Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR TREATING
AGING-ASSOCIATED IMPAIRMENTS USING CCR3-
INHIBITORS
I. Cross-Reference to Related Applications
Pursuant to 35 U.S.C. 119 (e), this application claims priority to the
filing date of United States
Provisional Patent Application No. 62/737,017 filed on September 26, 2018, the
disclosure of which is
herein incorporated by reference.
Introduction
The following is offered as background information only and is not admitted to
be prior art to the
present invention.
Aging is an important risk factor for multiple human diseases including
cognitive impairment,
cancer, arthritis, vision loss, osteoporosis, diabetes, cardiovascular
disease, and stroke. In addition to
normal synapse loss during natural aging, synapse loss is an early
pathological event common to many
neurodegenerative conditions and is the best correlate to the neuronal and
cognitive impairment associated
with these conditions. As such, aging remains the single most dominant risk
factor for dementia-related
neurodegenerative diseases such as Alzheimer's disease (AD) (Bishop, N.A. et
al., Neural mechanisms of
ageing and cognitive decline. Nature 464(7288), 529-535 (2010); Heeden, T. et
al., Insights into the ageing
mind: a view from cognitive neuroscience. Nat. Rev. Neurosci. 5(2), 87-96
(2004); Mattson, M.P., et al.,
Ageing and neuronal vulnerability. Nat. Rev. Neurosci. 7(4), 278-294 (2006)).
Aging affects all tissues
and functions of the body including the central nervous system, and a decline
in functions such as cognition
and motor activity can severely impact quality of life.
Treatment for cognitive and motor decline associated with neurodegeneration
has achieved limited
success in preventing and reversing impairment. It is therefore important to
identify new treatments for
maintaining cognitive integrity by protecting against, countering, or
reversing the effects of aging.
Unfortunately, drug treatments for cognitive and motor impairment face
significant hurdles. For example,
it is thought that for treatments such as small molecules to be effective in
treating cognitive decline and
motor activity, they must cross the blood-brain barrier (BBB). Transport
across the BBB is the exception
not the rule, and 98% of all small molecules do not cross it. (Pardridge,
William M, The Blood-Brain
Barrier: Bottleneck in Brain Drug Development, NeuroRx: The J. of the Am.
Society for Experimental
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NeuroTherapeutics, 2:3-14, 2005). As a result, therapies for neurodegenerative
diseases, such as
Alzheimer's disease, Huntington' s disease, Parkinson's disease, and
amyotrophic lateral sclerosis (ALS),
are limited; in fact, Alzheimer' s has one of the highest failure rates for
clinical development of any disease
area (99.6% failure in the last 20 years). (Id.; and Burke, M., Renewed focus
on dementia checked by drug
challenges, ChemistryWorld, Jul. 3, 2014). Those BBB-permeable drugs that are
approved for treatment
of cognitive disease like Alzheimer's, such as donepezil, exhibit temporary
effects and do not work on all
patients. (Banks, W.A., Drug Delivery to the Brain in Alzheimer's Disease:
Consideration of the Blood-
brain Barrier, Adv. Drug. Deliv. Rev. 64(7):629-39 (2012); and Burke, M.
supra). Similarly, levodopa
which treats Parkinson's disease symptoms like motor deficits and crosses the
BBB, eventually loses its
efficacy. An additional hurdle for treatments that do cross the BBB is that
making compounds more
lipophilic (which tends to make them more BBB-penetrating) also often results
in increased removal from
the blood, leading to decreased bioavailability. Hence, the increase in
lipophilicity offsets the plasma area
under the curve (AUC), minimizing brain uptake. (Pardridge, William M, supra).
Summary
The present invention overcomes these drawbacks. For example, Compound 1, a
compound of the
invention, has exhibited resistance in crossing the BBB in significant
concentrations yet unexpectedly
results in improvement of symptoms associated with age-related
neurodegeneration and age-related motor
decline. Thus, the present invention can act in a peripheral manner, forgoing
what was considered to be a
requirement for effective cognitive-acting pharmaceuticals, i.e. to work
directly on central nervous system.
And although certain embodiments of the invention may cross the BBB in
significant concentrations, their
ability to antagonize the CCL11/CCR3 pathway offers an alternative mechanism
of action to current
therapy for cognitive and motor defects.
The compounds of the invention act as antagonists of c-c motif chemokine
receptor 3 (CCR3), the
receptor for Eotaxin-1. Eotaxin-1 (CCL11) is a protein that is increased in
levels in blood plasma with
aging and which has been shown to cause decreases in cognitive function.
(Villeda et al., The aging systemic
milieu negatively regulates neurogenesis and cognitive function, Nature,
477(7362):90-94 (2011)).
Eotaxin/CC11 acts primarily on the G-protein coupled receptor CCR3 which is
expressed on eosinophils
in the periphery and on neurons and glial cells in the central nervous system.
(Xia, M, et al.,
Immunohistochemical Study of the fl-Chemokine Receptors CCR3 and CCR5 and
Their Ligands in the
Normal and Alzheimer's Disease Brains, Am. J. Pathol. 153(1);31-37 (1998)). In
Alzheimer's disease, it
has been observed that CCR3 levels are increased in the CNS. (Id.) The
cognitive effects of antagonizing
CCR3 in Alzheimer's disease and related disorders would thus be expected to be
mediated by centrally
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located eotaxin-1/CCR3 interaction. However, Compound 1 (a compound of the
invention), unexpectedly
resulted in improving cognitive function and stimulating neurogenesis despite
its inability to cross the BBB
in significant concentrations.
Methods of treating patients for aging-associated impairments,
neurodegenerative disease and
associated cognitive and motor decline are provided, including by way of
example and not limitation,
Alzheimer's disease, Parkinson's disease, Dementia with Lewy Bodies,
frontotemporal dementia,
Huntington disease, amyotrophic lateral sclerosis, multiple sclerosis,
glaucoma, myotonic dystrophy,
vascular dementia, progressive supranuclear palsy, and the like. Aspects of
the methods include modulation
of CCR3, the principal receptor of CCL11/eotaxin-1 through the administration
of a therapeutically
effective amount of CCR3 antagonists of the invention. The methods include
administering an effective
therapeutic dose of CCR3 antagonists to subjects or patients as well as
monitoring for specific clinical
endpoints. Also included are methods of treating neurodegenerative disease and
associated cognitive and
motor decline with an agent that acts peripherally, i.e. does not cross the
blood-brain barrier in significant
concentrations, yet exhibits improvement in the disease, such as improved
cognition or motor activity.
Methods of using a diagnostic or companion diagnostic test in connection with
the described aging-
associated impairments are also provided. By way of example and not
limitation, such diagnostics or
companion diagnostics include devices that can determine or detect the
presence of a subset of white blood
cells from a subject. The diagnostic or companion diagnostic device may also
determine or detect the
presence, relative or absolute concentration, relative or absolute number of
eosinophils from a subject's
blood or tissues. Such diagnostic or companion diagnostic devices may be used
in conjunction with
IV. Incorporation by Reference
All publications, patents and patent applications mentioned in this
specification are herein
incorporated by reference in their entirety to the same extent as if each
individual publication or patent
application was specifically and individually indicated to be incorporated by
reference.
V. Brief Description of the Figures
Figure 1A depicts the sum of doublecortin (DCX) positive cells in the
hippocampus of: 2-month-
old and 18-month-old C57B1/6 mice treated with IgG (n=18 for both groups); 18-
month-old mice treated
with Compound 1 for 2 weeks at either a high (n=16) or low dose (n=31); and 18-
month-old mice treated
with Compound 1 ("Cmpd 1") for 4 weeks at a low dose (n=16). Data shown are
the mean s.e.m., where
*P<0.05, ***P<0.001.
