Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/260918
PCT/US2022/031876
COMPOSITION AND METHOD FOR DUAL TARGETING IN TREATMENT OF
NEUROENDOCRINE TUMORS
TECHNICAL FIELD
100011 The present disclosure relates generally to compositions for targeting
and treating
neuroendocrine tumors. The composition in particular may include thyroid
hormone av133 integrin
receptor antagonists (referred to as "thyrointegrin antagonists") and
compounds that are targets of the
norepinephrine transporter (NET) or the catecholamine transporter (such as
benzyl guanidine ("BG") or
its derivatives).
BACKGROUND
[0002] The norepinephrinelcatecholamine transporter ("norepinephrine
transporter") is essential for
norepinephrine uptake at the synaptic terminals and adrenal chromaffm cells.
In neuroendocrine tumors,
the norepinephrine transporter is highly active and can. be targeted for
imaging and/or therapy with
localized radiotherapy. One of the most widely used theranostic agents
targeting the norepinephrine
transporter is meta-iodobenzylguanidine (MIBG), a guanidine analog of
norepinephrine. 1.231/1311-MIBG
theiranostics have been applied in the clinical evaluation and management of
neuroendocrine tumors,
especially in neuroblastoma, paraganglioma, and pheochromocytoma. 123I-MIBG
imaging has been used
in the evaluation of neuroblastoma, and 1311-MIBG for the treatment of
relapsed high-risk
neuroblastoma. however, the outcome remains sub-optimal. Positron Emission
Tomography (PET) tracers
targeting the norepinephrine transporter and its targets represent a better
option for the imaging and
assessment after treatment of neuroblastoma, paraganglioma / pheochromocytoma.
and carcinoids.
[0003] hitegfins are a super-family of cell surface adhesion receptors, which
control the attachment of
cells with the solid ex-tracellular environment, both to the extracellular
matrix (ECM), and to other cells.
Adhesion is of fundamental importance to a cell., it provides anchorage, cues
for migration. and signals
for growth and differentiation. Integrins are directly involved in numerous
normal and pathological
conditions, and as such are primary targets for therapeutic intervention.
Integrins are integral
transmembrane proteins, heterodimers, whose binding specificity depends on
which of the 14 a-chains
are combined with which of the 8 [f-chains. The integrins are classified in
four overlapping subfamilies,
containing the 01, f32, ID or av chains. A cell may express several different
integrins from each
subfamily. in the last several decades, it has been shown that intcgrins arc
major receptors involved in cell
adhesion, and so may be a suitable target for therapeutic intervention.
Integrin av133 regulates cell growth
and survival, since ligation of this receptor can. under some circumstances,
induce apoptosis in tumor
cells. Disruption of cell adhesion with anti-av133 antibodies, ROD peptides,
peptide mimetic or non-
peptide derivatives, and other integrin antagonists has been shown to slow
tumor growth.
[0004] Thyrointegrin antagonists have been shown to effect tumor angiogenesis
by interaction with
integrin avi33. The effect of thyrointegrin antagonists is described in U.S.
Pat. Pub. No. 2017/0348425
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
2
titled Non-Cleavable Polymer Conjugated with Alpha V Beta 3 (avI33) Integrin
Thyroid Antagonists, the
contents of which are incorporated by reference.
[0005] A composition comprising both a thyrointegrin antagonist compound and a
norepinepluine
transporter target compound would be well received in the art.
SUMMARY
[0006] According to an aspect. a composition comprises a compound of a general
formula:
/rt r
0
I T I
R,
4
rt2
or a salt thereof.; wherein RI, R2, R3, and R4 are each independently selected
from the group consisting
of hydrogen, iodine, fluorine, bromine, a methoxy group, a nitro group, an
amine group, and a nitrile
group; wherein R5, R6, R7, and R8 are each independently selected from the
group consisting of
hydrogen, iodine, and an alkane group; and ni > 0; n2> I; and Y includes an
amine.
[0007] According to another aspect, a method for dual targeting of tumor
cells, comprises
administering a composition comprising: a compound of a general formula:
2
itt3
Ft5
istkrikN. "
I
- coti
4 6
or a salt thereof; wherein RI, R2, R3, and R4 are each independently selected
from the group consisting
of hydrogen, iodine, fluorine, bromine, a methoxy group, a nitro group, an
amine group, and a nitrile
group; wherein R5, R6, R7, and R8 are each independently selected from the
group consisting of
hydrogen, iodine, and an alkane group; and n > 0; fl,? I; and Y includes an
amine.
[0008] According to another aspect, a composition comprises N-benzyl
guanidine; and
a thyrointegrin ovi:33 receptor antagonist; wherein the N-benzyl guanidine and
the thyrointegrin avii3
receptor antagonist arc connected by a linker.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The patent or application file contains at least one drawing executed
in color. Copies of this
patent or patent application publication with color drawing(s) will be
provided by the Office upon request
and payment of the necessary fee.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
3
[0010] Some of the embodiments will be described in detail with reference made
to the following
figures. in which like designations denote like members. wherein:
100111 Figure 1 depicts a general formula of an exemplary composition for use
in dual targeting of
neuroendocrine tumors;
10012] Figure 2a depicts another general formula of an exemplary composition
having a linker with a
monoamine;
[0013] Figure 2b depicts another general formula of an exemplary composition
having a linker with a
diamine;
[0014] Figure 2c depicts another general formula of an exemplary composition
having a linker with a
triazole;
[0015] Figure 3 depicts one exempla!), composition for use in dual targeting
of neuroendocrine tumors,
referred to as Composition 300, BG-PEG-TAT, or BG-P-TAT;
[0016] Figure 4a depicts an overview of a synthetic pathway for Composition
300 from Figure 3;
[0017] Figure 4b depicts a detailed schematic of the synthetic pathway of
Figure 4a;
[0018] Figure 4e depicts an overview of possible synthetic pathways for the
production of two other
exemplary compositions, referred to as Composition 201 (BG-PEG-MAT) and
Composition 202 (BG-
PEG-DAT), in which the production uses either a tosylate group or an aldehyde;
[0019] Figure 4d depicts an overview of alternative synthetic pathways for the
production of the
compositions shown in Figure 4c in which the production uses either a tosylate
group or an aldehyde;
100201 Figure 4e depicts a detailed schematic of the synthetic pathways of
Figures 4c and 4d that use
an aldehyde;
[0021] Figure 4f depicts a detailed schematic of the synthetic pathways of
Figures 4c and 4d using a
tosy late group;
[0022] Figure 5 depicts a graph showing no significant changes in body weight
of mice during multi-
day treatment with either a control or Composition 300 when administered at
different doses ranging from
1-10 mg/kg subcutaneously daily for 15 days:
[0023] Figure 6 depicts a graph showing dose-dependent decreases in tumor
volume over time (15
days) in the mice during the multi-day treatment at 1-10 mg/kg, subcutaneously
with Composition 300,
compared with increase in tumor volume for a control group;
[0024] Figure 7a shows images of mice in the control group with visible large
subcutaneous tumors,
along with abnormal animal head movements suggesting accompanying central
behavioral changes;
[0025] Figure 7b shows images of mice that have been treated with Composition
300 and demonstrate
significant reduction or absence of visible subcutaneous tumors (significant
shrinkage to elimination of
tumors) in a dose-dependent manner along with disappearance of the observed
abnormal animal head
movements;
[00261 Figure 8 is a graph of tumor weight as a function of dosage of
Composition 300 showing
significant tumor shrinkage to complete disappearance of the tumor;
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
4
[0027] Figure 9a are photographs of tumors showing relative tumor size and de-
vascularization as a
function of dosage of Composition 300;
10028] Figure 9b are photographs of tumors showing absolute tumor size as a
function of dosage of
Composition 300 demonstrating distinct tumor shrinkage to disappearance at the
10 mg/kg dosage level;
10029] Figure 10 is a graph of neuroblastoma cancer cell viability as a
function of dosage of
Composition 300 showing loss of cancer cell viability to complete loss at the
10 mg/kg dosage level;
10030] Figure 11 is a graph of neuroblastoma cancer cell necrosis as a
function of dosage of
Composition 300 showing increase in cancer cell necrosis from 80-100% at the 3
and 10 mg/kg doses;
[0031] Figure 12a is a graph of tumor weight shrinkage as a function of
treatment with different benzyl
guanidine derivatives including MIBG, BG, and polymer conjugated BG
administered subcutaneously
daily for 15 days at 3 mg/kg showing comparable shrinkage ranging from 40-50%
as compared to control
(PBS vehicle);
[0032] Figure 12b is a graph of tumor weight shrinkage as a function of
treatment with ben.zyl
guanidine. TAT derivative, or BG and TAT derivative co-administered versus BG-
P-TAT (Composition
