U.S. patent number 10,385,009 [Application Number 14/420,184] was granted by the patent office on 2019-08-20 for deuterated compounds and uses thereof.
This patent grant is currently assigned to BIOSTATUS LIMITED. The grantee listed for this patent is Biostatus Limited. Invention is credited to Rachel Jane Errington, Stephanie McKeown, Stefan Ogrodzinski, Laurence Patterson, Paul Smith.
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United States Patent |
10,385,009 |
Ogrodzinski , et
al. |
August 20, 2019 |
Deuterated compounds and uses thereof
Abstract
An anthraquinone compound of formula I (such as the compounds of
formulae II to X) and processes for making the same are provided.
Pharmaceutical compositions for use in the treatment of cancer,
optionally in combination with an agent capable of reducing the
level of oxygenation of a tumor, are also provided. Additionally,
an option for combination with chemotherapeutic and
radiotherapeutic modalities to enhance overall tumor cell kill is
provided. Methods for the detection of cellular hypoxia, both in
vivo and in vitro, are additionally provided. ##STR00001##
Inventors: |
Ogrodzinski; Stefan (Shepshed,
GB), Smith; Paul (Shepshed, GB), McKeown;
Stephanie (Shepshed, GB), Patterson; Laurence
(Shepshed, GB), Errington; Rachel Jane (Shepshed,
GB) |
Applicant: |
Name |
City |
State |
Country |
Type |
Biostatus Limited |
Shepshed, Leicestershire |
N/A |
GB |
|
|
Assignee: |
BIOSTATUS LIMITED (Shepshed,
Leicestershire, GB)
|
Family
ID: |
46935104 |
Appl.
No.: |
14/420,184 |
Filed: |
August 7, 2013 |
PCT
Filed: |
August 07, 2013 |
PCT No.: |
PCT/GB2013/052106 |
371(c)(1),(2),(4) Date: |
February 06, 2015 |
PCT
Pub. No.: |
WO2014/023956 |
PCT
Pub. Date: |
February 13, 2014 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20150307441 A1 |
Oct 29, 2015 |
|
Foreign Application Priority Data
|
|
|
|
|
Aug 8, 2012 [GB] |
|
|
1214169.3 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
1/18 (20180101); A61P 25/00 (20180101); A61P
17/00 (20180101); C07C 291/04 (20130101); A61P
1/04 (20180101); A61P 35/04 (20180101); A61P
5/28 (20180101); A61P 1/16 (20180101); A61P
13/12 (20180101); A61K 31/277 (20130101); A61P
11/00 (20180101); A61K 45/06 (20130101); G01N
31/225 (20130101); A61P 11/04 (20180101); A61P
27/02 (20180101); A61P 15/00 (20180101); A61P
11/02 (20180101); A61P 43/00 (20180101); A61P
1/02 (20180101); A61P 35/00 (20180101); A61K
31/136 (20130101); A61P 9/00 (20180101); A61P
13/08 (20180101); A61P 35/02 (20180101); C07C
225/36 (20130101); A61P 19/00 (20180101); A61P
13/10 (20180101); C07B 59/001 (20130101); C07C
2603/24 (20170501); C07B 2200/05 (20130101) |
Current International
Class: |
A61K
45/06 (20060101); G01N 31/22 (20060101); A61K
31/277 (20060101); A61K 31/136 (20060101); C07C
225/36 (20060101); C07B 59/00 (20060101); C07C
291/04 (20060101) |
Field of
Search: |
;552/236,237,238,240 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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H04502166 |
|
Apr 1992 |
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JP |
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2006501140 |
|
Jan 2006 |
|
JP |
|
WO 95/26325 |
|
Oct 1995 |
|
WO |
|
2006031719 |
|
Mar 2006 |
|
WO |
|
2006096458 |
|
Sep 2006 |
|
WO |
|
2011124927 |
|
Oct 2011 |
|
WO |
|
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|
Primary Examiner: Badio; Barbara P
Attorney, Agent or Firm: Leber IP Law Fujikawa; Shelly
M.
Claims
The invention claimed is:
1. A compound having one of Formula III or V: ##STR00014## wherein
each Y is independently selected from the group consisting of
hydrogen, hydroxy, halogeno, amino, C.sub.1-4 alkoxy and C.sub.2-8
alkanoyloxy.
2. The compound according to claim 1, wherein the compound is of
Formula VII or IX: ##STR00015##
3. The compound according to claim 1 wherein each Y is
independently selected from the group consisting of hydrogen,
hydroxy and halogeno.
4. The pharmaceutical composition comprising the compound according
to claim 1 together with a pharmaceutically acceptable buffer,
diluent, carrier, adjuvant or excipient.
5. A kit for detecting the oxygenation level of cells comprising
the compound according to claim 1.
6. The kit according to claim 5 further comprising a non-deuterated
form of a compound of Formula V.
7. A method of treating a pancreatic cancer tumour in a patient
comprising administering to the patient a therapeutically effective
amount of the compound according to claim 1.
8. The method according to claim 7, wherein administering the
therapeutically effective amount treats metastases or reduces
metastatic spread.
9. The method according to claim 7 further comprising administering
to the patient one or more of a chemotherapeutic agent and a
radiotherapeutic agent in combination with the therapeutically
effective amount of the compound.
10. The method according to claim 9 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent is/are
selected from the group consisting of anti-androgens (steroidal and
non-steroidal), vascular disrupting agents, anti-angiogenic agents,
anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors,
vasoconstricting agents, vasodilating agents, and radiotherapeutic
modalities.
11. The method according to claim 10 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent is at least
one anti-androgen.
12. The method according to claim 11 wherein the at least one
anti-androgen is selected from the group consisting of flutamide,
nilutamide, bicalutamide, finasteride, dutasteride, bexlosteride,
izonsteride, turosteride, epristeride, abiraterone and combinations
thereof.
13. The method according to claim 12 wherein the at least one
anti-androgen is bicalutamide.
14. The method according to claim 9 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent decreases
tumour oxygenation in vivo.
15. The method according to claim 14 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent lowers the
median oxygen level of the tumour to below 3%.
16. A method of treating a prostate cancer tumour in a patient
comprising administering to the patient a therapeutically effective
amount of the compound according to claim 1.
17. The method according to claim 16, wherein administering the
therapeutically effective amount treats metastases or reduces
metastatic spread.
18. The method according to claim 16 further comprising
administering to the patient one or more of a chemotherapeutic
agent and a radiotherapeutic agent in combination with the
therapeutically effective amount of the compound.
19. The method according to claim 18 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent is/are
selected from the group consisting of anti-androgens (steroidal and
non-steroidal), vascular disrupting agents, anti-angiogenic agents,
anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors,
vasoconstricting agents, vasodilating agents, and radiotherapeutic
modalities.
20. The method according to claim 19 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent is at least
one anti-androgen.
21. The method according to claim 20 wherein the at least one
anti-androgen is selected from the group consisting of flutamide,
nilutamide, bicalutamide, finasteride, dutasteride, bexlosteride,
izonsteride, turosteride, epristeride, abiraterone and combinations
thereof.
22. The method according to claim 21 wherein the at least one
anti-androgen is bicalutamide.
23. The method according to claim 18 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent decreases
tumour oxygenation in vivo.
24. The method according to claim 23 wherein the one or more of the
chemotherapeutic agent and the radiotherapeutic agent lowers the
median oxygen level of the tumour to below 3%.
25. A process for making the compound according to claim 1
comprising reacting an anthracene-9,10-dione with a deuterated
alkylenediamine under conditions suitable for the production of an
alkylaminoalkyl-aminoanthraquinone.
26. The process according to claim 25 further comprising the step
of reacting the alkylaminoalkylaminoanthraquinone with a
monoperoxyphthalate under conditions suitable for the production of
an N-oxide derivative of the alkylaminoalkylaminoanthraquinone.
27. The process according to claim 25 comprising reacting
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, with deuterated
N,N-dimethylethylenediamine under conditions suitable for the
production of
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyant-
hracene-9,10-dione.
28. The process according to claim 27 further comprising the step
of reacting the
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthra-
cene-9,10-dione with magnesium monoperoxyphthalate under conditions
suitable for the production of
1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-dihydro-
xy-anthracene-9,10-dione.
29. A method of detecting hypoxic cells in vitro or in vivo in a
group of cells, the method comprising: exposing a compound of
Formula V: ##STR00016## wherein each Y is independently selected
from the group consisting of hydrogen, hydroxy, halogeno, amino,
C.sub.1-4 alkoxy and C.sub.2-8 alkanoyloxy, to the group of cells;
analyzing the cells for the presence of a corresponding reduced
compound of Formula III: ##STR00017## determining the hypoxic cells
based on the presence of the corresponding reduced compound.
30. The method according to claim 29 in vitro.
31. The method according to claim 29 in vivo.
32. The method according to claim 31, further comprising:
surgically excising cells identified as being hypoxic.
33. The method according to claim 29 wherein the compound is used
in combination with a non-deuterated form of a compound of Formula
V.
34. The method according to claim 29 wherein the compound is
detected using a method selected from the group consisting of mass
spectrometry, nuclear magnetic resonance, infrared spectroscopy,
colorimetrically, Raman spectroscopy, nuclear magnetic resonance,
affinity capture methods, immunohistochemistry, flow cytometry,
microscopy and antibody-based detection methods.
Description
CROSS REFERENCE TO RELATED APPLICATION
The present application claims priority to Great Britain Patent
Application No. 1214169.3, filed Aug. 8, 2012, incorporated herein
in its entirety.
TECHNICAL FIELD
The present invention relates to novel anthraquinone compounds and
uses of the same, for example in the treatment of cancer.
BACKGROUND
The therapeutic advantage of an anticancer drug depends primarily
on the extent to which the agent shows selective activity for
tumour cells and the limiting toxicity towards non-target tissues.
Frequently the poor quality of the vasculature within the growing
tumour mass compromises the delivery of drugs, nutrients and
oxygen. It is recognised that tumours can have significantly lower
median oxygen levels (approximately 1% oxygen; pO2 7.5 mmHg)
compared to normal tissues (.about.5.5% oxygen; 42 mmHg)
(summarised from data presented by Brown and Wilson, 2004). In
addition, oxygenation levels can vary throughout the tumour due to
intermittent opening and closing of tumour blood vessels; poor
vascularisation, especially in the tumour core, contributes to
oxygen levels often being below 0.1% oxygen (1 mm Hg). Tumour cells
experiencing varying degrees of hypoxia, relative to normally
perfused tissues, can compromise treatment effectiveness and
contribute to the malignancy. Hypoxia-selective agents (e.g.
bioreductive drugs) comprise one class of agents that can be used
to target tumour cells in very low oxygen environments by virtue of
a selective activation to a cytotoxic form under reduced
oxygenation, addressing the problems of non-target tissue toxicity,
hypoxic cell drug resistance and cancer progression.
Poor oxygenation results in a relative state of hypoxia when
compared with normoxic conditions in which oxygenation has not been
compromised. Poor oxygenation within tumours can modify the
responses to treatment modalities and contribute to cancer
progression. Cells in such hypoxic areas are particularly resistant
to treatment with many of the conventionally used anticancer drugs;
this is attributed to poor drug delivery and/or lack of intrinsic
tumour cell sensitivity of viable but quiescent cells. Radiotherapy
is also less effective at very low oxygen levels since the
cytotoxicity of ionising radiation is enhanced by the presence of
oxygen (Radiobiology For The Radiologist, Hall E J, Giaccia A J,
Lippincott Williams & Wilkins, (2005)). Recent evidence shows
that tumour cells can adapt to low oxygen conditions and change the
pharmacodynamic responses to anticancer agents through the
induction of active cellular protective mechanisms (Vaupel and
Mayer 2007, Cancer Metastasis Rev 26(2): 225-239). Additionally, it
is recognized that tumour cells that survive hypoxic stress often
show a more malignant metastatic phenotype (Vaupel P, Metabolic
microenvironment of tumor cells: a key factor in malignant
progression, Exp Oncol 2010; 32, 125-127); this has significant
consequences for the patient. Following treatment with modalities
that target predominantly the better-oxygenated cells, the
stress-resistant hypoxic cells often repopulate the tumour with
cells that have an enhanced potential to spread to distant tissues.
The development of more malignant metastatic tumours is often the
precursor to a more significant disease-related morbidity and the
death of the patient.
An attractive approach is the use of a hypoxia activated prodrug
that is non-toxic towards adequately oxygenated cells found in
systemic tissues, but becomes activated or converted to a cytotoxic
form under reduced oxygenation conditions. N-oxide derivatives of
cytotoxic alkylaminoanthraquinones provide anthraquinone pro-drugs
that show almost no cytotoxicity. Importantly these prodrugs are
capable of being converted in vivo under the anaerobic/hypoxic
conditions found within neoplastic tissue. Specificity for the
tumour is ensured since systemic tissues, except for tumours,
almost never experience oxygen levels low enough to facilitate the
production of the cytotoxic drug.