Figure 1B depicts the sum of 5-bromo-2' -deoxyuridine (BrdU) positive cells in
the hippocampus
of: 2-month-old and 18-month-old C57B1/6 mice treated with IgG (n=19 for both
groups); 18-month-old
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mice treated with Compound 1 for 2 weeks at either a high (n=16) or low dose
(n=24). Data shown are the
mean + s.e.m., where
Figure 2A depicts the effect of Compound 1 on the time spent by young and old
C57B1/6 mice in
the novel (N) versus familiar/old ("F" or "0") arm during a Y-maze test. 2-
month-old mice were treated
for 2 weeks with IgG (n=18). 18-month-old mice were treated with: IgG for 2
weeks (n=18); Compound
1 at a high dose for 2 weeks (n=15); Compound 1 at a low dose for 2 weeks
(n=31); or Compound 1 at a
low dose for 4 weeks (n=16). Data shown are the mean s.e.m., where **P<0.05,
***P<0.01
Figure 2B depicts the effect of Compound 1 on the total number of visits made
by young and old
C57B1/6 mice into the novel (N) versus familiar (F) arm during a Y-maze test.
2-month-old mice were
treated for 2 weeks with IgG (n=18). 18-month-old mice were treated with: IgG
for 2 weeks (n=18);
Compound 1 at a high dose for 2 weeks (n=15); Compound 1 at a low dose for 2
weeks (n=31); or
Compound 1 at a low dose for 4 weeks (n=16). Data shown are the mean s.e.m.,
where *P<0.05,
**P<0.01, ***P<0.001.
Figure 3 depicts the effect of Compound 1 on bouts of entry ("number of
visits") into the novel
and familiar arms by C57B1/6 mice in the Y-maze test. The number of visits to
each arm was plotted for
each treatment group and subjected to paired t-test. 3-month-old mice (n=15)
received an infusion of
vehicle (veh) subcutaneously by Alzet mini-pump (0.5 L/hour) for four weeks,
with one replacement.
16.5-month-old mice were infused subcutaneously with either vehicle (veh,
n=16) or 50 mg/mL Compound
1 (n=16) by Alzet mini-pump (0.5 L/hour) for four weeks with one replacement.
Data shown are mean
s.e.m.; *P<0.05.
Figure 4A depicts the effect of Compound 1 on average time taken by C57B1/6
mice to find the
target hole in the Barnes Maze test. Average times were plotted for each
treatment group. 3-month-old
mice (n=18) received an infusion of vehicle (veh) subcutaneously by Alzet mini-
pump (0.5 L/hour) for
four weeks, with one replacement. 16.5-month-old mice were infused
subcutaneously with either vehicle
(veh, n=15) or 50 mg/mL Compound 1 (n=15) by Alzet mini-pump (0.5 L/hour) for
four weeks with one
replacement. Data shown are mean s.e.m.
Figure 4B depicts the effect of Compound 1 on the difference in latency to
find the target hole
between the last and first trial of the last day of the Barnes Maze test in
C57B1/6 mice. The differences
were plotted for each treatment group and the data subjected to an unpaired t-
test. 3-month-old mice (n=18)
received an infusion of vehicle (veh) subcutaneously by Alzet mini-pump (0.5
L/hour) for four weeks,
with one replacement. 16.5-month-old mice were infused subcutaneously with
either vehicle (veh, n=15)
or 50 mg/mL Compound 1 (n=15) by Alzet mini-pump (0.5 L/hour) for four weeks
with one replacement.
Data shown are mean s.e.m.
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Figure 5 depicts the effect of Compound 1 on neurogenesis in C57B1/6 mice by
detection of BrdU
positive cells from all sections in the dentate gyrus. The number of cells
positive for BrdU from the dentate
gyrus were plotted for each treatment group and the data subjected to an
unpaired t-test between the 16.5-
month-old groups. 3-month-old mice (n=17) received an infusion of vehicle
(veh) subcutaneously by Alzet
mini-pump (0.5 L/hour) for four weeks, with one replacement. 16.5-month-old
mice were infused
subcutaneously with either vehicle (veh, n=17) or 50 mg/mL Compound 1 (n=17)
by Alzet mini-pump
(0.5 L/hour) for four weeks with one replacement. Data shown are mean
s.e.m.; *P<0.05.
Figure 6 depicts the levels of Compound 1 detected in the CSF of C57BL/6 mice
in both young
(n=2) and old groups (n=3) (levels were both below 10 nM). Levels of Compound
1 were detected using
mass spectrometry.
Figure 7 depicts the distribution (measured as area under the curve (AUC)) of
Compound 1 in
C57BL/6J01aHsd mouse tissues over a time course. Compound was tagged with
carbon-14 label, and
measurements taken at 1, 24, and 168 hours.
Figure 8 depicts the pharmacokinetic profiles of Compound 1 after per os
(P.O.) dosing. Blood
plasma from male 2-month-old C57B1/6 mice receiving either 30 mg/kg or 150
mg/kg by oral gavage was
measured for Compound 1 at 20 minutes, 2 hours, 8 hours, and 12 hours after
administration. Plasma
concentration after drug administration was plotted over time.
Figure 9 depicts the effect of Compound 1 on exploratory preference during the
Open Field test in
young C57B1/6 mice. Exploratory preference was plotted as the ratio of time
spent in the periphery vs.
center and data was subjected to a one-way ANOVA. 2-month-old mice were
treated with: vehicle p.o.
b.i.d. (n=11); Compound 1 p.o. b.i.d. 30 mg/kg (n=12); vehicle p.o. b.i.d.
plus recombinant mouse CCL11
(eotaxin-1, or "rmE") i.p. (n=14); or Compound 1 p.o. b.i.d. 30 mg/kg plus
recombinant mouse CCL11
(n=14). Data shown are mean s.e.m.; "P<0.01.
Figure 10A depicts the effect of Compound 1 on the time spent in the novel
versus familiar arms
of the Y-maze test in young C57B1/6 mice. The time spent in the novel versus
familiar arms were plotted
for each treatment group and data was subjected to a paired t-test. 2-month-
old mice were treated with:
vehicle p.o. b.i.d. (n=13); Compound 1 p.o. b.i.d. 30 mg/kg (n=14); vehicle
p.o. b.i.d. plus recombinant
mouse CCL11 (eotaxin-1) i.p. (n=15); or Compound 1 p.o. b.i.d. 30 mg/kg plus
recombinant mouse CCL11
(n=15). Data shown are mean s.e.m.; "P<0.01.
Figure 10B depicts the effect of Compound 1 on the ratio of time spent in the
novel versus familiar
arm of the Y-maze test in young C57B1/6 mice. Ratios were plotted for each
treatment group and data was
subjected to an ANOVA Kruskal-Wallis test post-hoc. 2-month-old mice were
treated with: vehicle p.o.
b.i.d. (n=12); Compound 1 p.o. b.i.d. 30 mg/kg (n=14); vehicle p.o. b.i.d.
plus recombinant mouse CCL11
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(eotaxin-1) i.p. (n=15); or Compound 1 p.o. b.i.d. 30 mg/kg plus recombinant
mouse CCL11 (n=15). Data
shown are mean s.e.m.; *P<0.05.
Figure 10C depicts the effect of Compound 1 on the ratio of number of entries
made into the novel
versus familiar arm of the Y-maze test in young C57B1/6 mice. Ratios were
plotted for each treatment
group and data was subject to a t-test between vehicle and recombinant mouse
CCL11 treatment. 2-month-
old mice were treated with: vehicle p.o. b.i.d. (n=13); Compound 1 p.o. b.i.d.
30 mg/kg (n=14); vehicle p.o.
b.i.d. plus recombinant mouse CCL11 (eotaxin-1) i.p. (n=15); or Compound 1
p.o. b.i.d. 30 mg/kg plus
recombinant mouse CCL11 (n=15). Data shown are mean s.e.m.; *P<0.05.