300) all administered at 3 mg/kg, subcutaneously daily for 15 days (data
demonstrated 40-50% ttunor
shrinkage with BG, TAT, or BG co-administered with TAT versus 80% shrinkage
with BG-P-TAT
(Composition 300) as well as maximal loss of cancer cell viability with BG-P-
TAT);
[00331 Figure 13a are photographs of fluorescence images of various mice at 1
and 2 hours post-
administration of Cy5 labeled polymer conjugated TAT (Group 1), polymer
conjugated BG (Group 2),
an.d Polymer conjugated BG-TAT (Composition 300) (Group 3);
10034] Figure 13b are photographs of fluorescence images of the mice of Figure
13a at 4, 6, and 24
hours post-administration (data clearly showed distinct and highest intensity
accumulation (delineation
and imaging) in neuroblastoma tumor and its spread with Cy5-labeled polymer
conjugated BG-P-TAT
(Composition 300));
[00351 Figure 14 depicts another exemplary composition for use in dual
targeting of neuroendocrine
tumors, referred to as Composition 7a or dl-BG-P-TAT;
[0036] Figure 15 depicts another exemplary composition for use in dual
targeting of netu-oendocrine
tumors, referred to as Composition 7b or dIVI-BG-P-TAT;
[0037] Figure 16 depicts another exemplary composition for use in dual
targeting of new-oendocrine
tumors, referred to as Composition 15 or BG-P-PAT;
[0038] Figure 17 depicts an overview of a synthetic pathway for Compositions
7a and 7b from Figures
15 and 16;
[0039] Figure 18 depicts an overview of a portion of synthetic pathway for
Composition 15 from
Figure 16;
[0040] Figure 19 depicts an overview of another portion of synthetic pathway
for Composition 15 from
Figure 16;
100411 Figure 20 depicts respective binding affinity for exemplary
compositions;
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
[0042] Figure 21A depicts respective cellular uptake for exemplary
compositions;
[0043] Figure 21B depicts respective cellular uptake for exemplary
compositions;
100441 Figure 22 depicts a graph showing decreases in tumor volume over time
(20 days) in mice
during multi-day treatment at 3mg/kg, subcutaneously with Compositions 7a, 7b,
and 15, compared with
increase in tumor volume for a control group;
[00451 Figure 23 depicts a graph showing decreases in tumor weight over time
(20 days) in mice
during multi-day treatment at 3mg/kg, subcutaneously with Compositions 7a, 7b,
and 15, compared with
increase in tumor weight for the control group; and
[0046] Figure 24 depicts histology images showing the effects of Compositions
7a, 7b, and 15
compared with the control group.
DETAILED DESCRIPTION
[0047] A detailed description of the hereinafter-described embodiments of the
disclosed composition
and method is presented herein by way of exemplification and not limitation
with reference to the
Figures. Although certain embodiments are shown and described in detail, it
should be understood that
various changes and modifications might be made without departing from the
scope of the appended
claims. The scope of the present disclosure will in no way be limited to the
number of constituting
components. the materials thereof, the shapes thereof, colors thereof, the
relative arrangement thereof,
etc., and are disclosed simply as an example of embodiments or the present
disclosure. A more complete
understanding of the present embodiments and advantages thereof may be
acquired by referring to the
following description taken in conjunction with the accompanying drawings, in
which like reference
numbers indicate like features.
[0048] As a preface to the detailed description, it should be noted that, as
used in this specification and
the appended claims, the singular forms "a", "an" and "the" include plural
referents, unless the context
clearly dictates otherwise.
OVERVIEW
100491 Embodiments of the present disclosure describe new chemical
compositions, and methods of
synthesis thereof. The compositions disclosed and described herein may be
directed toward anti-
angiogenic agents, particularly thyrointegrin antagonists, which may be
capable of interacting with one or
more cell surface receptors of the integrin avii3 receptor family. The
compositions disclosed and
described herein may also be directed toward targets of the norepinephrine
transporter (also known as the
catecholamine transporter). Targets of the norepinephrine transporter may act
as neuroendocrine tumor
cell targeting agents.
[0050] 'the compositions disclosed and described herein may be directed toward
a composition
containing both a thyrointegrin antagonist and a norepinephrine transporter
target. Further, the
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
6
composition may use a polymer or other linker to link the thyrointegrin
antagonist and the norepinephrine
transporter target.
[00511 The norepinephrine transporter is a regulator of eatecholamine uptake
in normal physiology and
is highly expressed and over-active in neuroendocrine tumors like
neuroblastoma. Although the
norepinephrine analog, meta-iodobenzylguanidine (MIBG), is an established
substrate for the
norepinephrine transporter, analogs such as (123)1/(131)1- MIBG or analogs
having Fluoride (F18)
instead of Iodide (radioactive) may also be used for diagnostic imaging of
neuroblastoma and other
neuroendocrine tumors.
[0052] Investigations have demonstrated that various neuroblastoma cell lines
highly express the av133
integrin receptors (90-95%). However, high affinity av1i3 integrin receptor
antagonists showed limited
(40-50%) efficacy in term of tumor growth rate and cancer viability
inhibition. Similarly, benzyl
guanidine and its derivatives demonstrated limited anti-cancer efficacy of
neuroblastoma despite its
maximal (90-100%) uptake into neuroblastoma and other neuroendocrine tumors.
Furthermore, treatment
combinations of norepinephrine transporter targets such as benzyl guanidine or
its derivatives together
with thyrointegrin antagonists such as triazole tetraiodothyroacetic acid
derivatives did not exceed 50%
suppression of neuroblastoma growth and viability.
[00531 In contrast and unexpectedly, conjugation of norepinephrine transporter
targets such as benzyl
guanidine derivatives and thyrointegrin antagonists such as triazole
tetraiodoiluyoacetic acid derivatives
via different polymer linker such as Polyethylene Glycol (PEG) into a single
novel chemical entity
resulted in maximal uptake into neuroblastoma and other neuroendocrine tumors
along with maximal (80-
100%) suppression of tumor growth and viability at different doses. A
thyrointegrin antagonist
conjugated via a linker with a norepinephrine / catecholarnine transporter
target compound may provide a
composition that has a dual targeting effect for neuroendocrine tumor
targeting. For example, the
composition may comprise an alpha-V-beta-3 (avf53) integrin-thyroid hormone
receptor antagonist linked
to benzyl guanidine (or a benzyl guanidine derivative) according to one
embodiment of the invention.
[00541 The compositions described herein may be comprised of compounds, for
example a
thyrointegrin antagonist or derivative thereof covalently linked to a target
of the norepinephrine
transporter to form a a single chemical entity. The thyrointegrin antagonist
and the norepinephrine target
may be joined via a linker.
[0055] Reference may be made to specific thyrointegrin compounds and
norepinephrine compounds,
for example, tetrac, triac, and benzyl guanidine. These phrases include
derivatives of such compounds in
accordance with the full teachings of this disclosure, even where such
derivatives are not specifically
listed.
10056] Referring to the drawings, Figure 1 depicts an embodiment of a general
formula 100 comprising
a thyrointegrin antagonist 110 joined to a norepinephrine transporter target
120 via a linker 130. The
composition may be referred to as a thyrointegrin antagonist derivative
conjugated to a benzyl guanidine
derivative via the linker 130. or a thyrointegrin antagonist derivative
conjugated to a benzyl guanidine
derivative modified with the linker 130. Figure 1 depicts a carboxylic acid
form of the general formula
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
7
100. As would be apparent to one skilled in the art, a salt (e.g. a sodium
salt) of the general formula 100
may also be used.
[0057] The linker 130 comprises a spacer 132 and a polymer 131. The linker 130
resists
biodegradation such that the linker remains urtcleaved under physiological
conditions. In one
embodiment, the spacer 132 comprises a C112 unit and an adjacent repeating
linkage of methylene (CH2)
units which may be defmed by nl repeats wherein nl is an integer that is > 0.
In other embodiments, n1
may be > 1,? 2 or > 3. The linker 130 further comprises a moiety "Y."
Embodiments of the moiety
may in some instances be may be an amine. For example, the moiety Y of the
general formula may be a
divalent alkane having one amine group or a divalent alkane having two amine
groups as shown by the
examples of general formula 200a and 200b of Figures 2a and 2b. In another
embodiment, the moiety Y
may be a triazole as shown by the example of general formula 200c shown in
Figure 2c. The polymer
131 may comprise a polyether such as polyethylene glycol (PEG). Other polymers
may be used,
including chitosan, alginic acid, hyaluronic acid, and other polymers. In
embodiments using PEG as the
polymer 131, the polymer may have a molecular weight between 200 and 4,000g
per mole.