The anthraquinone N-oxide AQ4N (CAS#136470-65-0) is a prodrug that
is selectively bioreduced to AQ4, a potent DNA topoisomerase II
inhibitor, in hypoxic tumour cells. Previous publications have
taught the fundamental properties and in-vitro/in-vivo
characteristics of the prodrug AQ4N (for example, see U.S. Pat. No.
5,132,327).
The invention seeks to address the need for improved cancer
treatments by providing novel anthraquinone compounds with a
combination of preferable pharmacological and hypoxia-sensing
properties.
SUMMARY
The first aspect of the invention provides a compound of Formula
I
##STR00002## wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each
independently selected from the group consisting of hydrogen,
hydroxy, halogeno, amino, C.sub.1-4 alkoxy, C.sub.2-8 alkanoyloxy,
--NH-A-NHR, --NH-A-NR'R'' and --NH-A-N(O)R'R'' wherein A is an
alkylene group with a chain length of at least two carbon atoms
(between NH and NHR or N(O)R'R''), wherein R, R' and R'' are each
independently selected from C.sub.1-4 alkyl groups and C.sub.2-4
hydroxyalkyl and C.sub.2-4 dihydroxyalkyl groups in which the
carbon atom attached to the nitrogen atom does not carry a hydroxy
group and no carbon atom is substituted by two hydroxy groups, or
wherein R and R'' together are a C.sub.2-6 alkylene group which
with the nitrogen atom to which R' and R'' are attached forms a
heterocyclic group having 3 to 7 atoms in the ring, wherein at
least one of X.sub.1, X.sub.2, X.sub.3 and X.sub.4 is selected from
the group consisting of deuterated forms of --NH-A-NHR,
--NH-A-NR'R'' and --NH-A-N(O)R'R''.
Thus, the invention provides novel deuterated anthraquinone
compounds.
By "deuterated" we include that the compound comprises at least one
atom of deuterium or heavy hydrogen (i.e. D or .sup.2H). It will be
appreciated by persons skilled in the art that the compound may be
partially (i.e. selectively) or fully deuterated (i.e. containing
hydrogen present only in the form of deuterium).
By "selectively", in this context, we mean that some but not all
conventional .sup.1H hydrogen atoms are replaced with deuterium.
For example, one or more of substituent groups X.sub.1, X.sub.2,
X.sub.3 and X.sub.4 may be deuterated while the central
anthraquinone ring may be free of deuterium.
In one embodiment, the compound of the invention is selectively
deuterated within one or more of substituent groups --NH-A-NHR,
--NH-A-NR'R'' and/or --NH-A-N(O)R'R'' at positions X.sub.1,
X.sub.2, X.sub.3 and/or X.sub.4. Within each such substituent
group, it will be appreciated that A, R, R' and R'' may be fully
deuterated (i.e. thus containing no .sup.1H) or may be partially
deuterated.
In a preferred embodiment, the compound is deuterated only within
one or more of the terminal groups R, R' and R''. For example, R,
R' and/or R'' may represent: CD.sup.3; CH.sub.2CD.sup.3;
CD.sub.2CD.sup.3; CD.sub.2CH.sub.2CD.sup.3; and CD.sub.2CD.sub.2
CD.sub.2CD.sup.3.
The term "C.sub.1-4 alkyl" is intended to include linear or
branched alkyl groups comprising between one and four carbons.
Preferred alkyl groups which R, R' and/or R'' may independently
represent include C.sub.1 and C.sub.2 alkyl.
The term "lower alkylene" is to be construed accordingly.
The terms "C.sub.2-4 hydroxyalkyl" and "C.sub.2-4 dihydroxyalkyl"
are intended to include linear or branched alkyl groups comprising
between two and four carbons, to which are attached one or two
hydroxy groups, respectively. For example, R, R' and/or R'' may
independently represent: CH.sub.2CH.sub.2OH CH.sub.2CH(OH)CH.sub.3
CH.sub.2CH.sub.2CH(OH)CH.sub.2OH
The term "C.sub.1-4 alkoxy" is intended to include linear or
branched C.sub.1-4 alkyl groups bound to the core anthraquinone
(anthracene-9,10-dione) ring via oxygen. For example, R, R' and/or
R'' may independently represent: OCH.sub.3 OCH.sub.2CH.sub.3
OCH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.2CH.sub.2CH.sub.3
The term "C.sub.2-8 alkanoyloxy" is intended to include linear or
branched C.sub.2-8 alkanoyl groups bound to the core anthraquinone
(anthracene-9,10-dione) ring via oxygen. For example, R, R' and/or
R'' may independently represent: O(O)CCH.sub.3
O(O)CCH.sub.2CH.sub.3 O(O)CCH.sub.2CH.sub.2CH.sub.3
O(O)CCH.sub.2CH.sub.2CH.sub.2CH.sub.3
O(O)CCH.sub.2CH.sub.2CH(CH.sub.3)CH.sub.3
The term "hydroxy" is intended to represent --OH.
The term "halogeno" is intended to represent any halogen group,
such as --Br, --Cl and --F.
The term "amino" is intended to include primary amine groups, such
as --NH.sub.2.
It will be appreciated by persons skilled in the art that the
anthraquinone ring of the compounds may be substituted by X.sub.1,
X.sub.2, X.sub.3 and X.sub.4 at any of ring positions 1, 2, 3, 4,
5, 6, 7 or 8:
##STR00003##
In one embodiment of the first aspect of the invention, the
compound is substituted at ring positions 1, 4, 5 and 8, in
accordance with Formula II:
##STR00004##
In one embodiment, X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each
separately selected from the group consisting of hydrogen, hydroxy,
--NH-A-NHR, --NH-A-NR'R'', --NH-A-N(O)R'R'' and deuterated forms
thereof.
In one embodiment, X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each
separately selected from the group consisting of hydroxy,
--NH-A-NR'R'', --NH-A-N(O)R'R'' and deuterated forms thereof.
In one embodiment, X.sub.1 and X.sub.2 are both hydroxy and X.sub.3
and X.sub.4 are both --NH-A-N(O)R'R'' or deuterated forms
thereof.
In one embodiment, X.sub.1 and X.sub.2 are both hydroxy and X.sub.3
and X.sub.4 are both NH-A-NR'R'' or deuterated forms thereof.
In one embodiment, A is unbranched. For example, A may be
ethylene.
In one embodiment, R, R' and R'' are each independently selected
from the group consisting of --CH.sub.3, --CH.sub.2CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.3, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH(CH.sub.3)CH.sub.2OH,
--CH.sub.2CHOHCH.sub.2OH and deuterated forms thereof.
In one embodiment, one or two of X.sub.1, X.sub.2, X.sub.3 and
X.sub.4 are independently selected from the group consisting of
--NH--(CH.sub.2).sub.2--N(O)(CH.sub.3).sub.2,
--NH--(CH.sub.2).sub.2--N(O)(CH.sub.3)C.sub.2H.sub.5,
--NH--(CH.sub.2).sub.2--N(O)(C.sub.2H.sub.5).sub.2,
--NH--(CH.sub.2).sub.2--N(O) (CH.sub.2CH.sub.2OH).sub.2,
--NH--(CH.sub.2).sub.2--N(O)(CH.sub.2CH.sub.2CH.sub.2OH).sub.2,
--NH--(CH.sub.2).sub.2--N(O)CH(CH.sub.3)OH,
--NH--(CH.sub.2).sub.2--N(O)(CH.sub.2CHOHCH.sub.2OH).sub.2 and
deuterated forms thereof.
In one embodiment, one or two of X.sub.1, X.sub.2, X.sub.3 and
X.sub.4 are independently selected from the group consisting of
--NH--(CH.sub.2).sub.2--N(CH.sub.3).sub.2,
--NH--(CH.sub.2).sub.2--N(CH.sub.3)C.sub.2H.sub.5,
--NH--(CH.sub.2).sub.2--N(C.sub.2H.sub.5).sub.2,
--NH--(CH.sub.2).sub.2--N(CH.sub.2CH.sub.2OH).sub.2,
--NH--(CH.sub.2).sub.2--N(CH.sub.2CH.sub.2CH.sub.2OH).sub.2,
--NH--(CH.sub.2).sub.2--NCH(CH.sub.3)OH,
--NH--(CH.sub.2).sub.2--N(CH.sub.2CHOHCH.sub.2OH).sub.2 and
deuterated forms thereof.
In one embodiment, the compound of the invention comprises one
group --NH-A-N(O)R'R'' and one group --NH-A-NHR, the --NH-A-NHR
group being selected from --NH--(CH.sub.2).sub.2--NHCH.sub.3,
--NH--(CH.sub.2).sub.2--NHC.sub.2H.sub.5,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CH.sub.2CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH(CH.sub.3)CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CHOHCH.sub.2OH and deuterated
forms thereof.
In one embodiment, the compound of the invention comprises one
group --NH-A-NR'R'' and one group --NH-A-NHR, the --NH-A-NHR group
being selected from --NH--(CH.sub.2).sub.2--NHCH.sub.3,
--NH--(CH.sub.2).sub.2--NHC.sub.2H.sub.5,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CH.sub.2CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH(CH.sub.3)CH.sub.2OH,
--NH--(CH.sub.2).sub.2--NHCH.sub.2CHOHCH.sub.2OH and deuterated
forms thereof.
In preferred, but non-limiting, compounds of the invention:
(a) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--H and
X.sub.3=X.sub.4=--OH;
(b) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--OH, X.sub.3=--OH and
X.sub.4=--H;
(c) X.sub.1=--NH-A-N(O)R'R'' and X.sub.2=X.sub.3=X.sub.4=--OH;
(d) X.sub.1=X.sub.4=--NH-A-N(O)R'R'' and X.sub.2=X.sub.3=--OH;
(e) X.sub.1=X.sub.2=--NH-A-N(O)R'R'' and X.sub.3=X.sub.4=--OH;
(f) X.sub.1=X.sub.3=--NH-A-N(O)R'R'' and X.sub.2=X.sub.4=--OH;
(g) X.sub.1=--NH-A-NR'R'', X.sub.2=--H and
X.sub.3=X.sub.4=--OH;
(h) X.sub.1=--NH-A-NR'R'', X.sub.2=--OH at position 4, X.sub.3=--OH
and X.sub.4=--H;
(i) X.sub.1=--NH-A-NR'R'' and X.sub.2=X.sub.3=X.sub.4=--OH;
(j) X.sub.1=X.sub.4=--NH-A-NR'R'' and X.sub.2=X.sub.3=--OH;
(k) X.sub.1=X.sub.2=--NH-A-NR'R'' and X.sub.3=X.sub.4=--OH;
(l) X.sub.1=X.sub.3=--NH-A-NR'R'' and X.sub.2=X.sub.4=--OH;
and deuterated forms thereof.
In further preferred, but non-limiting, compounds of the
invention:
(a) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--NH-A-NHR, and
X.sub.3=X.sub.4=--OH;
(b) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--OH, X.sub.3=--NH-A-NHR and
X.sub.4=--OH;
(c) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=X.sub.3=--OH and
X.sub.4=--NH-A-NHR;
(d) X.sub.1=--NH-A-NR'R'', X.sub.2=--NH-A-NHR, and
X.sub.3=X.sub.4=--OH;
(e) X.sub.1=--NH-A-NR'R'', X.sub.2=--OH, X.sub.3=--NH-A-NHR and
X.sub.4=--OH;
(f) X.sub.1=--NH-A-NR'R'', X.sub.2=X.sub.3=--OH and
X.sub.4=--NH-A-NHR;
and deuterated forms thereof.
In further preferred, but non-limiting, compounds of the
invention:
(a) X.sub.1=X.sub.2=--NH-A-N(O)R'R'' and X.sub.3=X.sub.4=--OH;
(b) X.sub.1=X.sub.3=--NH-A-N(O)R'R'' and X.sub.2=X.sub.4=--OH;
(c) X.sub.1=X.sub.2=--NH-A-NR'R'' and X.sub.3=X.sub.4=--OH; and
(d) X.sub.1=X.sub.3=--NH-A-NR'R'' and X.sub.2=X.sub.4=--OH
wherein
both --NH-A-N(O)R'R'' are
--NH--(CH.sub.2).sub.2N(O)(CH.sub.3).sub.2 or
--NH--(CH.sub.2).sub.2N(O)(CH.sub.2CH.sub.2OH).sub.2, or deuterated
forms thereof and
both NH-A-NR'R'' are --NH--(CH.sub.2).sub.2N(CH.sub.3).sub.2 or
--NH--(CH.sub.2).sub.2N(CH.sub.2CH.sub.2OH).sub.2, or deuterated
forms thereof.