Figure 11A depicts the effect of Compound 1 on the average latency to find the
target hole during
the Barnes Maze test in young C57B1/6 mice. Average latencies were plotted as
units of time for each
treatment group on each trial. 2-month-old mice were treated with: vehicle
p.o. b.i.d. (n=13); Compound
1 p.o. b.i.d. 30 mg/kg (n=14); vehicle p.o. b.i.d. plus recombinant mouse
CCL11 (eotaxin-1) i.p. (n=15); or
Compound 1 p.o. b.i.d. 30 mg/kg plus recombinant mouse CCL11 (n=15). Data
shown are mean s.e.m.
Figure 11B depicts the effect of Compound 1 on the latency to find the target
hole during the
Barnes Maze test in young C57B1/6 mice. Latencies averaged over the last three
trials for each treatment
group were plotted and data was subjected to an unpaired t-test. 2-month-old
mice were treated with:
vehicle p.o. b.i.d. (n=13); Compound 1 p.o. b.i.d. 30 mg/kg (n=14); vehicle
p.o. b.i.d. plus recombinant
mouse CCL11 (eotaxin-1) i.p. (n=15); or Compound 1 p.o. b.i.d. 30 mg/kg plus
recombinant mouse CCL11
(n=15). Data shown are mean s.e.m.; *P<0.05.
Figure 12A depicts the effect of Compound 1 on memory for the novel arm in the
Y maze, by
number of entries made into the novel arm over the total entries. 24 month old
mice were treated with
Compound 1 p.o. b.i.d. 30 mg/kg or vehicle. Data shown are mean s.e.m.;
Figure 12B depicts the effect of Compound 1 on total distance travelled in the
Y maze, as a measure
of locomotor activity. 24 month old mice were treated with Compound 1 p.o.
b.i.d. 30 mg/kg or vehicle.
Data shown are mean s.e.m.; *P<0.05.
Figure 13A depicts the effect of Compound 1 on bouts of entry ("number of
visits") into the novel
and familiar arms by C57B1/6 mice in the Y-maze test in 24-month-old mice. The
number of visits to each
arm was plotted for each treatment group and subjected to a paired t-test.
Mice 23 months-old were dosed
with either vehicle control or Compound 1 subcutaneously BID (twice daily) for
21 days. Three weeks
later, mice were subjected to Y-maze testing. All mice received 5 consecutive
days of BrdU injection at
50 mg/kg IP immediately prior to start of treatment. Data shown are mean
s.e.m.; *P<0.05, "P<0.01,
***P<0.001 novel vs familiar arm by paired student's t-test.
Figure 13B depicts the effect of Compound 1 on the ratio of number of entries
made into the novel
versus familiar arm of the Y-maze test in 24-month-old mice. Ratios were
plotted for each treatment group
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and data was subject to a t-test between vehicle and Compound 1 treatment.
Mice 23 months-old were
dosed with either vehicle control or Compound 1 subcutaneously BID (twice
daily) for 21 days. Three
weeks later, mice were subjected to Y-maze testing. All mice received 5
consecutive days of BrdU injection
at 50 mg/kg IP immediately prior to start of treatment. Data shown are mean
s.e.m.; *P<0.05, **P<0.01,
***P<0.001 compared to control by student's t-test.
Figure 13C depicts the effect of Compound 1 on the time spent in the novel
versus familiar arms
of the Y-maze test in 24-month-old C57B1/6 mice. The time spent in the novel
versus familiar arms were
plotted for each treatment group and data was subjected to a paired t-test.
Mice 23 months-old were dosed
with either vehicle control or Compound 1 subcutaneously BID (twice daily) for
21 days. Three weeks
later, mice were subjected to Y-maze testing. All mice received 5 consecutive
days of BrdU injection at
50 mg/kg IP immediately prior to start of treatment. Data shown are mean
s.e.m.; *P<0.05, **P<0.01,
***P<0.001 novel vs familiar arm by paired student's t-test.
Figure 13D depicts the effect of Compound 1 on the ratio of time spent
("duration") in the novel
versus familiar arm of the Y-maze test in 24-month-old C57B1/6 mice. Ratios
were plotted for each
treatment group and data was subjected to a t-test. Mice 23 months-old were
dosed with either vehicle
control or Compound 1 subcutaneously BID (twice daily) for 21 days. Three
weeks later, mice were
subjected to Y-maze testing. All mice received 5 consecutive days of BrdU
injection at 50 mg/kg IP
immediately prior to start of treatment. Data shown are mean s.e.m.;
*P<0.05, **P<0.01,
compared to control by student's t-test.
Figure 13E depicts the effect of Compound 1 on the average velocity during the
Y-Maze test of
24-month-old C57B1/6 mice. Average velocities were plotted for each treatment
group and data was
subject to a t-test. Mice 23 months-old were dosed with either vehicle control
or Compound 1
subcutaneously BID (twice daily) for 21 days. Three weeks later, mice were
subjected to Y-maze testing.
All mice received 5 consecutive days of BrdU injection at 50 mg/kg IP
immediately prior to start of
treatment. Data shown are mean s.e.m.; *P<0.05, **P<0.01, ***P<0.001
compared to control by
student's t-test.
Figure 14A depicts the effect of Compound 1 on average time taken by 24-month-
old C57B1/6
mice to find the target hole in the Barnes Maze test. Average times were
plotted for each treatment group.
Mice 23 months-old were dosed with either vehicle control or Compound 1
subcutaneously BID (twice
daily) for 21 days. Three weeks later, mice were subjected to Barnes Maze
testing. Data shown are mean
s.e.m.; *P<0.05 compared to control by student's t-test.
Figure 14B depicts the effect of Compound 1 on velocity in 24-month-old
C57B1/6 mice in Barnes
Maze test, averaged over all trials for each treatment group. Mice 23 months-
old were dosed with either
vehicle control or Compound 1 subcutaneously BID (twice daily) for 21 days.
Three weeks later, mice
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were subjected to Barnes Maze testing. Data shown are mean s.e.m.; *P<0.05
compared to control by
student's t-test.
Figure 15A depicts the effect of Compound 1 on distance traveled in the Open
Field test in 24-
month-old C57B1/6 mice. Average distance traveled was plotted for each
treatment group. Mice 23
months-old were dosed with either vehicle control or Compound 1 subcutaneously
BID (twice daily) for
21 days. Three weeks later, mice were subjected to Open Field testing. Data
shown are mean s.e.m.
Figure 15B depicts the effect of Compound 1 on velocity in the Open Field test
in 24-month-old
C57B1/6 mice. Average velocity of the mice was plotted for each treatment
group. Mice 23 months-old
were dosed with either vehicle control or Compound 1 subcutaneously BID (twice
daily) for 21 days. Three
weeks later, mice were subjected to Open Field testing. Data shown are mean
s.e.m.
Figure 16A depicts the effect of Compound 1 on TNFalpha cytokine levels in the
plasma of 24-
month-old C57B1/6 mice. Mice 23 months-old were dosed with either vehicle
control or Compound 1
subcutaneously BID (twice daily) for 21 days. Levels of the inflammatory
cytokine trend lower in
Compound 1 treated mice than in vehicle treated mice.
Figure 16B depicts the effect of Compound 1 on IL6 cytokine levels in the
plasma of 24-month-
old C57B1/6 mice. Mice 23 months-old were dosed with either vehicle control or
Compound 1
subcutaneously BID (twice daily) for 21 days. Levels of the inflammatory
cytokine trend lower in
Compound 1 treated mice than in vehicle treated mice.
Figure 16C depicts the effect of Compound 1 on IL lbeta cytokine levels in the
plasma of 24-
month-old C57B1/6 mice. Mice 23 months-old were dosed with either vehicle
control or Compound 1
subcutaneously BID (twice daily) for 21 days. Levels of the inflammatory
cytokine trend lower in
Compound 1 treated mice than in vehicle treated mice.