[0058] The term thyroid antagonist describes a compound that has the ability
to inhibit or antagonize
one or more thyroid hormone receptors known by a person skilled in the art,
for example the integnin
family of thyroid hormone receptors, such as the thyroid hormone cell surface
receptor avi$3. The
thyrointegrin antagonist 110 may be an anti-angiogenic thyroid hormone or a
thyroid hormone receptor
antagonist. For example, the thyrointegrin antagonist 110 may be an alpha-V-
beta-3 (avp3) integrin-
thyroid hormone receptor antagonist.
[0059] Specific embodiments of the thyrointegrin antagonist 110 may include
tetratodothyroacetic acid
(tetrac), triiodothyroacetic acid (iliac), derivatives thereof and variations
thereof. Examples of one or
more variations of the thyrointegrin antagonist comprising tetrac and triac
may include, in some
embodiments Diaminotetrac (DAT) or Diaminotriac (DA'Fri) (hereinafter may be
referred to
interchangeably as "DAT"), Monoaminotetrac (MAT) or Monoaminotriac (MATri)
(hereinafter referred
to interchangeable as "MAT), Triazoletetrac (TAT) or Triazoletriac (TATri)
(hereinafter referred to
interchangeable as "TAT"), derivatives thereof or other thyroid antagonist
known by those skilled in the
art. Thyrointcgrin antagonists may be of the type described in U.S. Pat. Pub.
No. 2017/0348425 titled
Non-Cleavable Polymer Conjugated with Alpa V Beta 3 Integrin Th3Troid
Antagonists, the contents of
which are incorporated by reference.
[0060] Exemplary thyrointegrin antagonists based on the general structure 100
from Figure 1 are
shown below in Table 1.
100611 Table 1
Exemplary Thyrointegrin Antagonists
R5 R6 R7 R8
2
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
8
3 I H 14 H
4 H I H H
H H 1 I-I
6 H H H 1
7 1 I H H
-- ________________________ t ______
8 ' 1 H 1 H
9 If H I I
I I 1 H
11 H I I I
12 1 1 1 1
13 H H H H
14 H3C H H H
>---
H3C
H H3C\ _ H H
l¨
H3C
16 H 11 H3C> H
-----
H3C
17 H H H 1-
13C>._
i-13C;
18 H3Cs,. H3C \ ii II
¨ i---
H3C r
H3C
19 H3C H H3C H
>--- >--
H3C H3C
_______
H H H3C H3C
H3C>---- >---
H3C
21 1-13C H3C H3C H
>-- >---
H3C H3C H3C
/2 H H3C>....... H3C)
H3C)......._
H3C H3C H3C
23 H3C H3C H3C>.....,.... H3C
H3C
H3C>__
H3C H3C H3C H3C
24 CH3 H H H
113C-+CH3
CA 03220332 2023-11-24
WO 2022/260918
PCT/US2022/031876
9
25 H CH3 H H
H3C+CH3
26 H H CH3 H
H3C---1--CA-42
27 H H H CH3
H3C+CH3
28 CH3 CH3 Fl H
H3C¨f-CH3 H3C-+-CH3
29 CH H CH3 H
H3C¨h-CH3 H3C-1--CH3
30 H H CH3 CH3
H3C-1^-CH3 H3C----1---CH3
31 CH3 CH3 CH3 H
H3C--h-CH3 H3C CH3 H3C¨I---CH3
32 H CH3 CH3 CH3
H3C CH3 H3C-1¨CH3 H3C+CH3
33 CH.
3 CI-13 CH, CH,
H3C-1¨CH3 H3C+CH3 H3C-H¨CH3 H3C+CH3
100621 In some embodiments of the thyroimtegrin antagonist 110, the variables
depicted as RS, R6, R7,
and R8 may each independently be substituted for molecules such as hydrogen,
iodine, and alkalies. In
some embodiments, the alkanes have four or fewer carbons. For example, as
shown in Table 1, in some
embodiments of the thyrointegrin antagonist 110, the variables depicted as R5,
R6, R7, and R8 may each
independently be substituted for molecules of hydrogen, iodine, or alkane
groups such as isopropyl or
isobutyl. In the embodiments of Table 1, the alkanes have four or fewer
carbons.
[0063] The norepinephrine transporter target 120 may be a neuroendocrine tumor
cell targeting agent.
As an example, the norepinephrine transporter target 120 may be benzyl
guanidine or a benzyl guanidine
derivative. As a further example, the norepinephrine transporter target 120
may be N-benzyl guanidine or
a derivative thereof.
[0064] Exemplary norepinephrine transporter targets 120 based on the general t-
ormula 100 from Figure
I are shown below in Table 2.
[0065] Table 2
Exemplary Norepinephrine Transporter Targets
CA 03220332 2023- 11-24
WO 2022/260918 PCT/US2022/031876
14)
R1 R2 1(3 R4
1 F H H H
__________________________________________________ r-----
i H F H H
3 H H I, H
4 H H H F
5 Br t. H H H
6 H Br H Il
7 H H Br H
8 H H H Br
9 1 H H H
10 H 1 H H
11 H H 1 H
1/ H H H 1
13 OH H H H
14 H OH I H H
15 H H OH H
16 H H H OH
17 OMe H H H
18 II OMe H H
19 H H OMe H
20 H H H OMe
21 NO2 H Fl FI
22 H NO2 H H
23 H H NO2 H
24 H H H NO2
25 NH2 H ¨1----- H H
26 H NH2 .. i __ H H
27 H H NH2 H
28 H H H NH2
29 CN H H H
30 H CN H H
31 H H CN H
32 II H H CN
[0066] In some embodiments of the norepinephrine transporter target 120, the
variables depicted as RI,
1(2, R3, and R4 may be each independently be substituted for molecules such as
hydrogen, iodine,
fluorine, bromine, a methoxy group, a nitro group, an amine group, and a
nitrile group. For example, in
CA 03220332 2023-11-24
WO 2022/260918
PCT/US2022/031876
11
some embodiments of the norepinephrine transporter target 120, the variables
depicted as RI, R2, R3, and
R4 may be each independently be substituted for molecules of hydrogen, iodine,
fluorine, bromine, a
methoxy group, a nitro group, an amine group, and a nitrile group as described
above in Table 2.
Additional embodiments and substitutions may also be used. In one embodiment
at least one of RI, R2,
R3 and R4 is a radiolabel. Examples of suitable radiolabels include
1(123).1(131) and F(18). The
compound may be administered to humans of animals.
[00671 Any of the exemplary thyrointegrin antagonists 110 (along with any of
the other thyrointegrin
antagonist embodiments taught herein) may be joined via the linker 130 to any
of the exemplary
norepinephrine transporter targets 120 (along with any of the other
norepinephrine transporter target
embodiments taught herein) to form a composition.
[0068] As is clear from Table I and Table 2, there are a large number of
compounds that may be used
as the thyrointegrin antagonist 110 and a large number of compounds that may
be used as the
norepinephrine transporter target 120 in the composition. Further, the various
thyrointegrin antagonists
110 may be combined with various norepinephrine transporter targets 120,
resulting in a large number of
potential chemical structures for the composition described herein.
[0069] Embodiments of each of the compositions described herein may have
multiple types of utility
for treating a plurality of different diseases modulated by angiogenesis or
the inhibition thereof. Each of
the compositions described in the present disclosure, in view of presence of
the thyrointegrin antagonist
110 present in the described compositions, may have an affinity for targeting
the integrin receptor avii3
located on numerous types of cells found throughout the human body and various
animal bodies.
10070] Moreover, embodiments of each of the compositions described in the
current application may
have utility for treating a plurality of different diseases characterized by
activity of the norepincphrine
transporter. Each of the compositions described in the present disclosure, in
view of presence of the
norepinephrine transporter target 120 present in the described compositions,
may each have an affinity for
targeting numerous types of cells found throughout the human body and various
animal bodies that utilize
the norepinephrine transporter. Each of the compositions described in the
present disclosure may have
increased affinity for targeting cells demonstrating increased or above
average activity of the
norepinepltrine transporter, such as neuroendocrine tumor cells. A.s a more
specific example, the
composition may have increased affinity for targeting neuroblastoma,
pheochromocytoma, pancreatic
neuroendocrine tumor. and carcinoid tumor cells.