In further preferred, but non-limiting, compounds of the
invention:
(a) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--NH-A-NHR and
X.sub.3=X.sub.4=--OH;
(b) X.sub.1=--NH-A-N(O)R'R'', X.sub.2=--OH, X.sub.3=--NH-A-NHR and
X.sub.4=--OH;
(c) X.sub.1=--NH-A-NR'R'', X.sub.2=--NH-A-NHR and
X.sub.3=X.sub.4=--OH; and
(d) X.sub.1=--NH-A-NR'R'', X.sub.2=--OH, X.sub.3=--NH-A-NHR and
X.sub.4=--OH,
wherein
--NH-A-N(O)R'R'' is --NH--(CH.sub.2).sub.2N(O)(CH.sub.3).sub.2 or
--NH--(CH.sub.2).sub.2N(O)(CH.sub.2CH.sub.2OH).sub.2 or a
deuterated form thereof
--NH-A-NHR is NH--(CH.sub.2).sub.2NHCH.sub.3 or
NH(CH.sub.2).sub.2NHCH.sub.2CH.sub.2OH or a deuterated form
thereof
and NH-A-NR'R'' is --NH--(CH.sub.2).sub.2N(CH.sub.3).sub.2 or
--NH--(CH.sub.2).sub.2N(CH.sub.2CH.sub.2OH).sub.2 or a deuterated
form thereof.
The present invention relates to novel anthraquinone compounds and
uses of the same, for example in the treatment of cancer.
The present invention relates to novel anthraquinone compounds and
uses of the same, for example in the treatment of cancer.
In one embodiment, the compound is of Formula III or IV:
##STR00005## wherein Y are each independently selected from the
group consisting of hydrogen, hydroxy, halogeno, amino, C.sub.1-4
alkoxy and C.sub.2-8 alkanoxy, or a prodrug thereof.
By "prodrug", in this context, is included compounds which may
readily be converted in vivo to a compound of Formula III or IV. In
one embodiment, the conversion is triggered by the prodrug entering
an hypoxic environment, such as a solid tumour.
Examples of suitable prodrugs include N-oxide derivatives of the
compounds of Formula III or IV.
Thus, in one embodiment, the prodrug is a compound of Formula V or
VI:
##STR00006## wherein Y are each independently selected from the
group consisting of hydrogen, hydroxy, halogeno, amino, C.sub.1-4
alkoxy and C.sub.2-8 alkanoxy.
In one preferred embodiment, the compound is of Formula VII or
VIII:
##STR00007## or a prodrug thereof.
The present invention relates to novel anthraquinone compounds and
uses of the same, for example in the treatment of cancer.
The present invention relates to novel anthraquinone compounds and
uses of the same, for example in the treatment of cancer.
In a further preferred embodiment, the compound is prodrug of
Formula IX or X:
##STR00008##
In the compounds of Formulae III to X, it will be appreciated by
persons skilled in the art that one or more of the deuterium atoms
in one or more of the methyl groups attached to the nitrogen of the
terminal amino groups may be replaced by conventional hydrogen
(i.e. .sup.1H), provided that the compound comprises at least one
deuterium atom. For example, one, two, three or four of the methyl
groups may be --CH.sub.3, --CH.sub.2D or --CHD.sub.2. In one
embodiment, the methyl groups in the compound are either --CH.sub.3
or --CD.sub.3.
It will be further appreciated by skilled persons that certain
compounds of formulae I to X above may be counterbalanced by
counter-anions. Exemplary counter-anions include, but are not
limited to, halides (e.g. fluoride, chloride and bromide), sulfates
(e.g. decylsulfate), nitrates, perchlorates, sulfonates (e.g.
methane sulfonate) and trifluoroacetate. Other suitable
counter-anions will be well known to persons skilled in the art.
Thus, pharmaceutically, and/or veterinarily, acceptable derivatives
of the compounds of formulae I to X, such as salts and solvates,
are also included within the scope of the invention. Salts which
may be mentioned include: acid addition salts, for example, salts
formed with inorganic acids such as hydrochloric, hydrobromic,
sulfuric and phosphoric acid, with carboxylic acids or with
organo-sulfonic acids; base addition salts; metal salts formed with
bases, for example, the sodium and potassium salts.
In one embodiment, the compound is in the form of a halide salt,
for example a chloride salt.
It will be further appreciated by skilled persons that certain
compounds of formulae I to X may exhibit tautomerism. All
tautomeric forms and mixtures thereof are included within the scope
of the invention.
Compounds of formulae I to X may also contain one or more
asymmetric carbon atoms and may therefore exhibit optical and/or
diastereoisomerism. Diastereoisomers may be separated using
conventional techniques, e.g. chromatography or fractional
crystallisation. The various stereoisomers may be isolated by
separation of a racemic or other mixture of the compounds using
conventional, e.g. fractional crystallisation or HPLC, techniques.
Alternatively, the desired optical isomers may be made by reaction
of the appropriate optically active starting materials under
conditions which will not cause racemisation or epimerisation, or
by derivatisation, for example with a homochiral acid followed by
separation of the diastereomeric esters by conventional means (e.g.
HPLC, chromatography over silica). All stereoisomers are included
within the scope of the invention.
Various routes are available for the synthesis of the compounds of
the invention. One very convenient procedure for the preparation of
compounds having a group --NH-A-NR'R'' at the 1 and 4 positions
uses the appropriately substituted
2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione which is
condensed with the appropriate amine R''R'N--A--NH.sub.2, the 1,4
positions being activated in the leuco compound for reaction with
the amine. Such a condensation may conveniently be effected at a
temperature in a range of about 25.degree. C. or 35.degree. C. to
50.degree. C. or 60.degree. C. for one or more hours using a
solvent such as methanol, ethanol, water, dimethylformamide,
2-methoxyethanol, acetonitrile, nitrobenzene,
N,N,N'N'-tetra-methylenediamine or mixtures thereof. In some
instances a higher temperature and shorter reaction time may be
appropriate, for example with the compounds containing cyclic
groups NR'R''. The leuco derivative is then oxidized to the fully
aromatic anthracene-9,10-dione, conveniently using air oxidation or
oxidation with hydrogen peroxide, chloranil, sodium perborate or
manganese dioxide.
Although leuco compounds are primarily of interest for the
preparation of compounds substituted by two --NH-A-NHR'R'' groups,
it is possible to use them to prepare compounds containing more
than two such groups. Thus, by using
2,3-dihydro(leuco)-1,4,5,8-tetrahydroxyanthracene-9,10-dione and a
large excess of an amine --NH-A-NHR'R'' an
8-hydroxyanthracene-9,10-dione having three groups --NH-A-NHR'R''
at the 1, 4 and 5 positions may be prepared.
The leuco derivatives themselves are obtainable by heat treatment
of the corresponding fully aromatic
1,4-dihydroxyanthracene-9,10-dione, conveniently by heating at
above 90.degree. C. for 1 hour or more in a stream of nitrogen and,
if necessary, in the presence of a suitable reducing agent such as
sodium dithionite or zinc dust. Various anthracene-9,10-diones,
particularly hydroxyanthracene-9,10-diones, are commercially
available and various syntheses for such compounds are also
reported in the literature. One suitable procedure for their
preparation involves the reaction of an appropriately substituted
phthalic anhydride with hydroquinone in the presence of aluminium
chloride and sodium hydroxide at 180.degree. C. for one hour or
more. Anthracene-9,10-diones containing one form of substituent
group can be modified to provide other forms of substituent group
so that, for example, a dione containing an amino group can be
treated with sodium hydroxide/dithionite to yield the corresponding
hydroxy substituted compound.
Other suitable procedures for the preparation of intermediates for
oxidation to the N-oxide compounds of the invention include the
reaction of the appropriate chloro or fluoro substituted
anthracene-9,10-dione with the appropriate amine
R''R'N--A--NH.sub.2, for example by heating with a excess of the
amine at its reflux temperature for one or more hours. Certain of
these chloro- and fluoro anthracene-9,10-diones are known and
various syntheses for such compounds are also reported in the
literature. Thus, for example, a KF--NaF-mediated conversion of
3,6-dichlorophthalic anhydride to 3,6-difluorophthalic anhydride as
a precursor to making
1,4-difluoro-4,8-dihydroxyanthracene-9,10-dione (see Lee &
Denny, 1999, J. Chem. Soc., Perkin Trans. 1:2755-2758.
Additionally, for example,
1,5-dichloro-4,8-dihydroxyanthracene-9,10-dione may be prepared by
selective chlorination of 1,4,5,8-tetrahydroxyanthracene-9,10-dione
using a stoichiometric amount of sulphuryl chloride and controlled
temperature. This precursor may then be used to prepare an
intermediate having groups --NH-A-NR'R'' at the 1 and 5 positions
and hydroxy groups at the 4 and 8 positions, the hydroxy groups
conveniently being protected during the reaction with the amine
R''R'N--A--NH.sub.2. A similar approach is suitable for the
preparation of other chlorohydroxyanthracene-9,10-dione
intermediates.
Where the compound of the invention contains one or more groups
--NH-A-NHR in addition to the one or more groups --N-A-NR'R'' the
compound may conveniently be produced by reacting a suitable
precursor as discussed above with a mixture of amines
RN--A--NH.sub.2 and R''R'N--A--NH.sub.2, the resultant mixture of
products then being separated, for example by chromatography. Thus,
for example, 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione
on reaction with a mixture of 2-(2-hydroxyethylamino)ethylamine and
2-(diethylamino)ethylamine will yield a mixture of
1,4-bis{[2-(diethylamino)-ethyl]amino}anthracene-9,10-dione,
1,4-bis{[2-(2-hydroxyethyl-amino)-ethyl]amino}-anthracene-9,10-dione
and
1-(2-(diethylamino)ethyl]amino)-4-{[2-(2-hydroxyethylamino)-ethyl]amino}a-
nthracene-9,10-dione from which the last mentioned compound may be
separated, for example by chromatography. On oxidation, only the
tertiary nitrogen atom of the [2-(diethylamino)ethyl)] amino group
will be converted to N-oxide form.
Where one or more substituent groups is present it may be
appropriate, depending on the route of synthesis, to have these
present throughout in their final form or to generate the desired
groups at a later stage in the synthesis. Ether and ester groups X
may of course readily be prepared by modification of hydroxy groups
according to known procedures, precursors containing a hydroxy
group X more often being described in the literature than those
containing a corresponding ether or ester substituent.
It will be appreciated, however, that various alternative methods
for the preparation of the compounds and intermediates therefor may
be used as will be apparent in particular from the literature
relating to such intermediates. Further details of the preparation
of intermediates for the preparation of the compounds of the
present invention are to be found in U.S. Pat. No. 4,197,249 and GB
2,004,293B (the disclosures of which are incorporated herein by
reference).
Thus, a second aspect of the invention provides a process for
making a compound according to the first aspect of the invention
comprising reacting an anthracene-9,10-dione with a deuterated
alkylenediamine under conditions suitable for the production of an
alkylaminoalkylaminoanthraquinone.
Optionally, the process further comprises the step of reacting the
alkylaminoalkylaminoanthraquinone with a monoperoxyphthalate to
under conditions suitable for the production of an N-oxide
derivative of the alkylamino-alkylaminoanthraquinone.
In one embodiment, the process comprises reacting
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, 281-005 with
deuterated--N,N-dimethylethylene-diamine under conditions suitable
for the production of
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthra-
cene-9,10-dione.
In a further embodiment, the process comprises the step of reacting
the
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthra-
cene-9,10-dione with magnesium monoperoxyphthalate under conditions
suitable for the production of
1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-dihydro-
xy-anthracene-9,10-dione.
A third aspect of the invention provides a pharmaceutical
composition comprising a compound according to the first aspect of
the invention together with pharmaceutically acceptable buffer,
diluent, carrier, adjuvant or excipient.
By "pharmaceutically acceptable" we include a non-toxic material
that does not decrease the therapeutic effectiveness of the
compound of the invention. Such pharmaceutically acceptable
buffers, carriers or excipients are well-known in the art (see
Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro,
Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical
Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press
(2000), the disclosures of which are incorporated herein by
reference).
The term "buffer" is intended to mean an aqueous solution
containing an acid-base mixture with the purpose of stabilising pH.
Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS,
Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate,
glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO,
BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine,
HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO,
TAPS, TABS, TAPSO and TES.
The term "diluent" is intended to mean an aqueous or non-aqueous
solution with the purpose of diluting the agent in the
pharmaceutical preparation. The diluent may be one or more of
saline, water, polyethylene glycol, propylene glycol, ethanol or
oils (such as safflower oil, corn oil, peanut oil, cottonseed oil
or sesame oil).