Figure 16D depicts the effect of Compound 1 on IL5 cytokine levels in the
plasma of 24-month-
old C57B1/6 mice. Mice 23 months-old were dosed with either vehicle control or
Compound 1
subcutaneously BID (twice daily) for 21 days. Levels of the inflammatory
cytokine trend lower in
Compound 1 treated mice than in vehicle treated mice.
Figure 16E depicts the effect of Compound 1 on IL17 cytokine levels in the
plasma of 24-month-
old C57B1/6 mice. Mice 23 months-old were dosed with either vehicle control or
Compound 1
subcutaneously BID (twice daily) for 21 days. Levels of the inflammatory
cytokine trend lower in
Compound 1 treated mice than in vehicle treated mice.
Figure 17 depicts the effect of Compound 1 on activated microglia in 24-month-
old C57B1/6 mice.
Mice 23 months-old were dosed with either vehicle control or Compound 1
subcutaneously BID (twice
daily) for 21 days. Levels of CD68+ activated microglia trend lower in
Compound 1 treated mice than in
vehicle treated mice.
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Figure 18 depicts the effect of Compound 1 on the percentage of eosinophils in
complete white
blood cell (WBC) count, in 24-month-old C57BI/6 mice treated with control
saline or 30 mg/kg of
Compound 1 for 3 weeks BID, SQ. Compound 1 decreased an endogenous age-related
increase in blood
eosinophils.
Figure 19 depicts the effect of Compound 1 on the percentage of eosinophils in
complete white
blood cell (WBC) count, in 3-month-old hairless mice treated with control
saline or 30 mg/kg of Compound
1 for 2 weeks BID, PO. Compound 1 decreased an oxazolone-induced increase in
blood eosinophils.
Figure 20 shows the results of Compound 1 on a rotarod test for motor
coordination. Twenty-four-
month-old C57 mice were treated for 4 weeks with continuous infusion of
Compound 1 or vehicle by
osmotic pump. Treated mice succeeded more than vehicle-treated mice in a
binomial test, *P < 0.05.
Figure 21 shows the results of Compound 1 on the T maze test for memory.
Twenty-four-month-
old C57 mice were treated for 4 weeks with continuous infusion of Compound 1
or vehicle by osmotic
pump. Treated mice succeeded more than vehicle-treated mice in a binomial
test, *P < 0.05.
Figure 22A shows the results of Compound 1 on fecal output overnight. Twenty-
four-month-old
C57 mice were treated for 4 weeks with continuous infusion of Compound 1 or
vehicle by osmotic pump.
The weight of fecal pellets was measured overnight. Compound 1 treated mice
had significantly lower fecal
output compared to vehicle treated mice by student's t-test, *P < 0.05.
Figure 22B shows the results of Compound 1 on water drinking overnight. Twenty-
four-month-
old C57 mice were treated for 4 weeks with continuous infusion of Compound 1
or vehicle by osmotic
pump. The total water drank was measured overnight. Compound 1 treated mice
drank significantly more
water compared to vehicle treated mice by student's t-test, *P < 0.05.
Figure 22C shows the results of Compound 1 on food intake overnight. Twenty-
four-month-old
C57 mice were treated for 4 weeks with continuous infusion of Compound 1 or
vehicle by osmotic pump.
The total food eaten was measured overnight. There were no differences in
total food intake overnight
between the two groups.
Figure 23 depicts the effect of Compound 1 (Cmpd 1) on the numbers of CD68-
positive (CD68+)
activated microglia within the brains of three-month-old mice treated with LPS
and treated with Compound
1 for 18 days. Compound 1-treated mice exhibited decreased CD68+
immunoreactivity, and thus decreased
gliosis.
Figures 24A and 24B depict the effect of Compound 1 on anxiety in the Open
Field test in three-
month-old mice treated with LPS IP for 4 weeks and treated with Compound 1 per
orally BID (twice daily)
for 1 week. LPS treatment increased the preference for the periphery of the
Open Field, indicating increased
anxiety. Compound 1 treatment decreased the LPS-induced anxiety in the Open
Field. Data shown are mean
s.e.m; *P < 0.05 by student's t-test.
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Figures 25A and 25B depict the effect of Compound 1 on number of entries into
the novel and
familiar arms in the Y-maze test by 3-month-old C57B1/6 mice treated with LPS.
The number of visits to
each arm was plotted for each treatment group and subjected to a paired t-
test. Mice 3 months-old were
dosed with either vehicle or LPS IP for 6 weeks and dosed with vehicle control
or Compound 1 per orally
BID (twice daily) for 3 weeks. Compound 1-treated mice showed a significant
preference for the novel arm
while vehicle-treated mice did not. Data shown are mean s.e.m.; **P<0.01,
*P<0.05, novel vs familiar
arm by paired student's t-test.
Figures 26A and 26B depict the effect of Compound 1 on ILlbeta mRNA in the
brains from 3-
month-old C57B1/6 mice treated with LPS and/or Compound 1. Mice were dosed
with either vehicle
control or LPS IP for 7 weeks and dosed with vehicle or Compound 1 per orally
BID (twice daily) for 4
weeks. Tissues were harvested, and RNA was prepared from cortical brain
tissues. Levels of ILlbeta
mRNA were measured by qPCR and data are presented relative to vehicle control.
There was a trend
towards increased levels of ILlbeta mRNA with LPS treatment and a significant
decrease with Compound
1 treatment. Data shown are mean s.e.m; *P < 0.05 by student's t-test.
Figures 27A, 27B and 27C depict the effect of Compound 1 on microglia
activation in the
hippocampus from 3-month-old C57B1/6 mice treated with LPS and/or Compound 1.
Mice were dosed
with either vehicle control or LPS IP for 7 weeks and dosed with vehicle or
Compound 1 per orally BID
(twice daily) for 4 weeks. Tissues were harvested, and brain sections were
subjected to
immunohistochemistry for CD68, a marker for activated microglia. There was a
trend towards increased
levels of CD68 with LPS treatment and a robust trend towards decrease with
Compound 1 treatment. Data
shown are mean s.e.m.
Figures 28A, 28B and 28C depict the effect of Compound 1 on total microglia in
the hippocampus
from 3-month-old C57B1/6 mice treated with LPS and/or Compound 1. Mice were
dosed with either
vehicle control or LPS IP for 7 weeks and dosed with vehicle or Compound 1 per
orally BID (twice daily)
for 4 weeks. Tissues were harvested, and brain sections were subjected to
immunohistochemistry for Ibal,
a marker for microglia. There was a significant increase in Ibal with LPS
treatment and a trend towards
decrease with Compound 1 treatment. Data shown are mean s.e.m.; ***P<0.001,
*P<0.05, by student's
t-test.
Figures 29A and 29B depict the effect of Compound 1 on total astrocytes in the
hippocampus
from 3-month-old C57B1/6 mice treated with LPS and/or Compound 1. Mice were
dosed with either
vehicle control or LPS IP for 7 weeks and dosed with vehicle or Compound 1 per
orally BID (twice daily)
for 4 weeks. Tissues were harvested, and brain sections were subjected to
immunohistochemistry for GFAP,
a marker for astrocytes. There was a trend towards decrease with Compound 1
treatment. Data shown are
mean s.e.m.
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Figure 30 depicts the dosing regimen for three groups of C57BL/6 mice treated
with: (1) controls
for both LPS and Compound 1; (2) LPS plus vehicle control for Compound 1; or
(3) LPS plus Compound
1. All three groups were treated with controls, LPS, or Compound 1 for three
consecutive, simultaneous
days. The graph shows the results of histological analysis of the mouse brains
in an acute model of LPS-
induced inflammation. Microgliosis was measured by determining the percentage
of Iba-1 positive area in
the hippocampi. Data shown are mean s.e.m; *P< 0.05, ***P< 0.001; ordinary
one-way ANOVA was
used to test for statistical significance, with Dunnett's multiple comparisons
test post-hoc between treatment
groups.