100711 Still further, due to the composition's use of both a thyrointegrin
antagonist 110 and a
norepinephrine transporter target 120, the composition may have increased
utility and efficacy against
certain diseases and/or conditions. For example, neuroendocrine tumors are
susceptible to treatment with
thyrointegrin antagonists while also demonstrating increased activity of the
norepinephrine transporter.
The compositions described herein make use of both compounds for a dual
targeting effect in treatment of
neuroendocrine tumor cells. Further, the increased effect surpasses any
increase expected or achieved by
simultaneous separate treatment with a thyrointegrin antagonist and a
norepinephrine transporter target.
Further details regarding the beneficial utility is discussed below with
respect to experimental studies.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
12
[0072] As shown by the chemical structure of the general formula 100 of Figure
1, embodiments of the
chemical structure may include one or more variables defining the additional
features of the thyrointegrin
antagonist 110 of the general formula 100. For example, in some embodiments of
the thyrointegrin
antagonist 110, the variables depicted as R5, R6, R7, and R8 may be each
independently be hydrogen,
iodine, and alkanes as described above in Table 1.
[0073] There is thus a wide range of thyrointegrin antagonist compounds that
may be used as the
thyrointegrin antagonist 110 of the general formula 100. For example, as shown
in Figure 2a, the
thyrointegrin antagonist 110a may comprise a substitution of iodine for R5¨R8,
resulting in the formation
of a tetraiodothyroacetic acid (tetrac) derivative having a three-carbon
linker and a monoamine as the Y
moiety. General formula 200a may be referred to as monoamine-tetrac (MAT)
conjugated via PEG to
benzyl guanidine or a benzyl guanidine derivative. Likewise, in Figure 2b, the
tetrac molecule further
comprises a diamino Y moiety connected to the carbon linker. This general
formula 200b may be referred
to as diamino tetrac (DAT) conjugated via PEG to benzyl guanidine or a benzyl
guanidine derivative. In
the alternative embodiment of Figure 2c, the general formula 200c may comprise
a triazole moiety
connected to the single carbon of the carbon linker. This general formula 200c
may be referred to as
triazole tetrac (TAT) conjugated via PEG to benzyl guanidine or a benzyl
guanidine derivative.
[00741 Other thyrointcgrin antagonist compounds may also be used in forming
the compositions
described herein. For example, the general structure of the thyrointegrin
antagonists 110a, 110b, and
110c may be used wherein only R5¨R7 include iodine, thereby giving similar
triac derivatives. Further,
as shown in Table 1 above, similar structures may be used in which the
thyrointegrin antagonist
comprises a substitution of other elements or functional groups for any and/or
all of R5¨R8.
[00751 The norepinephrine transporter target 120 may comprise benzyl guanidine
or a benzyl guanidine
derivative. Embodiments of the chemical structure of the norepinepluine
transporter target 120 may
include one or more variables defining the additional features of the
norepinephrine transporter target 120
of the general formula 100 shown in Figure 1. For example, in some embodiments
of the norepinephrine
transporter target 120, the variables depicted as RI, R2, R3, and R4 may he
each independently be
substituted for molecules of hydrogen, iodine, fluorine, bromine, a methox-y
group, a nitro group, an
amine group, and a nitrile group as described above in Table 2.
[0076] Figure 3 depicts an exemplary Composition 300 of the general formula
100. Composition 300
comprises triazole tetrac conjugated to benzyl guanidine modified PEG.
Composition 300 may also be
referred to as BG-PEG-TAT or BG-P-TAT.
[0077] Synthesis of the compositions described herein is demonstrated below,
primarily with reference
to the exemplary composition shown in Figure 3, namely Composition 300.
Synthesis of similar
compositions, namely Composition 201 and Composition 202 (see Figure 4c.--40
are also provided as
examples and without limiting the disclosure to such compositions.
[0078] Example la: Synthesis of Exemplary Composition 300
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
13
[0079] This example provides a sample method for preparing Composition 300
shown in Figure 3.
Composition 300 is referred to as BG-PEG-TAT or BG-P-TAT. Composition 300 has
the chemical name
of 2-(4-(44(1-(20-(4-(guanidinome thy Dphenoxy)-3,6,9,12,15,18-hexaoxaicosyl)-
1H-1,2,3-triazol-4-
y1)methoxy)-3,5-cliiodophenoxy)-3,5-dilodoph.enyl)acetic acid, or [4-(4-{142-
(2-{242-(2-{242-(4-
Gutmidinomethyl-phenoxy)-edioxyl-ethoxy )-ethoxy)-ethoxy] -ethoxy }-ethoxy)-
ethyl]- 2,3]triazol-
4-y lmethoxy )-3,5-dliodo-phenoxy)-3,5-diiodo-phenyl]-acetic acid. The
molecular weight of
Composition 300 is 1284.44g/mol.
[0080] All commercially available chemicals were used without further
purification. All solvents were
dried and anhydrous solvents were obtained using activated molecular sieves
(0.3 or 0.4 1101 depending on
the type of solvent). All reactions (if not specifically containing water as
reactant, solvent or co-solvent)
were performed under Ar or N2 atmosphere, in oven-dried glassware. All new
compounds gave
satisfactory 'H NMR and mass spectrometry results. Melting points were
determined on an
Electrothermal MEL-TEMP* melting point apparatus and then on a Thomas HOOVER
Uni-mel
capillary melting point apparatus. Infrared spectra were recorded on a Thermo
Electron Nicolet Avatar
330 FT-IR apparatus. UV spectra were obtained from a SHIMADZU UV-1650PC UV-vis
spectrophotometer. The solution-state NMR experiments were all performed a
Bruker Advance II 800
MHz spectrometer equipped with a cryogenically cooled probe (TCD with z-axis
gradients (Bruker
BioSpin, Billerica, MA) at the Center for Biotechnology and Interdisciplinary
Studies, Rensselaer
Polytechnic Institute (RP1, Troy, NY). All tubes used were 5 mm outside
diameter. NMR data were
referenced to chloroform (CDC13; 7.27 ppm 'H, 77.20 ppm "C) or DMSO-d6 (5=
2.50 ppm, 38.92 ppm
"C) as internal reference. Chemical shifts 5 are given in ppm; multiplicities
are indicated as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet) and br (broad); coupling
constants, .1, are reported in Hz.
Thin layer chromatography was performed on silica gel plates with fluorescent
indicator. Visualization
was accomplished by UV light (254 and/or 365 rim) and/or by staining in eerie
ammonium molybdate or
sulfuric acid solution. Flash column chromatography was performed following
the procedure indicated in
J. Org. Chem. 43, 14, 1978, 2923-2925, with 230-400 mesh silica gel. High
resolution mass spectral
analysis was performed on either an Applied Biosystems API4000 LC/MS/MS or
Applied Biosystems
QSTAR XL mass spectrometers.
[0081] This example uses propargylated tetrac (PGT). Preparation of PCiT or a
derivative thereof from
tetrac is described in U.S. Pat. Pub. No. 2017/0348425 titled Non-Cleavable
Polymer Conjugated with
Alpha V Beta 3 integrin Thyroid Antagonists, the contents of which are
incorporated by reference.
[00821 Figure 4a depicts an overview of a synthetic pathway for Composition
300.
100831 Figure 4b depicts a detailed schematic of the synthetic pathway from
Figure 4a. Figure 4a
shows the scheme of synthesis of Composition 300 as an example of conjugation
of tetrac analogs to
benz,y1 guanine modified PEG via click chemistry. Other synthetic pathways may
be used.
[00841 The individual steps of the scheme of synthesis of Composition 300
shown in Figure 4b will be
described in more detail below in which the intermediary products arc referred
to by the number shown in
the click chemistry scheme.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
14
[0085] Synthesis ofheterobifunctional PEG. Although heterobifunctional linker
is commercial
available, for the ptuposcs of this example the following synthetic route for
preparation is used:
HO
%70, NaN3/rDMF dry, 70 C
Langmuir 2014, Q. 1130i-11300
Langmuir 2014, 30, 1130/ -11306 %40, psy DCM dry, 0 C
N3
[0086] Synthesis of Product 2 tert-butyl [(4-hydroxyphenyl)methyl]carbamate 2.
0
401
OH
[0087] Tert-butyl [(4-hydroxyphenyl)methyl]carbamate was synthesized according
to the protecting
method previously published {1) ACS Medicinal Chemistry Letters, 8(10), 1025-
1030; 2017. 2)
European Journal of Medicinal Chemistry, 126,384-407; 2017. 3) Tetrahedron
Letters, 47(46), 8039-
8042; 3006} the contents of which are hereby incorporated by reference.