The term "adjuvant" is intended to mean any compound added to the
formulation to increase the biological effect of the compound of
the invention. The adjuvant may be one or more of zinc, copper or
silver salts with different anions, for example, but not limited to
fluoride, chloride, bromide, iodide, thiocyanate, sulfite,
hydroxide, phosphate, carbonate, lactate, glycolate, citrate,
borate, tartrate, and acetates of different acyl composition. The
adjuvant may also be cationic polymers such as cationic cellulose
ethers, cationic cellulose esters, deacetylated hyaluronic acid,
chitosan, cationic dendrimers, cationic synthetic polymers such as
poly(vinyl imidazole), and cationic polypeptides such as
polyhistidine, polylysine, polyarginine, and peptides containing
these amino acids.
The excipient may be one or more of carbohydrates, polymers, lipids
and minerals. Examples of carbohydrates include lactose, glucose,
sucrose, mannitol, and cyclodextrines, which are added to the
composition, e.g., for facilitating lyophilisation. Examples of
polymers are starch, cellulose ethers, cellulose
carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl
cellulose, ethylhydroxyethyl cellulose, alginates, carageenans,
hyaluronic acid and derivatives thereof, polyacrylic acid,
polysulphonate, polyethylenglycol/polyethylene oxide,
polyethyleneoxide/polypropylene oxide copolymers,
polyvinylalcohol/polyvinylacetate of different degree of
hydrolysis, and polyvinylpyrrolidone, all of different molecular
weight, which are added to the composition, e.g., for viscosity
control, for achieving bioadhesion, or for protecting the lipid
from chemical and proteolytic degradation. Examples of lipids are
fatty acids, phospholipids, mono-, di-, and triglycerides,
ceramides, sphingolipids and glycolipids, all of different acyl
chain length and saturation, egg lecithin, soy lecithin,
hydrogenated egg and soy lecithin, which are added to the
composition for reasons similar to those for polymers. Examples of
minerals are talc, magnesium oxide, zinc oxide and titanium oxide,
which are added to the composition to obtain benefits such as
reduction of liquid accumulation or advantageous pigment
properties.
The compounds of the invention may be formulated into any type of
pharmaceutical composition known in the art to be suitable for the
delivery thereof.
In one preferred embodiment, the pharmaceutical compositions are
administered parenterally, for example, intravenously,
intracerebroventricularly, intraarticularly, intraarterially,
intraperitoneally, intrathecally, intraventricularly,
intrasternally, intracranially, intramuscularly or subcutaneously,
or they may be administered by infusion techniques. The
pharmaceutical compositions may also administered intra-tumourally
and/or peri-tumourally.
Such pharmaceutical compositions are conveniently used in the form
of a sterile aqueous solution which may contain other substances,
for example, enough salts or glucose to make the solution isotonic
with blood. The aqueous solutions should be suitably buffered
(preferably to a pH of from 3 to 9), if necessary. The preparation
of suitable parenteral formulations under sterile conditions is
readily accomplished by standard pharmaceutical techniques well
known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets of the kind
previously described.
In a further embodiment, the pharmaceutical compositions of the
invention may be in the form of a liposome, in which the agent is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids, which exist in
aggregated forms as micelles, insoluble monolayers and liquid
crystals. Suitable lipids for liposomal formulation include,
without limitation, monoglycerides, diglycerides, sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like.
Suitable lipids also include the lipids above modified by
poly(ethylene glycol) in the polar headgroup for prolonging
bloodstream circulation time. Preparation of such liposomal
formulations is can be found in for example U.S. Pat. No.
4,235,871, the disclosures of which are incorporated herein by
reference.
The pharmaceutical compositions of the invention may also be in the
form of biodegradable microspheres. Aliphatic polyesters, such as
poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of
PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides
have been widely used as biodegradable polymers in the production
of microspheres. Preparations of such microspheres can be found in
U.S. Pat. No. 5,851,451 and in EP 0 213 303, the disclosures of
which are incorporated herein by reference.
In a further embodiment, the pharmaceutical compositions of the
invention are provided in the form of polymer gels, where polymers
such as starch, cellulose ethers, cellulose carboxymethylcellulose,
hydroxypropylmethyl cellulose, hydroxyethyl cellulose,
ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic
acid and derivatives thereof, polyacrylic acid, polyvinyl
imidazole, polysulphonate, polyethylenglycol/polyethylene oxide,
polyethyleneoxide/polypropylene oxide copolymers,
polyvinylalcohol/polyvinylacetate of different degree of
hydrolysis, and polyvinylpyrrolidone are used for thickening of the
solution containing the agent. The polymers may also comprise
gelatin or collagen.
Alternatively, the compounds may simply be dissolved in saline,
water, polyethylene glycol, propylene glycol, ethanol or oils (such
as safflower oil, corn oil, peanut oil, cottonseed oil or sesame
oil), tragacanth gum, and/or various buffers.
It will be appreciated that the pharmaceutical compositions of the
invention may include ions and a defined pH for potentiation of
action of the active agent. Additionally, the compositions may be
subjected to conventional pharmaceutical operations such as
sterilisation and/or may contain conventional adjuvants such as
preservatives, stabilisers, wetting agents, emulsifiers, buffers,
fillers, etc.
The pharmaceutical compositions according to the invention may be
administered via any suitable route known to those skilled in the
art. Thus, possible routes of administration include parenteral
(intravenous, subcutaneous, and intramuscular), topical, ocular,
nasal, pulmonar, buccal, oral, parenteral, vaginal and rectal. Also
administration from implants is possible.
Alternatively, the pharmaceutical compositions may be administered
intranasally or by inhalation (for example, in the form of an
aerosol spray presentation from a pressurised container, pump,
spray or nebuliser with the use of a suitable propellant, such as
dichlorodifluoromethane, trichlorofluoro-methane,
dichlorotetrafluoro-ethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134A3 or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or
other suitable gas). In the case of a pressurised aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurised container, pump, spray or nebuliser
may contain a solution or suspension of the active polypeptide,
e.g. using a mixture of ethanol and the propellant as the solvent,
which may additionally contain a lubricant, e.g. sorbitan
trioleate. Capsules and cartridges (made, for example, from
gelatin) for use in an inhaler or insufflator may be formulated to
contain a powder mix of a compound of the invention and a suitable
powder base such as lactose or starch.
The pharmaceutical compositions will be administered to a patient
in a pharmaceutically effective dose. A `therapeutically effective
amount`, or `effective amount`, or `therapeutically effective`, as
used herein, refers to that amount which provides a therapeutic
effect for a given condition and administration regimen. This is a
predetermined quantity of active material calculated to produce a
desired therapeutic effect in association with the required
additive and diluent, i.e. a carrier or administration vehicle.
Further, it is intended to mean an amount sufficient to reduce and
most preferably prevent, a clinically significant deficit in the
activity, function and response of the host. Alternatively, a
therapeutically effective amount is sufficient to cause an
improvement in a clinically significant condition in a host. As is
appreciated by those skilled in the art, the amount of a compound
may vary depending on its specific activity. Suitable dosage
amounts may contain a predetermined quantity of active composition
calculated to produce the desired therapeutic effect in association
with the required diluent. In the methods and use for manufacture
of compositions of the invention, a therapeutically effective
amount of the active component is provided. A therapeutically
effective amount can be determined by the ordinary skilled medical
or veterinary worker based on patient characteristics, such as age,
weight, sex, condition, complications, other diseases, etc., as is
well known in the art. The administration of the pharmaceutically
effective dose can be carried out both by single administration in
the form of an individual dose unit or else several smaller dose
units and also by multiple administrations of subdivided doses at
specific intervals. Alternatively, the dose may be provided as a
continuous infusion over a prolonged period.
It will be appreciated that the compositions of the invention may
be formulated in unit dosage form, i.e. in the form of discrete
portions containing a unit dose or a multiple or sub-unit of a unit
dose.
Whilst the dosage of the compound used will vary according to the
activity of the particular compound and the condition being
treated, it may be stated by way of guidance that a dosage selected
in the range from 0.1 to 20 mg/kg per body weight per day,
particularly in the range from 0.1 to 5 mg/kg of body weight per
day, will often be suitable although higher doses than this, for
example in the range from 0.1 to 50 mg/kg of body weight per day
(or possibly even as high as described in U.S. Pat. No. 4,197,249)
may be considered in view of the lower level of toxic side effects
obtained with the compounds. This dosage regime may be continued
for however many days is appropriate to the patient in question,
the daily dosages being divided into several separate
administrations if desired. Thus, for example, in the case of
conditions such as advanced breast cancer, non-Hodgkin's lymphoma
and hepatoma, treatment for one day followed by a repeated dose
after an interval, such as 21 days, may be appropriate whilst for
the treatment of acute non-lymphocytic leukaemia, treatment over 5
consecutive days may be more suitable.
A fourth aspect of the invention provides a compound according to
the first aspect of the invention for use in medicine (clinical
and/or veterinary).
A fifth aspect of the invention provides a compound according to
the first aspect of the invention for use as a cytotoxin, or a
hypoxia activated prodrug thereof.
In one embodiment, the compound is for use in vivo as a cytotoxin,
or a hypoxia activated prodrug thereof.
By "hypoxia activated prodrug thereof" we include that the compound
is preferentially cytotoxic under, or following exposure to,
hypoxic conditions (i.e. exhibits greater cytotoxicity under, or
following exposure to, hypoxic conditions). For example, N-oxide
compounds of the invention, such as those of formulae V, VI, IX and
X, are relatively non-cytotoxic under normoxic conditions but are
readily reduced under hypoxic conditions to generate cytotoxic
compounds, such as those of formulae III, IV, VII and VIII.
In this context, "hypoxia" may be regarded as an oxygenation level
of 4% or lower (or .ltoreq.23 mmHg) when measured directly by
electrode methods. For example, the level of oxygenation may be
lower than 3.0%, 2.5%, 2%, 1.5%, 1% or 0.5 or 0.1%.
It will be appreciated by persons skilled in the art that the
hypoxia-induced activation of a compound's cytotoxic activity may
be determined either in vitro or in vivo.
For example, cytotoxicity may be determined in vitro at various
oxygenation levels measured by direct electrode methods.
Alternatively, the level of oxygenation in a tissue may be measured
indirectly, for example using histological sections probed with an
enzyme detection assay or by gene expression analysis.
For confirmation of hypoxia-activated cytotoxicity in vivo,
oxygenation levels in living tissue may be determined using both
the Helzel and OxyLite systems (for example, see Wen et al., 2008,
Radiat. Res. 169:67-75).
The results of blood flow and perfusion analyses may also infer the
existence of hypoxia in a given tissues. The application of agents
that modify blood flow or compromise blood vessel formation would
also on first principles be expected to reduce oxygenation in
affected tissues.
In particular, the invention provides a compound according to the
first aspect of the invention for use in the treatment of cancer in
mammals (most notably in humans).
For example, the compound may be for use in the treatment of a
cancer selected from the group consisting of bladder cancer, breast
cancer, bone cancer (primary and secondary, such as osteosarcoma
and Ewings sarcoma), brain cancer (including glioblastoma
multiforme and astrocytoma), cervical cancer, choriocarcinoma,
colon and rectal cancer, endometrial cancer, eye cancer,
gallbladder cancer, gastric cancer, gestational tumours, head and
neck cancer, kidney (renal cell) cancer, laryngeal cancer,
leukaemias (such as ALL, AML, CLL, CML and hairy cell leukaemias),
liver cancer, lung cancer, lymphomas (such as Hodkin's lymphoma and
non-Hodkin's lymphoma), melanoma, mesothelioma, mouth cancer,
myeloma, nasal and sinus cancers, nasopharyngeal cancer,
oesophageal cancer, ovarian cancer, pancreatic cancer, penile
cancer, prostate cancer, stomach cancer, testicular cancer, thyroid
cancer, uterine cancer, vaginal cancer, vulvar cancer and womb
cancer.
In one embodiment, the compound is for use in the treatment of a
solid tumour, such as various forms of sarcoma and carcinoma.
The compounds of the invention may be of particular use in the
treatment of a tumour that is naturally hypoxic, at least in part
(for example, having a median oxygen level of below 3%, e.g. lower
than 2.5%, 2%, 1.5%, 1% or 0.5%). An example of such tumours are
pancreatic cancer and prostate cancer, both typically exhibiting
low oxygen levels and a propensity for malignant progression.
The hypoxia-activated cytotoxicity of the prodrug compounds of the
invention allows the cytotoxicity to be targeted to the tumour
cells, reducing the risk of damage to healthy cells.
It is believed that hypoxia may play a role in facilitating the
malignant progression of certain cancers (for example, see
Rudolfsson & Bergh, 2009, Exp. Opin. Ther. Tar. 13:219-225). By
exerting a cytotoxic effect preferentially within the regions of
tumour hypoxia, the compounds of the invention may be able to
target cancer cells that are otherwise resistant to treatment, e.g.
by radiotherapy or conventional chemotherapeutic agents.