Figure 31 depicts the dosing regimen for three groups of C57BL/6 mice treated
with: vehicle
controls; vehicle treated LPS; or Compound 1. The graph shows the results of
histological analysis of the
mouse brains in an acute model of LPS-induced inflammation. Microgliosis was
measured by determining
the percentage of Iba-1 positive area in the hippocampi. Data shown are mean
s.e.m; *P< 0.05, ****P<
0.0001; ordinary one-way ANOVA was used to test for statistical significance,
with Dunnett's multiple
comparisons test post-hoc between treatment groups.
Figure 32 depicts the treatment study timeline for a mouse MPTP model of
Parkinson's Disease.
There were two arms of the study; the first arm tested gait and fine kinematic
motor function after 10 days
of treatment, while the second arm tested immune cell infiltration after 3
days of treatment.
Figure 33A displays a correlation heat map describing the degree of
correlation for ninety-seven
walking parameters in a gait and fine motor kinematic test of C57B1/6J mice
after ten days of Compound 1
treatment on study day 11. Ten principal components are presented, showing how
the original parameters
are correlated in datasets. The more intensity there is for each parameter,
the more strongly the parameter
is implicated in the corresponding principal component. Red is a positive
correlation and blue is a negative
correlation in relation to the 10 individual principal components (x-axis).
The left-side y-axis shows overall
groupings for the individual variables on the right-side y-axis. E.g., "ILC"
refers to intralimb coordination,
"Tail B" refers to tail base.
Figure 33B shows the fine motor skills and gait properties of C57B1/6J mice at
study day 11 after
days of Compound 1 treatment. It is illustrated as an overall gait analysis
score of MPTP treated
C57B1/6J mice. Differences between treatment groups in each of the 10
principal components were
combined in a composite score, and difference to vehicle-treated control group
is shown. There was a
significant difference in overall gait with MPTP treatment (* p < 0.05), and
there was no longer a significant
difference with Compound 1 treatment.
Figure 34 depicts forepaw toe clearance (one of the gait properties assessed
in the principle
component analysis) of C57B1/6J mice at study day 11 after 10 days of Compound
1 treatment. C57B1/6J
mice at study day 11 after 10 days of Compound 1 treatment. Data are presented
as mean + SEM (Group
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1: Vehicle + Vehicle, n = 15; Group 2: MPTP + Vehicle, n = 14; Group 3: MPTP +
Compound 1 (30
mg/kg), n = 13). Statistical significances: * p <0.05, Group 2: MPTP + Vehicle
vs. Group 1: Vehicle +
Vehicle (unpaired t-test).
Figure 35 depicts forepaw swing speed (one of the gait properties assessed in
the principle
component analysis) of C57B1/6J mice at study day 11 after 10 days of Compound
1 treatment. Data are
presented as mean + SEM (Group 1: Vehicle + Vehicle, n = 15; Group 2: MPTP +
Vehicle, n = 14; Group
3: MPTP + Compound 1 (30 mg/kg), n = 13). Statistical significances: * p
<0.05, Group 2: MPTP + Vehicle
vs. Group 1: Vehicle + Vehicle (unpaired t-test).
Figure 36 depicts ankle range of motion (one of the gait properties assessed
in the principle
component analysis) of C57B1/6J mice at study day 11 after 10 days of Compound
1 treatment. Data are
presented as mean + SEM (Group 1: Vehicle + Vehicle, n = 15; Group 2: MPTP +
Vehicle, n = 14; Group
3: MPTP + Compound 1 (30 mg/kg), n = 13). Statistical significances: * p
<0.05, Group 2: MPTP + Vehicle
vs. Group 1: Vehicle + Vehicle (unpaired t-test).
Figure 37 reports the acute effects of MPTP and Compound 1 on T-cell
trafficking into the brain
after 3 days of Compound 1 treatment. The total number of CD3 positive T-cells
counted in the substantia
nigra from 3 sections of 30 tim for each mouse are presented. Data shown are
mean s.e.m; ***P< 0.001;
one-way ANOVA, Sidak's multiple comparison test post-hoc.
Figures 38A and 38B report the acute effects of MPTP and Compound 1 on
microgliosis after 3
days of Compound 1 treatment. Figure 38A presents the degree of CD68-positive
area measured in the
striatum (n=7 for saline + vehicle; n=8 for MPTP + vehicle; n=7 for MPTP +
Compound 1). Figure 38B
presents the degree of CD68-positive area measured in the substantia nigra
pars compacta (n=7 for saline
+ vehicle; n=8 for MPTP + vehicle; n=8 for MPTP + Compound 1). Data shown are
mean s.e.m; **P<
0.01, ***P< 0.001, ****P< 0.0001; one-way ANOVA, Sidak's multiple comparison
test post-hoc.
Figure 39 depicts plasma eotaxin-1 levels in 6-month-old Line 61 synuclein-
overexpres sing
transgenic mice. Eotaxin-1 levels (CCL11) in Non-transgenic (NTg) versus
transgenic Line 61 synuclein
mice (Tg) were plotted at pg/mL concentrations.
Figure 40 reports the results from a wire suspension test on Line 61 synuclein
mice (transgenic
(Tg) and non-transgenic (nTg) aged-matched littermates.) The mean wire
suspension times per group are
shown, with animals from group C (non-transgenic, vehicle-treated) exhibiting
a significantly higher wire
suspension time. Animals from group A (transgenic, Compound-1 treated)
exhibited a significantly higher
wire suspension time compared to group B (transgenic, vehicle-treated). Data
are displayed as mean +
SEM of all animals per group; ***P<0.001; Dunn's post-test; *P<0.05 Mann
Whitney test for group A vs.
group B).
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Figure 41 reports the results from a grip strength test on Line 61 synuclein
mice (transgenic (Tg)
and non-transgenic (nTg) aged-matched littermates.) The mean maximum grip
force [g] per group is
shown. Animals from group A and C (transgenic, Compound 1-treated and non-
transgenic, vehicle-treated,
respectively) showed a significantly higher grip force compared to group B
(transgenic, vehicle-treated).
Data are displayed as mean + SEM of all animals per group. Groups were
compared to vehicle-treated
transgenic animals (group B); one-way ANOVA followed by Bonferroni's post-
test.
Figure 42 reports the number of mice from each treatment group that were
successfully able to
traverse the beam in a beam walk test. N = 15 mice in Group A, 14 mice in
Group B, and 15 mice in Group
C (groups described in Figure 40). All mice were able to traverse the easiest
beam in Trial 1, whereas no
mice from Treatment Group B were able to traverse the most difficult beam in
Trial 5. (Trial 1 = 13mm
rectangular beam; Trial 2 = 10 mm rectangular beam; Trial 3 = 28 mm
cylindrical beam; Trial 4 = 16 mm
cylindrical beam; Trial 5 = 11 mm cylindrical beam).
Figure 43 reports the results of five trials for beam walk slips for three
groups of mice (group A,
transgenic Compound 1-treated; group B, transgenic vehicle-treated; and group
C, non-transgenic vehicle-
treated). Only mice that traversed the beam entirely were included in the
analysis. Graphs represent the
mean number of slips [n] per group, and each graph represents one trial (1-5).
Data are mean + SEM of all
animals per group. Groups were compared to group B; one-way ANOVA followed by
Bonferroni's post-
test. Statistics for Trial 5 could not be performed, as no mice from Group B
were able to traverse the beam.
Figures 44A and 44B report eosinophil count from peripheral blood. Figure 44A
reports the
percentage of eosinophils (of all white blood cells) in peripheral blood from
three groups of mice (Tg Cmpd
1 = transgenic Compound 1-treated; Tg Veh = transgenic vehicle-treated; nTG
Veh = non-transgenic
vehicle-treated). Data were compared by t-test. Figure 44B reports the
absolute eosinophil count in
peripheral blood from three groups of mice (Tg Cmpd 1 = transgenic Compound 1-
treated; Tg Veh =
transgenic vehicle-treated; nTG Veh = non-transgenic vehicle-treated). Data
were compared by t-test.