Product 1, 4-
Hydroxybenzylarnine (0.62 g, 5 minol) slowly added with stirring to a solution
of di-tert-butyl
dicarbonate (1.2 g, 5.1 imnol) at room temperature. After the reaction mixture
was stirred for 8 h, the oily
residue was purified by column chromatography [SiO2- Et0Ac/hexanes (1:4)] to
afford 0.82 g of N-Boc-
4-hydroxybenzylamine as a colorless oil with 71% yield.
[00881 Synthesis of Product 3 etherification of tert-Butoxycarbony1-4-hydrox-
ybenzylamine to Bromo-
azido modified PEG(400) 3
o
3
[0089J Csan (867 mg, 2.67 mmol, 3 eq) was added with stirring to a solution of
tert-Butoxycarbony1-
4-hydroxybenzylamine (300 mg, 0.896 mmol, leg) in CAN (25 mL) at room
temperature. After the
reaction mixture was stirred for 30 min, Bromo-azido modified PEG(400) (445
mg, 1.05 mmol, 1.2 eq)
added to mixture and then temperature increased till reflux for 241i. It was
filtered to remove excess of
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
CsCO3. The solvents were removed under reduced pressure, and the oily residue
was purified by column
chromatography [SiO2: Et0Ac/hexanes (5:5)1 to afford product 3 as a yellow
oil. Yield: 433 mg, 87%.
[00901 Synthesis of Product 4. BOC de-protection
NA2
1101
4
100911 Product 3 (100 mg, 0.179 mmol, 3 eq) was dissolved in 3 ml anhydrous
1,4-dioxane and 3 ml
HCI (4N in dioxane) added to it and stirred at room temperature. After 24
hours, the solvent was removed
under reduced pressure, and the oily residue was purified to afford product 4
as a yellow oil in
quantitative yield (Yield: 73 g, 90%)
[00921 Synthesis of Product 5. Guanidination of Product 4
HN_Eloc
,I30C
xNH
5
[0093] Product 4 (85 mg, 0.17 mmol, 1 eq), N,Nr-Di-Boc-1H-pyrazole-l-
carboxamidine (54 mg, 17
mg, leg) was dissolved in 3-4 ml anhydrous diethylcarbodiimide "DCM" and then
triethyl amine "TEA"
(48 pl, 0.35 nimol, 2 eq) was added to the solution. The reaction mixture was
stirred at room temperature
for 12 li. After completion of the reaction the solvent was removed under
reduced pressure and the
residue dissolved in Et0Ac (30 m1). The organic phase washed with % 5 HC1 (25
ml) and brine (25 ml)
and then dried (Mg2SO4). The solvent was removed under reduced pressure to
yield product 5 which was
purified by column chromatography [SiO2: Et0Ac/hexanes (2:8)] Yield: 92 mg,
80%.
[0094] Synthesis of Product 6
FiN-BOC
BOC
r NH N-
6 0 y
OH
[00951 Product 5 (100mg, 1 eq) and 1 eq of PGT were dissolved in 20 nil THE
and stirred for 5 mm
then 0.5 eq of NaAscorbate and 0.5 eq of copperstdfatc in 2 ml water added to
mixture and stirred for 24
hours in 65 oC. After 24 hours, the solvents were removed under reduced
pressure, and Product 6 purified
in 65% yield.
[0096] Synthesis of Composition 300 (244-04( I -(20-(4-
(guanidinomethyl)phenoxy)-3,6,9,12,15,18-
hexaoxaicosyl)-1II-1,2,3-triazol-4-y1)methoxy)-3,5-diiodophenoxy)-3,5-
diiodophenyl)acetic acid)
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
16
H21
NH NH
0
OH
BG-PEGod-TAT
MW =1284.44 ghnol
7
[0097] Product 6 (50 mg) was dissolved in 3 ml anhydrous 1,4-dioxane and 3 ml
Ha (4N in dioxane)
added to it and stirred at 40C. After 24 hours, the solvent was removed under
reduced pressure, and the
oily residue was purified to afford Composition 300 as a yellow powder.
[0098] Other methods of synthesis may be used to reach Composition 300 or to
reach other
compositions having the general formula 100 shown in the Figure 1.
[0099] Example lb: Synthesis of Additional Exemplary Compositions
[0100] Figures 4c and 4d depict overviews of synthetic pathway for other
exemplary compositions, for
example Composition 201 following the general formula 200a and Composition 202
following the
general formula 200b, using either a tosylate group or an aldehyde.
[0101] Composition 201 may be referred to as BG-P-MA.T, BG-PEG-MA.T, or benzyl
guanidine
conjugated to monoaminotetrac via PEG. Composition 202 may be referred to as
BG-P-DAT, BG-PEG-
DAT, or benzyl guanidine conjugated to diaminotetrac via PEG. Benzyl guanidine
derivatives or other
norepinephrine transport targets may be used as described herein. Tetrac
derivatives or other
thyrointegrin antagonists may also be used as described herein, including but
not limited to iliac and trine
derivatives.
[01021 Figures 4e and 4f depict detailed schematics of the synthetic pathway
from Figures 4c and 4d.
Figures 4e and 4f shows the scheme of synthesis of Compositions 201 and 202 as
further examples of
conjugation of tetrac analogs to benzyl guanine modified PEG via click
chemistry. Again, other synthetic
pathways may be used.
METHODS OF USE
[0103] The compositions disclosed herein (including but not limited to the
exemplary compositions
such as Composition 300, Composition 201, and Composition 202) demonstrate
novel dual targeting in
treatment of cancer cells and tumors, particularly in treatment of
neuroendocrine tumors such as
neuroblastoma, pheochromocytoma, pancreatic neuroendoerine tumors, and
carcinoid tumors. Further,
the compositions show increased efficacy against neuroendocrine tumor cells
when compared with
thyrointegrin antagonist or norepinephrin.e transporter targets used or
administered separately, i.e., not
conjugated into a single composition.
[0104] The compositions may also be used for imaging of cancer cell/tumors.
For example. the
compositions described herein may be used to image neuroblastoma,
pheochromocytoma, pancreatic
neuroendocrine tumors, and carcinoid tumors. Imaging may be desirable for
diagnosis and/or for
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
17
treatment monitoring. Moreover, the compositions may be used for simultaneous
treatment and imaging.
For example, the compositions may demonstrate increased retention in the
targeted cancer cells/tumors,
allowing for enhanced treatment and more effective imaging.
[0105] Example 2: Effect on Subcutaneously Implanted Tumor in Female Nude Mice
[0106) The efficacy of Composition 300 (BG-P-TAT) was tested using
neuroblastoma SKNF2 cells
implanted into nude female mice.
[0107] Fifteen (15) female nude mice were implanted with twice with 10
cells/implant. The SKNF2
cell line was used with subcutaneous xenografts.
[01081 Eight (8) days following implantation, the mice were divided into four
groups receiving the
following treatment for 15 days:
Group Treatment Compound Dosage
Group I Control -- PBS
Group 2 Composition 300 (BG-PEG-TAT) I m glk g
Group 3 Composition 300 (BG-PEG-TAT) 3mg/kg
Group 4 Composition 300 (BG-PEG-TAT) I Omg/kg
10109] Following fifteen (15) days of treatment, tumors were collected in
order to evaluate
histopathology, and the following results were collected:
10110] Figure 5 shows the effect of the control and Composition 300 (BG-PEG-
TAT) treatment on
body weight of mice implanted with SKNF2 cell lines. As is shown, the body
weight was consistent
across all groups. Data demonstrate that daily treatment with Composition 300
(BG-PEG-TAT) at
different doses 1, 3 and 10 mg/kg daily for 15 days have no effect on animal
body weight versus control
animals.
[0111] Figure 6 shows the effect of Composition 300 (BG-PEG-TAT) treatment
versus control on
tumor volumes of mice implanted with SKNF2 cell lines. As shown, the control
group showed an
increase in tumor volume from approximately 825mm3to 1050rnm3over the 15 days
of treatment. All
groups receiving treatment with Composition 300 (BG-PEG-TAT) showed decreased
tumor size.
Further, the groups receiving treatment with Composition 300 (BC-PEG-TAT)
showed dose-dependent
decreases in tumor size, with the 10 mg/kg Group showing a tumor size
reduction from approximately
825m m3 to 100m in'.
[0112) Figures 7a--7b comprise photographs of mice front each treatment group
in which subcutaneous
tumors 70 can be visually compared. As shown in Figure 7a, the control group
shows large, clearly
visible tumors 70. Control animals also showed abnormal circling (head
rotation) 79, which was absent
in all treatment arms. The abnormal circling is believed to be an effect of
the tumor on the central
nervous sy stern.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
18
[0113] As shown in Figure 7b, the treatment groups show clear dose dependent
reductions in the size of
the tumors 70 to complete absence at the 10 mg/kg dose. As shown, in the 10
mg/kg treatment group
there is an absence of any visible tumor at the tumor location 70'.