Eradication of such resistant cells may, in turn, lead to a
reduction in metastasis.
Thus, in one embodiment, the compounds are for use in the treatment
or prevention of metastases (which may arise from the aetiology of
the cancer or as a consequence of treatment).
It will be appreciated by persons skilled in the art that the
compounds of the invention may be used on their own or in
combination with other cancer treatments (such as radiotherapeutic
modalities, e.g. radioisotopes and external beam radiation, and
chemotherapeutic agents; see below).
In one embodiment, the compounds are for use as a monotherapy (i.e.
without any other cancer treatments). However, it will be
appreciated that the cancer patient may also be receiving different
types of beneficial medication (such as a painkiller, sedative,
antidepressant, antibiotic, etc).
However, the compounds of the invention may alternatively be for
use in combination with one or more additional cancer treatments.
For example, the compounds may be used in combination with one,
two, three, four, five or more additional cancer treatments.
By "in combination" we include that the compound is administered to
a subject who is receiving one or more additional cancer treatments
in the same course of therapy. Thus, the term covers not only the
concomitant administration of the compound with one or more
additional cancer treatments (either as bolus doses or infusions)
but also the temporally separate administration of these cancer
treatments. For example, the compound may be administered within a
treatment schedule/cycle as defined by the patient's oncologist to
include one or more additional cancer treatments, administered
either before, concomitantly with or after the compound; for
example within ten weeks, nine weeks, eight weeks, seven weeks, six
weeks, five weeks, four weeks, three weeks, two week, ten days, one
week, five days, four days, three days, two days, one day, 12
hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1
hour, 45 minutes, 30 minutes, 20 minutes, 10 minutes or five
minutes. Each treatment cycle may be repeated on several occasions,
normally up to 6 cycles, but could be more or less than this number
depending on the nature of the cancer and its response to
treatment.
It will be appreciated by persons skilled in the art that the one
or more additional cancer treatments may be chemotherapeutic agents
or radiotherapeutic modalities.
In one embodiment, however, the one or more additional cancer
treatments comprise or consist of one or more chemotherapeutic
and/or radiotherapeutic modality.
Given the hypoxia-activated cytotoxicity of the prodrug compounds
of the invention, it is advantageous to administer them as part of
a combination treatment with one or more chemotherapeutic agents
and/or radiotherapeutic modalities capable of decreasing (at least,
transiently) tumour oxygenation levels in vivo. For example, the
one or more chemotherapeutic agents and/or radiotherapeutic
modalities may be capable of lowering the median oxygen level of
the tumour to below 3%, for example below 2.5%, 2%, 1.5%, 1%, 0.5%,
0.4%, 0.3%, 0.2% or below 0.1%.
It will be appreciated by skilled persons that a reduction in
tumour oxygenation levels may be achieved by a number of different
means, for example by the disruption of established tumour
vasculature, prevention of angiogenesis (new blood vessel
formation) and/or vasoconstriction.
Suitable cancer treatments may be selected from the group
consisting of anti-androgens (steroidal and non-steroidal),
vascular disrupting agents, anti-angiogenic agents, anti-VEGFR
agents, IL8 inhibitors, NO synthase inhibitors, vasoconstricting
agents, vasodilating agents and radiotherapy.
By "steroidal anti-androgens" we include cyproterone acetate.
By "anti-angiogenic agents" we include: (a) anti-VEGF antibodies or
antibody fragments such as bevacizumab, axitinib, pazopanib and
ranibizumab, pegaptanib sodium, tryptophanyl-tRNA synthetase,
AdPEDF, EYLEA, AG-013958, JSM6427, TG100801, ATG3, rapamycin,
endostatin; (b) drugs that block signalling within the cell such as
lapatinib, sunitinib, sorafenib, axitinib, pazopanib and AZ2171;
(c) tetrahydrocannabinol (THC) and cannabidiol; (d)
thiazolidinediones such as rosiglitazone, pioglitazone and
troglitazone (e) erlotinib, imatinib, gefitinib, dasatinib,
nilotinib, lapatinib; and (f) drugs that affect signals between
cells, such as thalidomide and lenalidomide.
By "vascular disrupting agents" we include small molecules (such as
taxanes, taxol, paclitaxel combretastatins, CA4P, Oxi4503,
aurostatins, dolostatins, colchine, azacolchicinol, ZD6126I,
MMP-activated colchicines, ICT2588, DMXAA, TZT1027 and AVE8062) and
biologicals (such as ADEPT, GDEPT and antibody drug-conjugates that
target the tumour vasculature).
By "IL8 inhibitors" we include repertaxin.
By "NO synthase inhibitors" we include N.sup.G-methyl-1-arginine
hydrochloride (546C88; 1-NMMA), NG-nitro-L-arginine (L-NNA),
L-nitroarginine methyl ester (L-NAME), LG-nitro-L-arginine
(L-NO-Arg) and 7-Nitro-Indazole (7-NI).
By "vasoconstricting agents" we include alpha 1 adrenoceptor
agonists (e.g. methoxamine, phenylephrine, oxymetazoline,
tetrahydralazine, xylometazoline), alpha 2 adrenoceptor agonists
(e.g. clonidine, guanabenz, guanfacine, .alpha.-methyldopa) and
vasopressin analogues (e.g. arginine vasopressin and triglycyl
lysine vasopressin).
By "vasodilating (`vascular steal`) agents" we include
alpha-adrenoceptor antagonists (alpha-blockers), angiotensin
converting enzyme (ACE) inhibitors, angiotensin receptor blockers
(ARBs), beta2-adrenoceptor agonists (.beta.2-agonists),
calcium-channel blockers (CCBs), centrally acting sympatholytics,
direct acting vasodilators, endothelin receptor antagonists,
ganglionic blockers, nitrodilators, phosphodiesterase inhibitors,
potassium-channel openers and renin inhibitors.
By "radiotherapy modalities" we include conventional external beam
radiation therapy (2DXRT), stereotactic radiosurgery (SRS),
stereotactic body radiation therapy (SBRT) and particle therapy
such as proton therapy; brachytherapy such as SAVI.TM.,
MammoSite.TM., Contura.TM., Proxcelan.TM., TheraSeed.TM. and
I-Seed.TM.; radioisotope therapy such as metaiodobenzylguanidine
(MIBG), iodine-131, hormone-bound lutetium-177 and yttrium-90
(peptide receptor radionuclide therapy).
In one preferred embodiment, the one or more cancer treatments
is/are non-steroidal anti-androgens, such as flutamide, nilutamide,
bicalutamide, finasteride, dutasteride, bexlosteride, izonsteride,
turosteride, epristeride and abiraterone.
Thus, in one embodiment, a compound according to the first aspect
of the invention is used in combination with bicalutamide in the
treatment of cancer, e.g. the prevention or reduction of
metastasis.
Thus, in one embodiment, a compound according to the first aspect
of the invention is used in combination with cancer
chemotherapeutic agents and/or radiotherapeutic modalities and/or
methods to reduce or increase the air being breathed by the
patients e.g. carbogen (with or without nicotinamide).
A related, sixth aspect of the invention provides the use of a
compound of the first aspect of the invention in the preparation of
a medicament for treating cancer.
Preferred embodiments of the sixth aspect of the invention are
described above in relation to the fifth aspect of the
invention.
A seventh aspect of the invention provides a method of treating
cancer in a patient comprising administering to the patient a
therapeutically effective amount of a compound of the first aspect
of the invention.
In one embodiment, the patient is mammalian (e.g. human).
Preferred embodiments of the seventh aspect of the invention are
described above in relation to the fifth aspect of the
invention.
An eighth aspect of the invention provides the use of a compound of
the first aspect of the invention as a marker of the oxygenation
level of cells. In particular, such compounds may be used as a
cellular hypoxic marker, either in vitro or in vivo.
In one embodiment, the cells are mammalian (e.g. human).
Exposure of the N-oxide forms of the compounds of the invention
(such as those of formulae V and VI) to hypoxic cells causes their
reduction to the corresponding amine form (such as those of
formulae III and IV), which can be readily detected by known
means.
The presence of the reduced compound (such as those of formulae III
and IV) can be used to detect hypoxic cells in vitro or in vivo.
The innate fluorescence properties retained by the reduced
compound(s) and the intracellular persistence of the reduced
compound(s) are advantageous for the discrimination, quantification
and localisation of cells that have been exposed to, or continue to
be exposed to hypoxic conditions.
For example, when acting as a cellular marker for hypoxia, the
reduced compound (such as those of formulae III and IV) maybe
detected using method(s) that identify chemical composition or
physical properties that include but are not limited to mass
spectrometry, infrared spectroscopy, colorimetry, Raman
spectroscopy, nuclear magnetic resonance or positron emission
tomography. Affinity capture methods would exploit the high
affinity binding potential of the reduced compound to DNA or
synthetic polynucleotide sequences.
Optical properties of the reduced compound(s) may be used to detect
compound in biological samples and include but are not limited to
flow cytometry and microscopy utilising the innate fluorescent
properties of the reduced compound. Secondary methods of detection
of reduced compound include but are not limited to a combination
with other molecular reporter compounds with the reduced compound
participating in resonant energy transfer reactions as either an
acceptor or donor. Other secondary methods of detection of reduced
compound include but are not limited to methods using antibody
based methods for molecular detection.
In one embodiment, the compounds of the invention are used to
identify hypoxic tumour cells in vivo, which may then be visualised
in situ or excised surgically.
In a further embodiment, a compound of the first aspect of the
invention is used as a cellular hypoxic marker in combination with
a non-deuterated form of a compound of the first aspect of the
invention.
By "in combination" in this context this includes that the
compounds may be applied to the cells (e.g. administered to a
patient) either concomitantly or sequentially (for example, within
24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30
minutes, 30 minutes, 10 minutes or less).
Thus, in a preferred embodiment, a compound of formulae IX or X is
used as a cellular hypoxic marker (in vivo or in vitro) in
combination with a compound as disclosed in U.S. Pat. No. 5,132,327
(for example, AQ4N).
A related, ninth aspect of the invention provides a kit of parts
for use in detecting the oxygenation level of cells comprising a
compound according to the first aspect of the invention.
Optionally, the kit further comprises a non-deuterated form of a
compound according to the first aspect of the invention (such as a
compound as disclosed in U.S. Pat. No. 5,132,327, for example
AQ4N).
Preferably, the compound(s) is/are provided in a sterile,
pyrogen-free form.
It will be appreciated that the kits of the invention may further
comprise one or more regents, control samples and/or
instructions.
DESCRIPTION OF THE DRAWINGS
Preferred, non-limiting examples which embody certain aspects of
the invention will now be described, with reference to the
following figures:
FIG. 1: The metabolites AQ4 and OCT1001 have similar cell cycle
arresting actions, under normal oxygenation conditions, indicating
that selective deuteration has not modified intrinsic biological
activity.
See Example B
FIG. 2: Similar hypoxia-enhanced cytotoxicity for AQ4N and
OCT1002
See Example B
FIG. 3: Exemplification of that the bioactivity of AQ4N and OCT1002
is dependent upon the degree of hypoxia
See Example B
FIG. 4: Hypoxia-dependent growth inhibition by AQ4N and OCT1002
arises from a similar mechanism of cell cycle arrest and is
dependent on the degree of hypoxia
See Example B
FIGS. 5 (A & B): Exemplification of shared bioactivity of AQ4N
and OCT1002 under hypoxic conditions for functional p53 (DoHH2) and
mutant p53 (SU-DHL-4) human B cell lymphoma cells
See Example B
FIG. 6: Intracellular accumulation of the OCT1001 far-red
fluorescent chromophore under hypoxia is responsive to OCT1002
pro-drug dose and oxygenation level
See Example B
FIG. 7: Deuteration does not affect the intrinsic capacity of the
metabolite (AQ4 or OCT1001) to accumulate within a cell
See Example B
FIG. 8: Accumulation of converted pro-drug OCT1001 correlates with
growth arrest
See Example
FIGS. 9 (A & B): Demonstration of intracellular fluorescence
following exposure to OCT1002 under hypoxic conditions and that
prodrug deuteration reduces intracellular accumulation but
increases persistence of the metabolite.