Figures 45A to 45G report the effect of Compound 1 on neuroinflammation.
Figure 45A reports
the CD68 positive area quantified in the hippocampus of: non-transgenic,
vehicle-treated mice; transgenic,
vehicle treated mice; and transgenic, Compound 1-treated mice (n=14, 12,
and15, respectively). Figure
45B reports the CD68 positive area quantified in the striatum of: non-
transgenic, vehicle-treated mice;
transgenic, vehicle treated mice; and transgenic, Compound 1-treated mice
(n=15, 11, and 16, respectively).
Figure 45C reports the Iba-1 positive area quantified in the hippocampus of:
non-transgenic, vehicle-
treated mice; transgenic, vehicle treated mice; and transgenic, Compound 1-
treated mice (n=14, 13, and 16,
respectively). Figure 45D reports the Ibal positive area quantified in the
striatum of: non-transgenic,
vehicle-treated mice; transgenic, vehicle treated mice; and transgenic,
Compound 1-treated mice (n=15, 11,
and 16, respectively). Data are mean +/- s.e.m.; *P<0.05. Figure 45E reports
the GFAP positive area
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quantified in the hippocampus of: non-transgenic, vehicle-treated mice;
transgenic, vehicle treated mice;
and transgenic, Compound 1-treated mice (n=14, 13, and 15, respectively).
Figure 45F reports the GFAP
positive area quantified in the striatum of: non-transgenic, vehicle-treated
mice; transgenic, vehicle treated
mice; and transgenic, Compound 1-treated mice (n=15, 13, and 15,
respectively). Figure 45G reports the
Iba-lpositive area quantified in the pars compacta of the substantia nigra of
non-transgenic, vehicle-treated
mice; transgenic, vehicle treated mice; and transgenic, Compound 1-treated
mice. Data are mean +/- s.e.m.;
*P<0.05.
Figures 46A and 46B report the effect of Compound 1 on circulating levels of
IL-4 and IL-6
cytokines in: non-transgenic, vehicle-treated mice; transgenic, vehicle
treated mice; and transgenic,
Compound 1-treated mice (n=14, 15, and 17, respectively). Figure 46A reports
the levels of IL-4 measured
in terminal cardiac plasma of all three groups. Figure 46B reports the levels
of IL-6 measured in terminal
cardiac plasma of all three groups. Data shown are mean +/- s.e.m; *P<0.05,
**13<0.01; one-way ANOVA,
Dunnett's multiple comparison test post-hoc.
Figures 47A to 47D show the effects of Compound 1 on EAE-induced markers in
the cerebella of
C57BL/6 mice. EAE resulted in an increase in CD3 and CD8-positive infiltrating
T-cells in the cerebellum.
Figure 47A shows an increase in CD3 positive infiltrating T-cells in the
cerebellum which is significantly
reduced following treatment with Compound 1 for 9 days. Figure 47B shows an
increase in CD8 positive
infiltrating cytotoxic T-cells in the cerebellum which is significantly
reduced following treatment with
Compound 1 for 9 days. Figure 47C shows a significant increase Iba-1 positive
area in the cerebellum after
EAE, which was significantly reduced in the cerebellum after 9 days of
treatment. Figure 47D shows a
significant increase CD68 positive area in the cerebellum after EAE, which was
significantly reduced in
the cerebellum after 9 days of treatment. Data shown are mean +/- s.e.m;
*P<0.05, **13<0.01; one-way
ANOVA, Dunnett's multiple comparison test post-hoc.
Figure 48 depicts the concentrations of human eotaxin-1 in a proteomic screen.
Relative
concentrations of human eotaxin-1 were measured in a commercially-available
affinity-based assay
(SomaLogic). Plasma samples from each of 18, 30, 45, 55, and 66-year-old
donors were plotted.
Figure 49A depicts the effect of Compound 1 on inhibition of eosinophil shape
change. Whole
blood from humans treated with Compound 1 was incubated with recombinant
eotaxin to trigger eosinophil
shape change. Inhibition of shape change was plotted against plasma Compound 1
concentrations.
Figure 49B depicts the effect of Compound 1 on CCR3 internalization. Whole
blood from humans
treated with Compound 1 was incubated with recombinant eotaxin to trigger CCR3
internalization and
labeled with anti-CCR3 antibody. Inhibition of CCR3 internalization by
Compound 1 was plotted against
plasma Compound 1 concentrations.
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VI. Detailed Description
Aspects of the invention include methods of treating aging-associated
impairments/neurodegenerative diseases. The aging-associated impairment may
manifest in a number of
different ways, e.g., as aging-associated cognitive impairment and/or
physiological impairment, e.g., in the
form of damage to central or peripheral organs of the body, such as but not
limited to: cell injury, tissue
damage, organ dysfunction, aging-associated lifespan shortening and
carcinogenesis, where specific organs
and tissues of interest include, but are not limited to skin, neuron, muscle,
pancreas, brain, kidney, lung,
stomach, intestine, spleen, heart, adipose tissue, testes, ovary, uterus,
liver and bone; in the form of
decreased neurogenesis, etc.
In some embodiments, the aging-associated impairment is an aging-associated
impairment in
cognitive ability in an individual, i.e., an aging-associated cognitive
impairment. By cognitive ability, or
"cognition", it is meant the mental processes that include attention and
concentration, learning complex
tasks and concepts, memory (acquiring, retaining, and retrieving new
information in the short and/or long
term), information processing (dealing with information gathered by the five
senses), visuospatial function
(visual perception, depth perception, using mental imagery, copying drawings,
constructing objects or
shapes), producing and understanding language, verbal fluency (word-finding),
solving problems, making
decisions, and executive functions (planning and prioritizing). By "cognitive
decline", it is meant a
progressive decrease in one or more of these abilities, e.g., a decline in
memory, language, thinking,
judgment, etc. By "an impairment in cognitive ability" and "cognitive
impairment", it is meant a reduction
in cognitive ability relative to a healthy individual, e.g., an age-matched
healthy individual, or relative to
the ability of the individual at an earlier point in time, e.g., 2 weeks, 1
month, 2 months, 3 months, 6 months,
1 year, 2 years, 5 years, or 10 years or more previously. Aging-associated
cognitive impairments include
impairments in cognitive ability that are typically associated with aging,
including, for example, cognitive
impairment associated with the natural aging process, e.g., mild cognitive
impairment (M.C.I.); and
cognitive impairment associated with an aging-associated disorder, that is, a
disorder that is seen with
increasing frequency with increasing senescence, e.g., a neurodegenerative
condition such as Alzheimer' s
disease, Parkinson's disease, Dementia with Lewy Bodies, frontotemporal
dementia, Huntington's disease,
amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic
dystrophy, vascular dementia, and
the like.
By "treatment" it is meant that at least an amelioration of one or more
symptoms associated with
an aging-associated impairment afflicting the subject is achieved, where
amelioration is used in a broad
sense to refer to at least a reduction in the magnitude of a parameter, e.g.,
a symptom associated with the
impairment being treated. As such, treatment also includes situations where a
pathological condition, or at
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least symptoms associated therewith, are completely inhibited, e.g., prevented
from happening, or stopped,
e.g., terminated, such that the adult mammal no longer suffers from the
impairment, or at least the symptoms
that characterize the impairment. In some instances, "treatment", "treating"
and the like refer to obtaining a
desired pharmacologic and/or physiologic effect. The effect may be
prophylactic in terms of completely
or partially preventing a disease or symptom thereof and/or may be therapeutic
in terms of a partial or
complete cure for a disease and/or adverse effect attributable to the disease.
"Treatment" may be any
treatment of a disease in a subject, and includes: (a) preventing the disease
from occurring in a subject
which may be predisposed to the disease but has not yet been diagnosed as
having it; (b) inhibiting the
disease, i.e., arresting its development; or (c) relieving the disease, i.e.,
causing regression of the disease.