[0114] Figure 8 shows the effect of the control and Composition 300 (130-1'EG-
TAT) treatment on
tumor weight of mice implanted with SKNF2 cell lines. As can be seen, the
treatment groups show a
dose-dependent reduction of tumor weight in comparison with the control group.
Data showed 60%, 80%
and 100% tumor shrinkage at the 1, 3, ad 10 mg/kg doses, respectively.
[0115] Figure 9a and Figure 9b shows the effect of the control and Composition
300 (BO-PEG-TAT)
treatment on vasculature and tumor size of mice implanted with SKNF2 cell
lines. As can be seen, the
control group demonstrated significant increases in size of the tumors 70 as
increased vascularization.
Vascularized areas 90 of the control group tumors 70 are clearly visible. In
contrast, the treatment groups
show a dose-dependent reduction in size of the tumors 70, including tumor
shrinkage at the 10 mg/kg
dose. Tumor vasculature was also clearly diminished as shown. In fact, as
shown in Figure 9b, with
respect to the 10 mg/kg group, there was only necrotic skin 75 at the location
of the implanted tumor 70'
(see Figure 7b) to be removed for histopathological examination; the treatment
demonstrated tumor
shrinkage at this dose.
10116.1 Figure 10 shows the effect of the control and Composition 300 (BG-PEG-
TAT) treatment on
tumor cell viability of mice implanted with SKNF2 cell lines. As can be seen,
the treatment groups show
a dose-dependent reduction in tumor cell viability. 70-75% cell viability was
shown in control with 20-
30% necrosis in the center of the tumor. In contrast, Composition 300 (BG-PEG-
TAT) treatment at
different doses showed loss of cell viability to 50%, 20, and 0.00% at 1, 3,
and 10 mg/kg, daily treatment
for 15 days, respectively. The 10mwkg group demonstrated a total lack of
viable tumor cells following
fifteen (15) days of treatment.
[0117] Figure 11 shows the effect of the control and Composition 300 (BG-PEG-
TAT) treatment on
tumor cell necrosis of mice implanted with SKNF2 cell lines. As can be seen,
the treatment groups show
a dose-dependent increase in tumor cell necrosis. The 10 mg/kg group
demonstrated a tumor cell necrosis
rate approaching 100%, the 3ing/kg group demonstrated a tumor cell necrosis
rate of approximately 80%,
and the 1 mg/kg demonstrated a tumor cell necrosis rate of approximately 50%.
[0118] Example 3: Comparative Examples:
[0119] Figures 12a and 12b shown the effect of the control and treatment with
BG, BG derivatives,
thyrointegrin antagonists such as 1AT derivatives, and combinations (co-
administration) thereof, versus
Composition 300 (BG-P-TAT) on tumor cell necrosis of mice implanted with SKNF2
cell lines.
[0120] In summary, known thyrointegrin antagonists for treatment of tumor
cells achieve substantially
inferior results when compared with Composition 300 (BG-P-TAT). For example,
triazole tetrac
derivatives delivered subcutaneously daily for three (3) weeks at 3mg/kg has
been shown to reduce tumor
growth by approximately 40-50% and reduce tumor viability by approximately 40-
50%. Similarly,
triazole tetrac derivatives have also been shown to reduce tumor growth by
approximately 40-50% and
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
19
reduce tumor viability by approximately 40-50%. Further, even a combination
treatment of two triazole
tetrac derivatives in combination delivered subcutaneously daily for three (3)
weeks at 3mglk.g only
achieves a reduction of 40-50% for tumor growth and tumor viability. Similar
results are obtained with
treatments using benzyl guanidine and benzyl guanidine derivatives. Further,
even co-administration of
benzyl guanidine and thyrointegrin antagonists fails to demonstrate increased
efficacy over the 40-50%
mark.
[0121] In contrast, treatment with Composition 300 (BG-P-TAT) resulted in 80%
reduction in tumor
where the viability of residual tumor was reduced by 80%.
[0122] Comparative Example 3a: Effect of TAT Derivative on Tumor Weight:
[0123] The avI33 integrin receptor antagonists (thyrointegrin antagonists)
showed limited (40-50%)
efficacy in term of tumor growth rate and cancer viability inhibition in the
case of neuroendocrine tumors
such as neuroblastoma, pheochromocytoma, pancreatic neuroendocrine tumors, and
carcinoid tumors.
For example, the graph of Figure 12b includes the effect of a triazole tetrac
derivative (referred to as
TAT) on tumor weight when compared with a control group (phosphate-buffered
saline "PBS"). The
specific derivative tested was beta cyclodextrin triazole tetrac. As shown,
the 3 mg/kg dosage resulted in
approximately 40-50% reduction of tumor weight.
10124] Comparative Example 3b: Effect of Benzyl Guanidine and Derivatives on
Tumor Weight:
[0125] Similarly, benzyl guanidine and its derivatives demonstrate limited (40-
50%) efficacy in term of
tumor growth rate and cancer viability inhibition in the case of
neuroendocrine tumors such as
neuroblastoma, pheochromocytoma, pancreatic neuroendocrine tumors, and
carcinoid tumors. For
example, the graph in Figure 12a includes the effect of .benzyl guanidine (BG)
and benzyl guanidine
derivatives (such as MIBG and a polymer conjugated benzyl guanidine
(specifically PLGA-PEG-BG,
referred to as polymer-BG) on tumor weight when compared with a control group
(PBS). The treatment
compounds demonstrated limited anti-cancer efficacy of neuroblastoma despite
its maximal (90-100%)
uptake into neuroblastoma and other neuroendocrine tumors.
[0126] Comparative Example 3c: Effect of Co-Administration of Separate
Norepinephrine Transporter
Target and Thy rointegrin Antagonist:
[0127] Furthermore, treatment combinations comprising co-administration of
norepinephrine
transporter targets such as benzyl guanidine or derivatives together with
thyrointegrin antagonists such as
triazole tetraiodothyroacetic acid derivatives did not exceed 40-50%
suppression of neuroblastoma
growth and viability. For example, benzyl guanidine co-administered with a
tetrac derivative (BG-4-TAT)
did not surpass the 40-50% efficacy demonstrated by individual treatment with
either compound as
shown in Figure 12b (BG-FTAT). Again, beta cyclodextrin triazole tetrac was
the tetrac derivative used.
[0128] Comparative Example 3d: Effect of Composition 300 (BG-P-TAT) (Benzyl
guanidine
conjugated to TAT via PEG):
[0129] Again, treatment with Composition 300 (BG-P-TAT) resulted in
significant improvement in the
effect on tumor weight compared with both the control and other types of
treatments as shown in Figure
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
12b. Composition 300 achieves approximately 80% reduction in tumor. Further,
the viability of residual
tumor was reduced by 80%. In fact, Composition 300 (TAT conjugated to BG)
demonstrated a
significant increase in efficacy over even co-administration of TAT and BG
separately (BG-FTAT).
[0130] The comparative examples from Figures 12a and 12b are summarized in the
following Table 3:
10131] Table 3
comparative Tumor Growth Suppression
and Tumor Survival Suppression Effect
Treatment Compou nd/Composit ion Dosage Percentage of
Percentage of
Tumor Growth Tumor
Survival
Suppression
Suppression
Benzyl guanidine (BG) 3mg/kg 40-50%
40-50%
Metaiodobenzylguanidine (MIBG) 3mg/kg 40-50%
40-50%
Benzyl guanidine with Polymer (PLGA- 3mg/kg 40-50%
40-50%
PEG-GB) (Polymer-BG)
Triazole Tetrac Derivative 1 (beta 3mg/kg 40-50%
40-50%
cyclodextrin triazole tetrac) crAD
Co-Administration of Benzyl guanidine 3mg/kg 40-50%
and Triazole Tar= Derivative 1
(13G+TAT)
Composition 300 (BG-P-TAT) 3mg/kg 80-90%
80-90%
[0132] Example 4: Imaging of Subcutaneously Implanted Tumor in Athymic Female
Mice.
[0133) Athymic female mice were implanted twice each with I O'cells/implant.
The SKNFI cell line
was used with subcutaneous xenografts.