See Example B
FIG. 10: Effect of bicalutamide on the oxygenation of 22Rv1
prostate tumours grown as xenografts
See Example C
FIG. 11: Effect of bicalutamide on blood vessels in 22Rv1 tumour
xenografts
See Example C
FIG. 12: Effect of bicalutamide only or AQ4N single dose or OCT1002
single dose on 22Rv1 xenografts in mice
See Example C
FIG. 13: Combined effect of AQ4N single dose or OCT1002 single dose
on 22Rv1 xenografts in mice treated daily with bicalutamide
See Example C
FIG. 14: Effect of OCT1002 on LNCaP xenografts in mice treated
with/without bicalutamide
See Example C
FIG. 15: OCT1002 is reduced in hypoxic LNCaP tumour cells in
vivo
See Example C
FIG. 16: OCT1002 reduces the metastatic spread of LNCaP tumours to
the lungs
See Example C
DETAILED DESCRIPTION OF THE EMBODIMENTS
Examples
Example A: Synthesis of Alkylaminoalkylaminoanthraquinones and
their N-Oxides
(a) Preparation of
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione
##STR00009##
A mixture of 4,7-difluoroisobenzofuran-1,3-dione (8.50 g, 46.2
mmol), hydroquinone (5.64 g, 51.3 mmol), aluminium trichloride
(36.9 g, 277 mmol) and sulfolane (10 mL) was stirred together for
16 hours at 165.degree. C. The reaction was effectively a melt as
the mixture does not become a viscous red syrup until
.about.150.degree. C. To minimise the risk of a sudden exotherm and
evolution of HCl gas, the reaction was stirred in portions, cooled
in an ice bath and stirred again until mixing was sufficient. Only
then was the mixture heated.
The mixture was poured carefully into ice and 2M HCl added (50 mL).
The mixture was stirred, then filtered, washing the resultant
slurry with further 2M HCl. The solid was re-slurried a further 3
times with 2M HCl to reduce the aluminium content of the product. A
final slurry was washed with ether twice; drying in a round bottom
flask at 60.degree. C. until constant weight afforded
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (9.82 g, 35.6 mmol,
77% yield).
.sup.1H NMR (DMSO-d.sup.6) was clean and consistent with the
desired material.
(b) Preparation of
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthr-
acene-9,10-dione
##STR00010##
A suspension of deuterated-d6-dimethylamine hydrochloride (18.4 g,
210 mmol) and 2-bromoacetonitrile (14.63 ml, 210 mmol) in anhydrous
THF (250 mL) in a round bottom flask was cooled to -10.degree. C.
with vigorous stirring and treated portion-wise with potassium
carbonate (58.1 g, 420 mmol). After addition of the base, the
reaction was fitted with a reflux condenser and balloons and
allowed to warm slowly to 5.degree. C. over 2 hours. TLC (1:1
EtOAc/Iso-Hexanes) indicated the presence of product. The mixture
was stirred at room temperature over a weekend.
The residue was diluted with DCM (250 mL) and filtered, washing
with copious amounts of DCM. The mother liquors were degassed with
N.sub.2 for 1 hour, then reduced in volume by half on the rotavap.
Then a 4M dioxane solution of hydrogen chloride (52.5 ml, 210 mmol)
was added, precipitating a white solid and the mixture allowed to
stand for 10 minutes before being filtered, washing with DCM to
afford deuterated-d6-dimethylacetonitrile (21.73 g, 172 mmol, 82%
yield).
.sup.1H NMR (400 MHz, d.sub.6-DMSO) .delta.: 4.47 (2H, s) was
consistent with the desired material.
(c) Preparation of deuterated-d6-N,N-dimethylethylenediamine
##STR00011##
To a stirred suspension of deuterated-d6-dimethylacetonitrile
(21.72 g, 172 mmol) in Et.sub.2O (200 mL) at 0.degree. C. was added
d/w a 1M ether solution of lithium aluminium hydride (515 ml, 515
mmol) via dropping funnel over 1.5 hours. After the addition, the
cooling bath was removed. After a further 1.5 hr, the reaction was
quenched at 15.degree. C. (no higher than 18.degree. C.) with
sodium sulfate decahydrate (0.5 eq rel. to LiAlH4, 80 g) cautiously
(delayed reaction) over 1.5 hours. The mixture was left to stir for
1 hour and subsequently filtered, washing with ether. The filtrate
was stored overnight in the dark. The ether was removed on the
rotavap at .about.40.degree. C. with no vacuum to afford
deuterated-d6-N,N-dimethylethylenediamine (15.89 g, 160 mmol, 93%
yield was clean and consistent with the desired material but
contained .about.0.25 eq ether).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 2.76 (2H, t), 2.33 (2H,
t)
(d) Preparation of
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthr-
acene-9,10-dione ("OCT1001")
##STR00012##
A solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, (4.9
g, 17.74 mmol) in pyridine (35 mL) was treated with
deuterated-d6-N,N-dimethylethylenediamine, (16.57 ml, 142 mmol) as
a steady stream. The mixture was warmed to 40.degree. C. and
allowed to stir for 24 hours under a flow of nitrogen. The reaction
was taken off heat and cooled in an ice-bath. A chilled mixture of
ammonium hydroxide (30%, 30 mL) and brine (30 mL) were added and
the mixture stirred in an ice-bath for 2 hours. After this time the
mixture was filtered washing with a 10% ammonium hydroxide solution
(130 mL). The solid was air-dried for 30 minutes, then transferred
to a tared flask and dried under vacuum at 60.degree. C. until
constant weight (.about.2 h).
The bulk material was purified by flash chromatography (Biotage,
120 g) loading in DCM (through cotton wool plug) eluting with 6
then 10% MeOH (containing 1% NH.sub.3)/DCM to give
1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthra-
cene-9,10-dione (2.01 g, 4.73 mmol, 26.7% yield).
The product was analysed by LCMS (m/z 425.3 (M+H).sup.+ (ES.sup.+);
423.2 (M-H)- (ES)-, at 0.90 and 1.03 min (product smears on
column), 100%.
.sup.1H NMR (CDCl.sub.3) was clean and consistent with the desired
material .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 13.51 (2H, s),
10.40 (2H, br t), 7.17 (2H, s), 7.11 (2H, s), 3.47 (4H, q), 2.66
(4H, t).
(e) Preparation of
1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydr-
oxyanthracene-9,10-dione ("OCT1002")
##STR00013##
A suspension containing magnesium monoperoxyphthalate, MMPP (3.10
g, 6.27 mmol) in methanol (8 mL) was added dropwise to a stirred
solution of 281-041 (1.90 g, 4.48 mmol), AQ4 in methanol (8 mL) and
DCM (30 mL) cooled to -11.degree. C. After the addition was
complete, the reaction solution was allowed to warm to 0.degree. C.
and stirred overnight in the dark (warmed to room temperature
during this time). Pre-cooled EtOAc (30 mL) and EtOH (6 mL) were
added the reaction mixture at 0.degree. C. This mixture was allowed
to stir for 30 minutes then a 4M solution of hydrogen chloride
(4.48 ml, 17.90 mmol) in dioxane was added dropwise at
approximately -10 to -15.degree. C. The resulting slurry was then
stirred for 10 minutes then filtered, washing with EtOH/Water (9:1,
100 mL), MeOH/EtOAc (1:1, 100 mL) and EtOAc (60 mL) and dried under
vacuum (on rotavap) at 40.degree. C. for 2 hours (constant weight)
to afford
1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]-amino)-5,8-di-hyd-
roxyanthracene-9,10-dione (2.15 g, 3.99 mmol, 89% yield) as a dark
blue powder.
The product was analysed by LCMS (standard 4 min. method, agilent),
m/z 458.2 (M+H).sup.+ (ES.sup.+), at 3.07 min, 98.3% purity @ 254
nm. .sup.1H NMR (400 MHz, D.sub.2O) .delta.: 6.73 (2H, br s), 6.43
(2H, br s), 3.76 (4H, br s), 3.58 (4H, br s).
.sup.1H NMR (D.sub.2O) was consistent with the desired
material.
Example B: In Vitro Properties of
1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydr-
oxyanthracene-9,10-dione and Its Active Metabolite
(a) The metabolites AQ4 and OCT1001 have similar cell cycle
arresting actions, under normal oxygenation conditions, indicating
that selective deuteration has not modified intrinsic biological
activity. A549 human lung cancer cells were cultured using
conventional methods for adherent cells and exposed for 4 days to
0, 1, 3 or 10 nM agents under standard cell culture conditions of
5% carbon dioxide in air at 37 deg C. Harvested cells were
permeabilised and stained with the DNA fluorescent dye ethidium
bromide and cell cycle distributions determined by conventional
flow cytometry. FIG. 1 (flow cytometry) shows similar increases in
the G2 peaks of the DNA content distributions between 3-10 nM
(indicating cell cycle arrest) for cells exposed to exogenous
metabolites
1,4-bis-{[2-(dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dio-
ne ("AQ4") and
1,4-bis-{[2-(deuterated-d6-dimethylamino)-ethyl]amino)-5,8-dihydroxy-anth-
racene-9,10-dione ("OCT1001"). (b) Similar Hypoxia-Enhanced
Cytotoxicity for AQ4N and OCT1002 Human T cell leukemia cells
(Jurkat) were cultured using conventional methods for suspension
cultures in air or under 1% oxygen conditions for 4 days in the
presence of a range of concentrations of either AQ4N or OCT1002.
The relative cell number was determined using a conventional
Coulter Counter particle counting method. FIG. 2 shows that the
compounds tested require hypoxic conditions for the inhibition of
cell proliferation. Thus,
1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene--
9,10-dione ("AQ4N") and
1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hyd-
roxy-anthracene-9,10-dione ("OCT1002") both exhibit pronounced
cytostatic activity under conditions of hypoxia (1% oxygen). As a
control it is shown that hypoxia does not modify the cytostatic
action of a direct acting DNA topoisomerase inhibitor (VP-16),
achieving similar levels of prolonged cytostatic action. (c)
Exemplification of that the Bioactivity of AQ4N and OCT1002 is
Dependent Upon the Degree of Hypoxia A549 human lung cancer cells
were cultured using conventional methods for adherent cells and
exposed for 4 days to varying concentrations of either AQ4N and
OCT1002 agents under standard cell culture conditions of 5% carbon
dioxide in air (normoxia) at 37 deg C., or under conditions of
reduced oxygen (1% and 3%). Data are plotted as relative population
doublings determined by cell detachment and Coulter Counter
particle counting of cell densities at the start and end of the
exposure period. FIG. 3 shows that for the compounds tested, namely
1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-
-9,10-dione ("AQ4N") and
1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hyd-
roxyanthracene-9,10-dione ("OCT1002"), growth inhibition is
dependent upon the degree of hypoxia and drug concentration, with
the two agents showing similar responses. (d) Hypoxic Sensitisation
by AQ4N and OCT1002 A549 human lung cancer cells were used in this
experiment; culture conditions were as described in (c) above. Cell
cycle analysis was performed as described in (a) above. FIG. 4
shows that the compounds tested, namely
1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-
-9,10-dione ("AQ4N") and
1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hyd-
roxy-anthracene-9,10-dione ("OCT1002"), generate similar cell cycle
arrest (determined by flow cytometry) within the bioactive drug
dose range. The degree of late cell cycle arrest is increased as
oxygenation levels are reduced. (e) Exemplification of Shared
Bioactivity of AQ4N and OCT1002 Under Hypoxic Conditions for p53
Functional and Mutant p53 Human B Cell Lymphoma Cell Lines Human B
cell lymphoma cells were cultured using conventional methods for
suspension cultures in air, 1% or 3% oxygenation conditions for 4
days in the presence of a range of concentrations of either AQ4N or
OCT1002. The relative cell numbers were determined using a
conventional Coulter Counter particle counting method. FIG. 5(A)
shows that the compounds tested are equally and selectively
cytotoxic in hypoxic conditions against DoHH2 human B cell lymphoma
cells (bcl2 overexpressing; p53 wt) grown in suspension and exposed
to prodrugs for 4 days under 21% (circles), 3% (triangles) or 1%
O.sub.2 (squares). Thus,
1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene--
9,10-dione ("AQ4N") and
1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hyd-
roxy-anthracene-9,10-dione ("OCT1002") both exhibit pronounced
cytostatic activity under conditions of hypoxia (1% oxygen), with
the growth inhibition being sensitive to the degree of hypoxia.
