to any one of claims 1-71.
82. The method of claim 81, wherein the hyperproliferative disease is a cancer.
83. The method of claim 8 1 or claim 82, wherein the cancer is breast cancer, lung cancer, colon
cancer, an endometrial tumor, cervical cancer, ovarian cancer, mammary cancer, prostate
cancer, liver cancer, melanoma, lymphoma, or myeloma.
84. The method claim 83, wherein the cancer is breast cancer.
85. The method of any one of claims 81- 83, further comprising administering to the patient a
second therapy.
86. The method of claim 85, wherein the second therapy is an immunotherapy or surgery.
87. The method of claim 86, wherein the second therapy is an immunotherapy.
88. The method according to any one of claims 85 and 86, wherein the immunotherapy comprises
administering a pharmaceutically effective amount of trastuzumab, adoptively transferred T
lymphocytes, or a therapeutic antibody.
89. The method of claim 88, wherein the therapeutic antibody selectively binds HER2/neu.
90. The method of claim 88, wherein the immunotherapy is trastuzumab.
91. The method of claim 88, wherein the immunotherapy comprises administering adoptively
transferred T lymphocytes to the patient.
92. The method of claim 91, wherein the adoptively transferred T lymphocytes are engineered to
express a chimeric antigen receptor (CAR) to the patient.
93. The method of claim 88, wherein the immunotherapy comprises administering an immune
modulating antibody to the patient.
94. The method of claim 93, wherein the immune modulating antibody is an anti-PD-1, anti-4-1-
BB, anti-GITR, anti-TIM3, anti-LAG3, anti-TIGIT, anti-CTLA-4 or an anti-LIGHT antibody.
95. The method of claim 85, wherein the second therapy is radiotherapy or a chemotherapeutic
agent.
96. The method of claim 86, wherein the second therapy is surgery.
97. The method of claim 95, wherein the second therapy is a chemotherapeutic agent.
98. The method of claim 97, wherein the chemotherapeutic agent is anthracyclines, taxanes,
methotrexate, mitoxantrone, estramustine, doxorubicin, etoposide, vinblastine, carboplatin,
vinorelbine, 5-fluorouracil, cisplatin, topotecan, ifosfamide, cyclophosphamide, epirubicin,
gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine, pemetrexed, melphalan,
capecitabine, oxaliplatin, BRAF inhibitors, and TGF-beta inhibitors.