[01341 Group 1 consisted of three mice and were treated with PEG-TAT-dye
(Cy5). Group 2 consisted
of three mice and were treated with PEG-BG-dye (Cy5). Group 3 consisted of
three mice and were
treated with TAT-PEG-BG-dyc (Cy5) wherein the TAT and BG were covalently
linked with a PEG linker
as compound 300. The treatment groups are shown below:
Group Treatment Composition
Group 1 PEG modified txiazole tetrac derivative with Cy5
dye
Group 2 PEG modified benzyl guanidine derivative with
Cy5 dye
CA 03220332 2023-11-24
WO 2022/260918
PCT/US2022/031876
21
Group 3 Composition 300 with Cy5 dye
10135] Fluorescence imaging (Cy 5) was conducted 1 hour, 2 hours, 4 hours, 6
hours, and 24 hours
post-administration. Imaging results are shown in Figures 13a and 13b, in
which the tumor location is
circled in yellow and the Cy5 dye appears as red. As shown in these figures,
there was a dramatic
increase in the fluorescence signal when the TAT and BG were covalently linked
and Composition 300
showed marked improvement in both uptake into the SKNE1 neuroblastoma tumors
and retention time
within the tumor when compared with either a triazole tetrac derivative alone
or a benzyl guanidine
derivative alone.
[01361 Neuroblastoma tumor cells were used in the treatment example discussed.
Those skilled in the
art would appreciate these examples are valid models for treatment of other
tumor types, particularly
other neuroendocrine tumors. Further, any tumor or disease state demonstrating
increased activity of the
norepinephrine transporter in which thyrointegrin moderated antiangiogenic
activity would be desired
may be treated by the disclosed compositions.
[01371 In light of these examples, the compositions described herein show
increased efficacy against
tumor cells, particularly neuroendocrine tumors. These compositions may be
used to treat
neuroendocrine tumors such as neuroblastoma, pheochromocytoma, pancreatic
neuroendocrine tumors,
and carcinoid tumors, for example by injectable, topical, sublingual, oral,
and other routes of
administration.
ADDITIONAL EXEMPLARY COMPOUNDS
[0138] As discussed above, compositions based on the general structure 100 may
include variations at
RI through R8 and/or variations in the linker 130, for example, variations in
the spacer 132, the polymer
131, and/or the moiety Y. Exemplary embodiments including such variations are
discussed in more detail
below. These exemplary embodiments are not meant to limit the disclosure to
any of the specifically
presented embodiments. Instead, the descriptions of the various embodiments
have been presented for
purposes of illustration, and are not intended to be exhaustive or limited to
the embodiments disclosed.
Many modifications and variations will be apparent to those of ordinary skill
in the art without departing
from the scope and spirit of the described embodiments.
[0139] Figure 14 depicts an exemplary Composition 7a of the general formula
100. Composition 7a
comprises triazole tetrac conjugated to benzyl guanidine modified PEG wherein
iodo groups have been
chosen as substituents on the benzyl guanidine aromatic ring. Composition 7a
may also be referred to as
dl-BG-PEG-TAT or dl-BG-P-TAT.
[01401 Figure 15 depicts an exemplary Composition 7b of the general formula
100. Composition 7b
comprises triazole tetrac conjugated to benzyl guanidine modified PEG wherein
rnethoxy groups have
been chosen as substituents on the benzyl guanidine aromatic ring. Composition
7b may also be referred
to as dM-BG-PEG-TAT or dM-BG-P-TAT.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
22
[0141] Figure 16 depicts an exemplary Composition 15 of the general formula
100. Composition 15
comprises tetrac conjugated to benzyl guanidine modified PEG wherein the amine
of the moiety Y is
piperazine. Composition 15 may also be referred to as BG-PEG-PAT or BG-P-PAT
wherein PAT refers
to pipe razine tetrac.
[01421 Synthesis of these compositions is demonstrated below.
[0143] Example 5: Synthesis of Compositions 7a and 7b
10144] The synthesis of dl-BG-P-TAT (7a) and dM-BG-P-TAT (7b) was accomplished
as described in
Scheme 1. Amine groups of iodo and methoxy substituted 4-hydroxy benzyl amine
were protected with
di-tert-butyl di-carbonate. Compounds 2a and 2h were characterized with 'H-
NMR. The peak observed at
1.49 ppm was assigned to tert-butyloxycarbonyl (Boc) protons. In the next
reaction. compounds 2a and
2b were reacted with commercially available Br-PEG6-N1 in the presence of
K2CO3 and ACN under
reflux conditions to get compound 3a and 3h with 90% and 85% yields,
respectively. The 1H-NMR
spectra of compounds 3a and 3b exhibited peaks of PEG protons between 3.40 and
3.97 ppm. Then,
amino groups were deprotected in 4 N HCI (in dioxane) and the product was
confirmed by disappearance
of Boc-proton signals at 1.48 and 1.49 ppm in the 311 NMR spectra of 4a and
4b. In the next step, N,N1-di-
Boc-1H-pyrazole-l-carboxamidine was reacted with compounds 4a and 4b to
acquire Boc-protected
guanidine compounds 5a and 5b. The '11-NMR spectra of compounds 5a and 5b
clearly showed peaks at
1.49-1.52 and 150-1.52 ppm, respectively, which can be assigned to two
separate Boc groups' protons.
NH2 NHBoc NHBoc NH2 N, HBoc
Lcrs: b c
YOH H '~"--1113
X X 6 X 6
6
1 2a: Xr- I 3a: Xs. I 46: X= I Sa: X= I
2b: X=-OCH3 312: X=-OCH3 4h: X=-OCH3 013: X.-
0043
NHBoc NH2
N_
Htl)11--Q10X 6
is: X=
[0145] fib: Xis -OCH3 7b: Xs -00-
i3
Scheme 1. Synthesis diagram of compounds 7a and 7b. a) Boc20, 6 h; b) K2CO3,
ACN, Br-PEG6-N3,
reflux, 24 h; c) HC1 (4 N in dioxane), rt, 4 h, d) DCM, TEA, N,N1-di-Boc-1H-
pyrazole-l-carboxamidine,
rt, 12 h; e) POT, THF :water 4:1, CuSO4, Na Ascorbate, rt, 24 h; 0 HCI N in
dioxane), rt, 24 h.
Scheme 1 is shown in Figure 17.
[0146] Then, azide-containing compounds 5a and 5b were conjugated with
propargylated tetrac,
(PGT)36, which is terminal alkyne-containing tetrac, in a click reaction by
forming a triazole ring to get
compounds Ga and 6b. CuSO4/Na Ascorbate (0.3eq:0.6eq) in THE :water (4:1) was
used to generate Cu+
in situ at room temperature. The characteristic singlet peak of triazole ring
protons appeared at 8.59 and
8.60 ppm in the 11-1-NMR spectra of compounds 6a and 6b, respectively. Lastly,
protecting Boc groups
were removed in 4 N HCI (in dioxane), and the resulting product was purified
with reverse phase column
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
23
chromatography with MeOH:water (70:30) to get compounds 7a and 7b. The '1-1-
NMR (Figure S21, S23),
'C-NMR, and mass spectra of compounds 7a and 7b confirmed their structure.
[0147] Example 6: Synthesis of Composition 15
[0148] The synthesis of BU-P-PAls 15 was accomplished as described in Scheme
2. First, the amino
group of 4-hydroxybenzyl amine 8 was protected with Boc group. Then, Br-PEG7-
0H was reacted with
the phenolic OH group of 9 in the presence of K2CO3 and ACN at reflux
temperature to get 10, and it was
characterized with '1-1-NMR by observing PEG proton peaks at 3.6-3.8 ppm.
NI H2 NHBoc "Mac NHBoc NHBoc
d
1C0-1¨'11 Lat)H3' ab'"4,,A,021 Woo 1:7"4"A`Nr)"'"12-
c-1
9 10 11 12
11):31)
NH2 NNBoc
----- , , Bodejlrao
I "4/-01)131I :0?.0
'
lisCe
13 14 g I
tiletiNH2 ii_0,,Itn,j,,zieri:r0136
7
1.10
is
[0149] Scheme 2. Synthesis diagram of compound 15. a) Boc20, 6 h; b) K2CO3,
ACN, Br-PEG7-0H
reflux, 24 11; c) Tos-C1, DCM, TEA, O'C-rt, 2 li; d) compound 19 (see Scheme
3), ACN, K2C.0360'C, 18
e) HC1 (4 N in dioxane), rt, 4 h; 0 DCM, TEA, N,Ni-di-Boc-1H-pyrazole-i-
carboxamidine. it 12 h; g)
dioxane :water, conc. HC1, rt, 24 h. Scheme 2 is shown in. Figure 18.