Treatment may result in a variety of different physical manifestations, e.g.,
modulation in gene expression,
increased neurogenesis, rejuvenation of tissue or organs, etc. Treatment of
ongoing disease, where the
treatment stabilizes or reduces the undesirable clinical symptoms of the
patient, occurs in some
embodiments. Such treatment may be performed prior to complete loss of
function in the affected tissues.
The subject therapy may be administered during the symptomatic stage of the
disease, and in some cases
after the symptomatic stage of the disease.
In some instances where the aging-associated impairment is aging-associated
cognitive decline,
treatment by methods of the present disclosure slows, or reduces, the
progression of aging-associated
cognitive decline. In other words, cognitive abilities in the individual
decline more slowly, if at all,
following treatment by the disclosed methods than prior to or in the absence
of treatment by the disclosed
methods. In some instances, treatment by methods of the present disclosure
stabilizes the cognitive abilities
of an individual. For example, the progression of cognitive decline in an
individual suffering from aging-
associated cognitive decline is halted following treatment by the disclosed
methods. As another example,
cognitive decline in an individual, e.g., an individual 40 years old or older,
that is projected to suffer from
aging-associated cognitive decline, is prevented following treatment by the
disclosed methods. In other
words, no (further) cognitive impairment is observed. In some instances,
treatment by methods of the
present disclosure reduces, or reverses, cognitive impairment, e.g., as
observed by improving cognitive
abilities in an individual suffering from aging-associated cognitive decline.
In other words, the cognitive
abilities of the individual suffering from aging-associated cognitive decline
following treatment by the
disclosed methods are better than they were prior to treatment by the
disclosed methods, i.e., they improve
upon treatment. In some instances, treatment by methods of the present
disclosure abrogates cognitive
impairment. In other words, the cognitive abilities of the individual
suffering from aging-associated
cognitive decline are restored, e.g., to their level when the individual was
about 40 years old or less,
following treatment by the disclosed methods, e.g., as evidenced by improved
cognitive abilities in an
individual suffering from aging-associated cognitive decline.
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In some instances where the aging-associated impairment is aging-associated
motor impairment or
decline, treatment by methods of the present disclosure slows, or reduces, the
progression of aging-
associated impairment or decline. In other words, motor abilities in the
individual decline more slowly, if
at all, following treatment by the disclosed methods than prior to or in the
absence of treatment by the
disclosed methods. In some instances, treatment by methods of the present
disclosure stabilizes the motor
abilities of an individual. For example, the progression of motor decline in
an individual suffering from
aging-associated motor decline is halted following treatment by the disclosed
methods. As another
example, motor decline in an individual, e.g., an individual 40 years old or
older, that is projected to suffer
from aging-associated motor decline, is prevented following treatment by the
disclosed methods. In other
words, no (further) motor impairment is observed. In some instances, treatment
by methods of the present
disclosure reduces, or reverses, motor impairment, e.g., as observed by
improving motor coordination or
abilities in an individual suffering from aging-associated motor decline. In
other words, the motor abilities
of the individual suffering from aging-associated motor decline following
treatment by the disclosed
methods are better than they were prior to treatment by the disclosed methods,
i.e., they improve upon
treatment. In some instances, treatment by methods of the present disclosure
abrogates motor impairment.
In other words, the motor coordination or abilities of the individual
suffering from aging-associated motor
decline are restored, e.g., to their level when the individual was about 40
years old or less, following
treatment by the disclosed methods, e.g., as evidenced by improved motor
coordination or abilities in an
individual suffering from aging-associated motor decline.
In some instances, treatment of an adult mammal in accordance with the methods
results in a change
in a central organ, e.g., a central nervous system organ, such as the brain,
spinal cord, etc., where the change
may manifest in a number of different ways, e.g., as described in greater
detail below, including but not
limited to molecular, structural and/or functional, e.g., in the form of
enhanced neurogenesis.
Methods of treating dysfunction caused by neurodegenerative disease are
provided, the method
comprising administering compounds from the formulae discussed below. An
embodiment of the invention
comprises a method of improving cognition or motor activity in subjects with
brain-associated, cognitive-
associated, or motor disease, the method comprising administering a
therapeutically effective amount of a
compound from the chemical formulae discussed below. Additional embodiments of
the invention
comprise a method of increasing neurogenesis in subjects with brain- or
cognitive-associated disease, the
method comprising administering a therapeutically effective amount of a
compound from the chemical
formulae discussed below. Additional embodiments of the invention comprise a
method of alleviating or
treating symptoms of brain- or cognitive-associated disease, the method
comprising administering a
therapeutically effective amount of a compound from the chemical formulae
below. Additional
embodiments of the invention include methods of alleviating symptoms of
central nervous system-
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associated disease, the method comprising administering a therapeutically
effective amount of a primarily
peripheral-acting agent from the chemical formulae below. Further additional
embodiments of the
invention include methods of improving motor activity in a subject with age-
related motor dysfunction, the
method comprising administering a therapeutically effective amount of a
compound from the chemical
formulae discussed below. The methods of the invention may also comprise
monitoring improvement in
age-related disease, including for example, improvement in cognition, motor
activity, neurogenesis and the
like in a subject diagnosed with one or more such disease or dysfunction.
Additional embodiments of the invention include administering a
therapeutically effective amount
of a compound wherein the compound is in the form of the co-crystals or salts
of the formulae discussed
below. Further embodiments of the invention include administering a
therapeutically effective amount of
a compound wherein the compound is in the form of individual optical isomers,
a mixture of the individual
enantiomers, a racemate or enantiomerically pure compounds. Additional
embodiments of the invention
also include administering a therapeutically effective amount of a compound
wherein the compound is in
the form of the pharmaceutical compositions and formulations further discussed
below.
Additional embodiments of the invention that treat aging-associated motor
impairment or decline
include modifying agents which inhibit the eotaxin/CCR3 pathway. Such
modifying agents include not
only compounds from the formulae discussed below, but other eotaxin and CCR3
inhibiting agents.
Modifying agents that are contemplated include by way of example and not of
limitation: the compounds
of the formulae discussed below and other CCR3 small molecule inhibitors (e.g.
bipiperdine derivatives
described for example in U.S. Patent No. 7,705,153, cyclic amine derivatives
described for example in U.S.
7,576,117, and the CCR3 antagonists described in Pease JE and Horuk R, Expert
Opin Drug Discov (2014)
9(5):467-83, all herein incorporated by reference in their entirety); anti-
eotaxin antibodies; anti-CCR3
antibodies; aptamers that inhibit either eotaxin or CCR3 expression or
function (methods of producing such
aptamers include U.S. Pat. Nos. 5,270,163, 5,840,867, 6,180,348); antisense
oligonucleotides or siRNAs
that inhibit expression or function of either eotaxin or CCR3 (e.g. described
in U.S. Pat. No. 6,822,087);
soluble CCR3 receptor protein (e.g. decoys); and the like.
Methods of using a diagnostic test or companion diagnostic test in connection
with the described
aging-associated impairments are also provided. One embodiment of such
diagnostic or companion
diagnostic test is an in vitro diagnostic test. An embodiment of the in vitro
diagnostic test is a companion
device used with a particular therapeutic. Embodiments of the particular
therapeutic include for example
and not limitation a CCR3 small molecular inhibitor, an anti-CCR3 antibody,
small molecule inhibitors to
the CCR3 ligand Eotaxin-1, an anti-Eotaxin-1 antibody, and antisense RNA to
either Eotaxin-1 or CCR3.
By way of example and not limitation, such diagnostics or companion
diagnostics include
determination or detection of the presence of a subset of white blood cells
from a subject. The diagnostics
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or companion diagnostics may also be the determination of presence, relative
or absolute concentration,
relative or absolute number of eosinophils from a subject's blood, tissues, or
other such samples. Blood
may be obtained for example, by venipuncture or other similar methods. Other
samples may include by
way of example and not limitation, sputum, cerebrospinal fluid or tissue
biopsies.