Likewise, FIG. 5(B) shows that the prodrugs AQ4N and OCT1002 are
equally selectively cytotoxic in hypoxic conditions against
SU-DHL-4 human B cell lymphoma cells (bcl2 overexpressing; p53
mutant) grown in suspension and exposed to prodrugs for 4 days
under 21% (circles), 3% (triangles) or 1% O.sub.2 (squares). Again,
the growth inhibition is sensitive to the degree of hypoxia. (f)
Reciprocity Between an Imposed pO.sub.2 Level and the Degree of
End-Product Generation OCT1002 and AQ4N show reciprocity between an
imposed pO.sub.2 level and the degree of end-product generation in
the biologically relevant range of hypoxia with low or undetectable
levels of conversion under normoxia (and undetectable levels of
AQ4N or OCT1002 showing that the metabolites are the primary
persistent anthraquinone forms) Relative to AQ4N, the deuterated
variant OCT1002 shows a reduction in overall capacity for
reduction/accumulation (HPLC analysis) within moribund cells, under
protracted exposure conditions showing a reduction of `redundant
targeting` in a human lung cancer cell line. In this case redundant
targeting of a prodrug refers to the over-generation of the
cytotoxic form beyond that required for cell inactivation since
conversion of the prodrug can continue even when cell cycle arrest
has occurred. The consequences of over-generation will be increased
deleterious effects of the converted form when released from the
initial target cell. This undesirable bystander effect on nearby
tissue not initially subject to hypoxic conditions will comprise
non-target normal and tumour cells. Damage to normal cells is
clearly undesirable. Suboptimal exposure of non-target tumour cells
through a bystander effect may compromise their responses to other
agent(s) delivered in combination or generate selective conditions
for the development of drug resistance. Table 1 shows a comparison
of HPLC analysis of metabolite generation following exposure of
human A549 cells to AQ4N and OCT1002 under varying degrees of
hypoxia and concentration (data derived from two determinations)
where 21% is taken to represent normal oxygenation conditions. Data
show the consistent reduction in the generation of OCT1001 compared
with AQ4 in cells exposed to the conditions indicated and washed
prior to assay for the presence of prodrug or their metabolites.
Data also shows that the molecular forms present in cells
experiencing hypoxia are the metabolites and not parent
prodrugs.
TABLE-US-00001 TABLE 1 Dose of Humidified Relative prodrug
oxygenation pmoles metabolite generated prodrug (OCT1002 conditions
per 10.sup.5 cells.sup.a reduction to or AQ4N) pO.sub.2 mm range
range metabolite nM .times. days % O.sub.2 Hg AQ4 OCT1001 AQ4
OCT1001 OCT1001/AQ4 30 1% 7.1 9.25 5.64 1.46 1.20 0.61 30 3% 21.4
0.78 0.49 0.05 0.06 0.62 30 21% 142.2 <0.10 0.10 0.03 0.02 1.02
100 1% 7.1 >42.95 16.17 6.59 8.16 <0.38 100 3% 21.4 5.58 1.93
1.13 0.16 0.35 100 21% 142.2 0.23 0.11 0.08 0.03 0.50 .sup.aNo AQ4N
or OCT1002 detected in any sample indicating that either all
prodrug forms are depleted by undergoing metabolism or that, by the
method used, such forms are not readily retained within cells.
(g) Intracellular Accumulation of the OCT1001 Far-Red Fluorescent
Chromophore Under Hypoxia is Responsive to OCT1002 Prodrug Dose and
Oxygenation Level Adherent A549 cells were cultured by conventional
methods and exposed to 0, 30 or 100 nM OCT1002 for 4 days in air,
1% or 3% oxygenation levels. Detached cells were analysed far red
fluorescence intensity using conventional flow cytometry and 633 nm
wavelength excitation (1.times.10.sup.4 cells analysed). FIG. 6
shows mean fluorescence intensity increases in a linear function of
pro-drug dose and is dependent upon oxygenation levels. This
provides a convenient fluorometric, single live cell analytical
method for analyzing cell population experience of prevailing
pO.sub.2 levels. (h) Deuteration does not Affect the Intrinsic
Capacity of the Active Metabolite (OCT1001) to Accumulate A549
human lung cancer cells were used in this experiment, as described
in (g) above. Under normoxia conditions, similar levels of
accumulation of OCT1001 and AQ4 were observed within cells (see
FIG. 7). Thus, the overlaid histograms for the population
distribution of fluorescence in cells exposed to AQ4 or OCT1001
under normoxia shows similar cellular accumulation potential. (i)
Accumulation of Converted Pro-Drug OCT1001 Correlates with Growth
Arrest (Increasingly Moribund Cells) A549 human lung cancer cells
were used in this experiment, as described in (g) above, with the
exception that light side scatter (488 nm wavelength) was collected
versus fluorescence intensity (>695 nm wavelength). FIG. 8 shows
collected flow cytometry data for A549 cells exposed to 0, 30 and
100 nM OCT1002 under 21%, 3% and 1% oxygen over 4 days. Plotting
all data points reveals that increasing light side scatter
parameter (reflecting the expansion of cell size and complexity
associated with growth arrest) correlates with the increase in
fluorescence intensity (indicating co-accumulation of OCT1001). (j)
Demonstration of Intracellular Fluorescence Following Exposure to
OCT1002 Under Hypoxic Conditions and that Prodrug Deuteration
Reduces Intracellular Accumulation but Increases Persistence of the
Metabolite. A549 cells were cultured using conventional methods and
allowed to attach to the glass substrate in chamber slides and
exposed to OCT1002 under hypoxia. Fluorescence imaging of live
cells used conventional confocal fluorescence microscopy using
red-line laser excitation. FIG. 9a shows that the far red
fluorescence detected in cells is intracellular (background
fluorescence not detectable in control cultures) with evidence of
regions of cytoplasmic accumulation. The data exemplify the single
cell hypoxia sensing properties of the deuterated pro-drug at the
single-cell level. Given the confirmation of intracellular
fluorescence associated with conversion of OCT1002 to OCT1001 under
hypoxia, A549 human lung cancer cells were further used to assess
differential accumulation or retention of the metabolites using
flow cytometry as described in (g) above. Following exposure to
AQ4N or OCT1002 under 1% oxygen, cells were detached for analysis,
or washed and incubated for 24 h in drug free medium and held under
normal oxygenation conditions prior to detachment and analysis by
flow cytometry Flow cytometry data in FIG. 9b shows the reduced
cellular accumulation (after 4 day exposure) but also reduced loss
(after 24 h post exposure recovery) of intracellular fluorescence
attributable to the metabolite OCT1001, compared with the
fluorescence attributable to the metabolite AQ4, following exposure
of A549 cells to pro-drugs OCT1001 and AQ4 in A549 under hypoxia.
Thus, deuteration changes the in situ intracellular compartment
loading/retention of hypoxia converted forms of OCT1002.
Conclusions
The above studies demonstrate the in vitro properties of an
exemplary deuterated compound of the invention (the N-oxide
prodrug, OCT1002, and its active metabolite, OCT1001). (a) Evidence
of primary biological activity following reduction of the prodrug
in hypoxia that elicits growth arrest in different tumour cell
types; (b) For an equally effective toxicity for the reduced drug
(OCT1001) the toxicity of OCT1002 to cells in normoxia is
significantly less. (c) Reciprocity between pO.sub.2 level and
end-product generation in the biologically relevant range of
hypoxia; (d) The ability of cellular fluorescence to report in situ
generation of of metabolite providing for the sensing and reporting
of hypoxic environments; (e) A distinct molecular/atomic signature
provided by site-specific deuteration that can be used to trace
prodrug conversion and metabolism by physico-chemical methods; and
(f) Prodrug deuteration results in reduced accumulation of the
reduced form under hypoxia but increased persistence/retention of
the reduced form upon removal of external drug and re-oxygenation.
This property demonstrated in moribund cells confirms both reduced
redundant targeting of the deuterated form and convenient signal
persistence for hypoxia sensing applications.
Example C--Effect of OCT1002 on Tumour Growth and Metastasis In
Vivo
Given the hypoxia-activated cytotoxicity of the prodrug compounds
of the invention, it may be advantageous to administer them as part
of a combination treatment with one or more chemotherapeutic agents
and/or radiotherapeutic modalities capable of decreasing (at least,
transiently) tumour oxygenation levels in vivo. Bicalutamide
(marketed as Casodex, Cosudex, Calutide, Kalumid) is an oral
non-steroidal anti-androgen used in the treatment of prostate
cancer including as monotherapy for the treatment of earlier stages
of the disease. 22Rv1 is a human prostate carcinoma epithelial cell
line (Sramkoski R M, Pretlow T G 2nd, Giaconia J M, Pretlow T P,
Schwartz S, Sy M S, Marengo S R, Rhim J S, Zhang D, Jacobberger J W
A new human prostate carcinoma cell line, 22Rv1. In Vitro Cell Dev
Biol Anim. 1999 July-August; 35(7):403-9). The cell line expresses
prostate specific antigen (PSA). Growth is weakly stimulated by
dihydroxytestosterone and lysates are immunoreactive with androgen
receptor antibody by Western blot analysis.
(i) Effect of Bicalutamide on the Oxygenation of 22Rv1 Prostate
Tumours Grown as Xenografts
Male SCID mice (>8 weeks) bearing 22Rv1 prostate tumours of
100-150 mm.sup.3 were treated daily for 28 days by oral gavage with
either vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day
in vehicle). Before commencement of treatment (day 0) pO2 (mmHg)
was measured using an Oxylite oxygen electrode probe; this was
repeated on the days indicated.
TABLE-US-00002 Day of Mean p0.sub.2 .+-. SD Significance
Significance Treatment Treatment (mmHg) (to vehicle) (to day 0)
Vehicle only 0 15.277 .+-. 11.254 7 14.741 .+-. 4.290 14 3.165 .+-.
3.275 21 2.660 .+-. 1.889 28 3.546 .+-. 1.563 Bicalutamide 0 15.277
.+-. 11.254 ns (2 mg/kg/ 7 1.996 .+-. 1.989 <0.05 <0.05 day)
14 0.486 .+-. 0.107 ns <0.05 21 1.291 .+-. 0.291 ns <0.05 28
11.905 .+-. 0.861 <0.01 ns
Table 3 shows mean pO2 values.+-.SD. Also shown are statistical
comparisons of the bicalutamide group compared to control and to
day 0 values; ns=not significant. 22Rv1 cells grow as a solid
tumour on the backs of SCID mice. Tumour oxygenation was measured
over 28 days in vehicle and bicalutamide (2 mg/kg/day) treated mice
(see Table 3 above). Bicalutamide caused a drop in tumour
oxygenation (as shown in FIG. 10); from .about.15.3 mmHg (2%
oxygen) to 2.0 mmHg (0.3% oxygen) at day 7 and to 0.5 mmHg (0.1%
oxygen) at day 14. This drop persists for approximately 2 weeks
before recovering to almost normal somewhere beyond 21 and 28 (at
which time it is not significantly different from the starting
level of oxygenation). The faster-growing, vehicle-treated,
controls showed no significant drop in oxygen levels up to day 7.
However, during the subsequent week (probably related to tumour
size) the median oxygen levels drop to about 3 mmHg (0.4% oxygen)
and do indicate recovery. Conclusion Hypoxia exists in the 22Rv1
solid tumour model. The addition of bicalutamide alters the
patterns of oxygen levels indicated by the tumour. Hypoxia is
clearly relevant to the 22Rv1 model and the response of such a
model to monotherapy (.+-.bicalutamide); and the potential role of
OCT1002 in a combination treatment. (ii) Effect of Bicalutamide on
Blood Vessels in 22Rv1 Tumour Xenografts Dorsal skin folds were
secured using window chambers onto the backs of male SCID mice
(>8 weeks). 22Rv1 tumour fragments were implanted and allowed to
vascularise for 7 days before commencement of treatment. Animals
were treated daily via oral gavage with either vehicle (0.1% DMSO
in corn oil) or bicalutamide (2 mg/kg in vehicle). Anaesthetised
mice were injected i.v. with FITC-labelled dextran immediately
prior to imaging with a confocal microscope. Each image is
representative of a minimum of 5 animals per treatment group. 22Rv1
tumours were grown in window chambers/dorsal skin flaps on the
backs of SCID mice. Tumour fragments were imaged (see FIG. 11)
before treatment began (A) vehicle and (E) bicalutamide
pre-treatment groups and then after 7, 14 and 21 days of treatment,
(B-D) vehicle only (F-H) bicalutamide (10.times. magnification).
Within 7 days tumour fragments showed the development of extensive
small vessels indicated as day 0 of the experimental period (see
FIG. 11). In vehicle-treated tumours vessel density showed a slight
change by day 14 and by day 21 the small vessel numbers were
reduced. In bicalutamide-treated tumours, loss of small vessels was
seen at days 7 and 14 with some recovery by day 21. This is
consistent with oxygen electrode data i.e., fall and then recovery
of oxygenation. Conclusions Vehicle has no effect on blood vessels
for at least 7 days. By day 14 there is a slight pruning of vessels
which is clearly seen by day 21. This vessel loss, although not as
dramatic as seen in the bicalutamide treated tumours (at days 7 and
14; Ming et al., 2007), may be due to vascular collapse and
necrosis seen at this time in this fast growing vehicle-treated
tumour. The oxygen levels drop somewhat earlier, i.e. sometime
between days 7 and 14 (see FIG. 10). In bicalutamide-treated 22Rv1
tumours there is a marked early loss of tumour vasculature (by day
7). The data provide evidence that bicalutamide causes a profound
drop in tumour oxygenation through an anti-vascular effect; this
may be direct or alternatively it could be due to inhibition of the
production of pro-angiogenic factors by the tumour cells. By day
21, the small vessels have returned which is consistent with the
increased level of oxygenation seen in FIG. 10. (iii) Effect of
Bicalutamide Only or AQ4N Single Dose or OCT1002 Single Dose on
22Rv1 Xenografts in Mice. Male SCID mice (>8 weeks) bearing
22Rv1 xenograft tumours of 100-150 mm3 were treated for 28 days.