[0150] A different method was used to introduce a tetrac unit on the PEG
(Scheme 3). First, carboxylic
acid group of tetrac 16 was converted to methyl ester in Me0H and SOC12 to get
17. Then it was reacted
with tert-butyl 4-(3-(methanesulfonyloxy)propyl)piperazine-l-carbox-ylate
hydrochloride 18 and Cs2CO3
as a base in ACN, followed by treatment with HCl (4 N in dioxane) solution to
deprotect the Boc group.
The structure of resulting compound 19 was characterized with IH-NMR. Aromatic
protons of tetrac were
observed at 7.32 and 8.04 ppm and piperazine protons were observed at 2.77 and
2.94 ppm.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
24
0
a
ticr-A'r H 1-1.s.S 0
HO
17
r'm
Elac--N liNCN01)Icr 41),44.0
H3C0
18 19
[0151] Scheme 3. Synthesis diagram of compound 19. a) SOC12. Me0H; b) 17, ACN,
Cs2CO:;,60 C,
18 h; c) 4 N HC1 in dioxane, 2 h. Scheme 3 is shown in Figure 19.
[01521 Compound 19 was introduced (Scheme 2) to a PEG unit after the
tosylation reaction of PEG-
011 10 in the presence of K2CO3 and ACN to give compound 12. The 'II-NMR
spectrum of 12 (Figure
S40) confirmed the structure by observing tetrac and N-Boc benzylamine
aromatic proton peaks at 7.18-
7.79 and 6.88-7.20, respectively. After N-Boc deprotection of compound 12,
free amine of 13 was used
with N,N'-di-Boc-1H-pyrazole-l-carboxamidine in DCM and TEA as a base to
introduce Boc-protected
guanidine group and afforded compound 14. Finally, methyl ester and Boc
protection groups were
hydrolyzed with cone. HC1 in dioxane :water to give desired compound 15. IH-
NMR (Figure S46) and
the mass spectrum of 15 confirmed its structure. Purities of final synthesized
products 7a, 7b, and 15 were
confirmed to be >95% by HPLC.
[0153] Compounds dl-BG-P-TAT (7a), d114-BG-P-TAT (7b), and BG-P-PAT (15)
showed relatively
higher binding affinity towards purified integrin av133 receptor with lower
1Cso values 1.1 tiM, 0.5 nM,
and 0.3 nM, respectively, compared to 10.3 nM for BG-P-TAT. Thus, Compound 15
BG-P-PAT shows
approximately a 30-fold increase in binding affinity relative to 13G-P-TAT.
Figure 20 shows the
respective binding percentage towards purified integrin av03 receptor.
[0154] Further, the compounds displayed in vitro cellular uptake (SK-N-F1
neuroblastoma cells)
similar to BG-P-TAT. The uptake is shown graphically in Figures 21A and 21B.
[0155] Molecular docking studies were also carried out for Compounds 7a, 7b,
and 15. The molecular
docking results show a bent structure of the molecules at the binding site.
The interaction and docking
analysis revealed that 15 has the best interaction rate with high binding
energy -14.4 kcal/mol and forms 9
hydrogen bonds with integrin 133 subunit 7a and 7b had binding energies of -
6.1 kcal/mol and -7.8
kcal/mol, respectively, and 7a formed 6 hydrogen bonds (1 with as' domain and
5 with 133 domain) and 7b
formed 6 hydrogen bonds (1 with av domain, 4 with 03 domain and 1 with Mn
atom). Energy values for
7a, 7b, and 15 with binding energies and residues involved in interactions are
listed in Table 4. The 30-
fold higher av133 binding dimity of 15 versus the close analog BG-P-TAT may be
due to additional
hydrogen bonds of the BG portion of 15 in with Asp-127 and Asp-126, which may
be a result of the
longer linker chain in BG-P-PAT, allowing the BG portion easier access to this
domain than the BG in
BG-P-TAT, as well as additional hydrogen bonding of the piperazine nitrogen.
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
Table 4: Binding energies of compounds with integrin ay133
Docking
Score Bond
Distance
Compound (kcal/mol) Interacting Residues
(A)
A-chain B-chain
(ov.subunit) (11J-subunit)
dl-BG-P-TAT (7a) -6.1 Tyr 178 2.9
Tyr-166
Arg 214 2.8
Ser 334 2.5
Ser 337 2.5
Lys 125 3.7
dM-BO-P-TAT (7b) -7.8 Tvr 178 3.2
=
Arg 216
2.2,2.2,4.5
Ser 334 2.4
Mn 4.8
BO-P-PAT (15) -14.4 Asp 126 3.5
Asp 127 3.5,
4.2
Arg 214 4.1
Asn 215 3.8
Ala 218 3.2
Asp 251
3.5.3.4
Lys 253 4.5
Thr 311 3.4
Asn 313 3.3
METHODS OF USE
1.0156) Example 7: Effect on Subcutaneously Implanted Tumor in Female Nude
Mice
[01571 The efficacy of Compositions 7a (dl-BG-P-TAT). 7b (dM-BG-P-TAT). and 15
(BG-P-PAT)
were tested using neuroblastoma SICNFI cells implanted into nude female mice
similar to the examples
discussed above for Composition 300 (BO-P-TAT).
[01581 Following twenty (20) days of treatment at 3mgfkg (7 days for
Composition 7a due to skin
irritation and discomfort) tumors were collected in order to evaluate
histopathology, and the following
results were collected:
CA 03220332 2023- 11-24
WO 2022/260918
PCT/US2022/031876
26
[0159] Figure 22 shows the effect of Compositions 7a (dl-BG-P-TAT), 7b (dM-BG-
P-TAT), and 15
(BG-P-PAT) versus control on tumor volumes of mice implanted with SKNF1 cell
lines. A.s shown, both
completed treatment groups (Compositions 7b (dM-BG-P-TAT) and 15 (BG-P-PAT))
showed decreased
tumor vohune compared with the control. The control group showed an increase
from 175mm' to
1000mm3 over twenty days while the completed treatment groups showed no
substantial increase in
tumor volume.
[0160] Figure 23 shows the effect of Compositions 7a (dl-BG-P-TAT), 7b (dM-BG-
P-'FA'T), and 15
(BG-P-PAT) versus control on tumor weight of mice implanted with SKNF1 cell
lines. As can be seen,
the completed treatment groups show a reduction of tumor weight in comparison
with the control group.
Data showed a 90% decrease in tumor weight for Composition 15 (BG-P-PAT) and
an 86% decrease in
tumor weight for Composition 7b (dM-BG-P-TAT). Even the halted treatment group
for Composition 7a
(di-BCi-P-TAT) showed a 67% decrease in tumor weight.
[0161] Further, to compare the histopathological changes in tumors of
untreated and treated groups,
tumors were harvested, fixed, and stained with hematoxylin and eosin (H&E).
Necrosis at low
magnification of tumors from animals treated with compounds 7a, 7b, and 15
versus control is seen
clearly as shown in Figure . The staining showed large areas of necrosis,
fibrosis, and cell debris with
approximately 98% (Composition 15 1.3G-P-PA.1), 85% (Composition 7b dM-1.3G-P-
TAT), and 70%
(Composition 7a dl-BG-P-TAT) . On the other hand, tumors from the untreated
group had mostly viable
tumor cells. At higher magnification (40X), the tumor treated with Composition
15 BG-P-PAT showed
large areas of necrosis replaced with normal tissue. (Again, Compound 7a dl-BG-
P-TAT was
administered for only 7 days versus 20 days for the other two regimes).
[0162] Neuroblastoma tumor cells were used in the treatment examples
discussed. Those skilled in the
art would appreciate these examples are valid models for treatment of other
tumor types, particularly
other neuroendocrine tumors. Further, any tumor or disease state demonstrating
increased activity of the
norepinephrine transporter in which thyrointegrin moderated antiangiogenic
activity would be desired
may be treated by the disclosed compositions.
[0163] In light of these examples, the compositions described herein show
increased efficacy against
tumor cells, particularly neuroendocrine tumors. These compositions may be
used to treat
neuroendocrine tumors such as neuroblastoma, pheochromocytoma, pancreatic
neuroendocrine ttunors,
and carcinoid tumors, for example by injectable, topical, sublingual, oral,
and other routes of
administration.
10164] The descriptions of the various embodiments of the present invention
have been presented for
purposes of illustration, but are not intended to be exhaustive or limited to
the embodiments disclosed.
Many modifications and variations will be apparent to those of ordinary skill
in the art without departing
from the scope and spirit of the described embodiments. The terminology used
herein was chosen to best
explain the principles of the embodiments, the practical application or
technical improvement over
technologies found in the marketplace, or to enable others of ordinary skill
in the art to understand the
embodiments disclosed herein.
CA 03220332 2023- 11-24