Treatment included Vehicle (0.1% DMSO in corn oil) or bicalutamide
(2 mg/kg/day in vehicle) both administered daily via oral gavage.
Alternatively, at day 7 of the experimental period AQ4N or OCT1002
(50 mg/kg in sterile PBS) was administered intraperitoneally as a
single dose. Tumour volumes were measured using callipers every
other day. Data analysis to determine the time dependent effect of
treatment(s) on tumour volume was performed. Tumour volume was
normalised to day 6 (ie pre-produg addition). Time series and
regression analysis was undertaken. Tumour growth is normalised to
day 6, so that overall tumour growth, and patterns can be compared
FIGS. 12 (A and B). Despite the lack of sensitivity to bicalutamide
in vitro, the 22Rv1 tumours show a small but significant slowing of
growth. Classical cross-sectional comparison of growth delay showed
that mice treated with vehicle required 14.0.+-.0.3 days to reach
four times the volume at the start of treatment. Bicalutamide
treatment (2 mg/kg/day) increased this to 18.5.+-.0.8 days; thus
this was a growth delay of 4.5 days. Graphical regression fits
indicate that 22RV1 tumours treated with bicalutamide only show a
delay in growth (during days 10-20), despite continuing daily
exposure to bicalutamide; the tumours exhibit an overall
exponential growth pattern (R.sup.2=0.9915) to day 24. Addition of
AQ4N given as a single dose (50 mg/kg) on day 7, a different growth
pattern was observed compared to that of the bicalutamide treatment
alone, regression fitting showed a non-linear polynomial growth
pattern (R.sup.2=0.9948). Addition of OCT1002 given as a single
dose (50 mg/kg) on day 7; tumours treated with this single dose
were capable of maintaining a polynomial (x.sup.2) growth rate
pattern, this was also a non-linear pattern (R.sup.2=0.9978).
OCT1002 treated tumours showed an overall reduced rate of growth
over the remaining period of the experiment (beyond day 22)
compared to the bicalutamide only and AQ4N only treated tumours.
Cumulative growth over the entire period (progressive area under
the curve), indicates this difference (FIG. 12B). (iv) Combined
Effect of AQ4N Single Dose or OCT1002 Single Dose on 22Rv1
Xenografts in Mice Treated Daily with Bicalutamide Male SCID mice
(>8 weeks) bearing 22Rv1 xenograft tumours of 100-150 mm.sup.3
were treated for 28 days. Vehicle (0.1% DMSO in corn oil) and
bicalutamide (2 mg/kg/day in vehicle) treatments were administered
daily via oral gavage. AQ4N or OCT1002 (50 mg/kg in sterile PBS)
was administered intraperitoneally as a single dose at day 7.
Tumour volumes were measured using callipers every other day.
Animals were culled once the tumour burden reached .gtoreq.800
mm.sup.3. Tumour growth is normalised to day 6, so that overall
tumour growth, and patterns can be compared (FIGS. 13 (A and B).
Bicalutamide treatment alone (2 mg/kg/day) is discussed above; it
exhibits a overall exponential growth pattern (R.sup.2=0.9915) to
day 24. Bicalutamide treatment was combined with an AQ4N single
dose (50 mg/kg) given on day 7, a modified growth pattern was
observed compared to that of the bicalutamide treatment alone,
regression fitting showed a non-linear polynomial growth pattern
(R.sup.2=0.9982), with divergence of growth to bicalutamide alone
apparent at beyond day 20. Bicalutamide treatment treatment was
combined with an OCT1002 single dose (50 mg/kg) given on day 7; a
different modified growth pattern was observed regression fitting
showed a linear tumour growth response (R.sup.2=0.9955), with
divergence of growth to bicalutamide alone apparent at beyond day
14. Conclusions The combined treatment indicates two critical
features.
(i) the first is an earlier effective tumour growth inhibition of
OCT1002 on the bicalutamide treated tumours compared to AQ4N;
(ii) the second indicates a sustained tumour growth inhibition
(indicated by a maintained linear response); that reflects a
persistence OCT1002 and tumour growth inhibition. Thus with OCT1002
administered at the time when hypoxia/low oxygen levels were
achieved; an early and sustained effect was obtained. The
combination of OCT1002 with bicalutamide was more effective at
inhibiting tumour growth as compared to AQ4N with bicalutamide. (v)
Effect of OCT1002 on LNCaP Xenografts in Mice Treated with/without
Bicalutamide Male SCID mice (>8 weeks) bearing LNCaP xenograft
tumours of 100-150 mm.sup.3 were treated for 28 days. Vehicle (0.1%
DMSO in corn oil) and bicalutamide (2 mg/kg/day in vehicle)
treatments were administered daily via oral gavage. OCT1002 (50
mg/kg in sterile PBS) was administered intraperitoneally as a
single dose at day 7. Tumour volumes were measured using callipers
every other day. Growth curves are the mean of .gtoreq.5 animals in
bicalutamide and vehicle treatment groups; bicalutamide+OCT1002
group (n=5 until day 14; then n=3) and vehicle+OCT1002 (n=5 until
day 13; n=1).+-.s.e. Table 6 below shows the growth delays
calculated for the time to reach twice the treatment size.
Bicalutamide causes a 5.1 day delay in LNCaP tumour growth compared
to vehicle. When OCT1002 (50 mg/kg single dose on day 7) was given
in combination with vehicle (daily administration) there was no
appreciable effect on tumour growth (Table 6 below). Bicalutamide
(daily for 28 days) initially slows tumour growth until day 12-14.
Tumour growth then recovers and the tumours are the same size as
the vehicle-treated tumours by day 28 (Table 6 below). Tumours
treated with a single dose of OCT1002 reduced the growth rate in
combination with bicalutamide and this was significantly different
from control at all times between days 14 and days 28 at the
termination of the experiment (FIG. 15). Conclusions Administration
of OCT1002 at day 7 had no significant effect on LNCaP tumour
growth. This shows that the better-oxygenated tumours (i.e. as
compared to bicalutamide-treated tumours) there is low toxicity of
OCT1002 and that this better-oxygenated fraction of cells is
predominant in contributing to growth in vehicle-treated control
tumours. Combination of a single dose of OCT1002 with bicalutamide
blocked the increase in growth rate observed in the
bicalutamide-treated group. OCT1002 is very effective at blocking
tumour growth from 12 days onwards where, for bicalutamide alone,
there is a delay and then recovery. The initial slowing and then
recovery after day 14 of LNCaP tumour growth, during daily
treatment with bicalutamide, is consistent with the drop and then
recovery of tumour oxygenation and blood vessels (Ming et al.,
2012, supra.).
TABLE-US-00003 TABLE 6 Time to 2x start volume Growth Treatment
(days) Delay (days) Vehicle Only 11.2 .+-. 1.88 Bicalutamide 16.2
.+-. 1.94 5 .+-. 3.82 OCT1002 only 13 .+-. 0.89 1.8 .+-. 2.77
OCT1002 + 25.5 .+-. 3.22 14.3 .+-. 5.1 Bicalutamide
(vi) OCT1002 Prodrug is Converted to Metabolites in Hypoxic LNCaP
Tumour Cells In Vivo Methods A dorsal skin flap (window chamber)
was attached to the dorsum of male SCID mice and a 1 mm.sup.3
LNCaP-Luc tumour fragment inserted; this was left to vascularise
for 7 days. Mice were then treated orally for 21 days with either
vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day).
Seven days after induction of (a) vehicle or (b) bicalutamide mice
were dosed intraperitoneally with OCT1002 (50 mg/kg). Two hours
after injection of OCT1002 mice were injected intravenously with
FITC-dextran. Images were captured using a confocal laser scanning
microscopy to show blood vessels (green) and OCT1001 (blue)
patterns in the tumour. (Magnification 10.times. with 3.times.
digital zoom) (pixel resolution). Images were also acquired at day
0 (i.e. 7 days after tumour fragment implantation), 14 and 21. Only
FITC-dextran was administered on days 0, 14 and 21. (c) Full panel
of images 0, 7, 14 and 21 days. Control mice were treated orally
for 21 days with vehicle (0.1% DMSO in corn oil): vascularisation
was maintained throughout. By 7 days the tumour fragment was
vascularised (day 0 of experiment shown in FIG. 15C green). In mice
treated with vehicle+OCT1002 at day 7: the converted compound
OCT1001 (blue) is in a few areas where vascularisation is poor
(FIG. 15A). Mice treated with bicalutamide (2 mg/kg/day in
vehicle): vascularisation was reduced at days 7. On day 7, two
hours after intraperitoneal injection of a single dose of OCT1002
(50 mg/kg) large quantities of converted compound (OCT1001; blue)
can be seen across the whole tumour fragment (FIG. 15B). Mice
treated with bicalutamide (2 mg/kg/day in vehicle): vascularisation
was reduced at days 7 and 14, this recovered by day 21 (Ming et
al., 2012, supra.). Tumours were re-examined at days 14 and 21.
OCT1001 (blue) is still localised to the tumour at day 14; by day
21 the amount of compound was considerably lower (FIG. 15C).
Conclusions OCT1002, administered intraperitoneally, distributed
widely throughout the tumour fragments localised in the skin fold
on the backs of the mice. Distribution was extensive even when the
vasculature was significantly decreased (i.e. by the bicalutamide
treatment at days 7 and 14). OCT1001 was found predominantly where
the oxygen levels are low (i.e. areas of poor vascularisation);
small areas were seen in the control also (indicating that hypoxia
can occur in untreated tumours but to a lesser extent. Extensive
localisation of OCT1001 was still observed at day 14 of
bicalutamide treatment showing that the compound remains for at
least 7 days. By day 21, tumour blood vessels show some recovery
and OCT1001 levels are lower although still above background. The
persistence of the reduced product, OCT1001, for >7 days shows
that the half-life of the converted compound is long. However it
may be less than AQ4 since by day 21 the OCT1001 signal is very
much decreased. This may be due to the different cellular binding
properties of OCT1001 as compared to AQ4 and potentially will
provide a rationale for less cumulative systemic toxicity which
might be caused through persistence of small amount of reduced
compound in marginally hypoxic peripheral tissues. This should not
affect the primary efficacy of OCT1002/OCT1001 at the predominant
site of metabolism (i.e. the hypoxic cells in tumours) since large
amounts are seen throughout the hypoxic tumour fragment which
persists for greater than 7 days. (vii) OCT1002 Reduces the
Metastatic Spread of LNCaP Tumours to the Lungs Methods Male SCID
mice (>8 weeks) bearing LNCaP-luc xenograft tumours of 100-150
mm.sup.3 were treated for 28 days (the luciferase-expressing cells
had similar characteristics to parental LNCaP cells; Ming et al.,
2012, supra.). Vehicle (0.1% DMSO in corn oil) and bicalutamide (2
mg/kg/day in vehicle) treatments were administered daily via oral
gavage. OCT1002 (50 mg/kg in sterile PBS) was administered
intraperitoneally as a single dose at day 7. On day 28 of
treatment, animals were injected i.p. with a solution of
D-luciferin (150 mg/kg in PBS) 15 mins prior to imaging. Animals
were then killed and a range of tissues were removed for the
detection of bioluminescence using the IVIS imaging system
(Xenogen, USA). Images were taken for 5 minutes and quantification
of bioluminescence was achieved by drawing a region of interest
around the area and measuring total flux in photons/second
(ph/sec). A range of tissues were excised, however only the lungs
and tumour showed measurable bioluminescence. The mean.+-.s.e of
bioluminescence in the lungs is shown in FIG. 16; bicalutamide and
vehicle treatment groups (n=10); bicalutamide+OCT1002 group (n=3).
and vehicle+OCT1002 (n=1). *Bicalutamide vs bicalutamide+OCT1002
(p=0.024). Mice treated with vehicle showed some metastatic spread
to the lung. OCT1002, single dose day 7, had no effect on this
spread. Bicalutamide appeared to increase the extent of metastatic
spread although the result did not reach significance. Combination
of OCT1002 with bicalutamide showed that OCT1002 significantly
reduces the metastatic spread to the lungs caused by bicalutamide.
(P=0.024) Conclusions OCT1002 given as a single dose at day 7 was
able to reduce significantly the metastatic spread to the lungs
caused by bicalutamide treatment.
* * * * *