Methods of detecting or determining the presence, absolute or relative
concentration, or relative or
absolute number of white blood cells such as eosinophils, neutrophils,
lymphocytes, basophils, and
monocytes are known to those having ordinary skill in the art. Methods to
determine the presence, absolute
or relative concentration, or relative or absolute number of eosinophils are
also known to those having
ordinary skill in the art. See Walsh GM, Eosinophils¨Methods and Protocols
ISBN:9781493910151,
which is herein incorporated by reference in its entirety. Such methods
include, by way of example and not
limitation: (1) complete blood count with 5-part differential using a
hematology analyzer (See, e.g. O'Neil
et al., Laboratory Hematology 7:116-124 (2001) and the U.S. Centers for
Disease Control laboratory
methods (available at https://wwwn.cdc.govinchs/data/nhanes/2013-
2014/1abmethods/CBC_H_MET_
COMPLETE_BLOOD_COUNT.pdf); (2) quantitative eosinophil count in sputum using
smear preparation
stained by hematoxylin and eosin (H and E) (See, e.g., Bandyopadhyay A, et al.
Lung India. 2013 Apr-Jun;
30(2): 117-123); (3) flow cytometry quantification of eosinophils using
antibodies specific to various
immune cells such as CD45, CD125, CD193, F4/80, and Siglec-8 (Abcam, San
Francisco, CA) ((See, e.g.,
Yu Y-R A et al., Am J Respir Cell Mol Biol. 2016 Jan; 54(1): 13-24 and
Cossarizza A, et al., Eur. J.
Immunol. 2017 47:1584-1797); (4) eosinophil autofluorescence quantified using
flow cytometry (See, e.g.
Guenther G et al., J Immunol May 1, 2015, 194 (1 Supplement) 206.12 and Thurau
AM et al., Cytometry
23:159-58 (1996)); (5) Isolation of eosinophils from whole blood using Dextran
70, Ficoll-Paque and
antibody-based magnetic colloid cell separation (See, e.g. Munoz NM, et al.,
Nature Protocols, 2613-20
(2006); Akuthota P, et al., Curr Protoc Immunol 98(1): pp. 7.31.1-7.31.8,
2012; and Akuthota P, et al.,
Methods Mol Biol 1178:13-20 (2014)); (6) Histological staining by general
stain (eosin or similar dye) or
by antibody-based stain against eosinophil-specific protein (Major Basic
Protein (MBP), Eosinophil
Cationic Protein (ECP), Eosinophil Peroxidase (EPO), Eosinophil Derived
Neurotoxin (EDN), SiglecF (or
5ig1ec8) among others) (See, e.g. Koller DY et al., Allergy 54(10):1094-9
(1999) and Akuthota P., Capron
K., Weller P.F. (2014) Eosinophil Purification from Peripheral Blood. In:
Walsh G. (eds) Eosinophils.
Methods in Molecular Biology (Methods and Protocols), vol 1178. Humana Press,
New York, NY); (7)
Histological staining of processes associated spatially with eosinophils such
as degranulation, Eosinophil
Extracellular Traps (EETs) and/or specific proteins among those (Galectin 10
for example).
The diagnostic or companion diagnostic devices may incorporate such methods to
determine the
presence, absolute or relative concentration, or relative or absolute number
of eosinophils. By determining
the he presence, absolute or relative concentration, or relative or absolute
number of eosinophils, the devices
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may be used with other methods of the invention. By way of example and not
limitation, such other
methods may include the methods of treating aging-associated
impairments/neurodegenerative disease
recited herein. In some embodiments, the aging-associated impairment is an
aging-associated impairment
in cognitive ability in an individual, i.e., an aging-associated impairment.
Such aging-associated
impairments may include by way of example and not limitation a
neurodegenerative condition such as
Alzheimer's disease, Parkinson's disease, Dementia with Lewy Bodies,
frontotemporal dementia,
Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,
glaucoma, myotonic dystrophy,
vascular dementia, and the like. Such aging-associated impairments can include
cognitive impairments
and/or motor impairment or decline.
An embodiment of the invention includes treating a subject diagnosed with an
aging-associated
impairment with a therapeutically effective amount of at least one of the
compounds of the invention
disclosed herein in conjunction with a diagnostic or companion diagnostic
device. Another embodiment of
the invention includes treating said subject with a therapeutically effective
amount of Compound 1
disclosed herein in conjunction with a diagnostic device. Said embodiments of
the devices may be used
for example to determine or detect the eosinophil presence, absolute or
relative concentration, or absolute
or relative number in a blood or tissue sample of a subject. Another
embodiment performs a step of
detecting or determining the eosinophil presence, absolute or relative
concentration, or absolute or relative
number in a blood or tissue sample of a subject before, during, or after
treatment.
The experimental figures herein report that the Parkinson's Disease mammalian
model of synuclein
overexpression results in decreased eosinophils, which are restored to levels
in non-transgenic mice with
Compound 1 treatment, suggesting beneficial immune modulation in this model of
Parkinson's disease.
This also suggests that determining eosinophil levels in Parkinson's Disease
patients treated with
Compound 1 can be a biomarker for the disease, including determining the level
of progression, stasis, or
regression of the disease, as well as treatment efficacy. (See, e.g. Figures
44A and 44B).
Another embodiment performs the step of detecting or determining the
eosinophil presence,
absolute or relative concentration, or absolute or relative number in a blood
or tissue sample of a subject
diagnosed with Parkinson's Disease before, during, or after treatment. A
further embodiment comprises
determining the presence, absolute or relative concentration, or absolute or
relative number in a blood or
tissue sample of a subject diagnosed with Parkinson's Disease before being
treated with a compound of the
invention such as Compound 1 in order to obtain a baseline concentration or
number. A further embodiment
subsequently performs a step of detecting or determining levels of eosinophils
after treatment in order to
compare such levels to the baseline concentration or number of eosinophils so
as to monitor the efficacy of
treatment. Comparison of such levels may show an increase or decrease in the
number or concentration of
eosinophils compared to baseline. Another embodiment of the invention
comprises a step of monitoring
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the progress of the disease or impairment by comparing the number or
concentration of eosinophils, where
if the number is increased after treatment, there is improvement in the
progression of the disease. In another
embodiment, improvement may comprise a 1-5% increase over baseline in the
number or concentration of
eosinophils in a subject's blood or tissue, a 5-10% increase; a 11-15%
increase; a 16-20% increase; a 21-
25% increase; a 26-30% increase; a 31-35% increase; a 36-40% increase; a 41-
45% increase; 46-50%
increase; a 51-55% increase; a 56-60% increase; a 61-65% a 66-70% increase; a
71-75% increase; a 76-
80% increase; an 81-85% increase; an 86-90% increase; a 91-95% increase; a 96-
100% increase; and
increases of 1-1.5x, 1.5-2x, 2-2.5x, 2.5-3x, 3-3.5x, 3.5-4x, 4-4.5x, 4.5-5x, 5-
5.5x, 5.5-6x, 6-6.5x, 6.5-7x,
7-7.5x, 7.5-8x, 8-8.5x, 8.5-9x, 9-9.5x, 9.5-10x, and greater than 10x.
Another embodiment of the invention includes diagnosing Parkinson's Disease by
determining the
baseline in the number or concentration of eosinophils in a subject's blood or
tissue and comparing that to
standard numbers or concentrations of eosinophils in a population without
Parkinson's Disease who have
normal eosinophil counts. It is known in the art that an eosinophil count is
the number of eosinophils in
the body. For adults, a normal eosinophil count is between 30 and 350 but can
be up to 500 cubic
millimeters (mm3) in the blood. (See Medical News Today, Dec 2018 available
at:
https://www.medicalnewstoday.com/articles/323868.php which is herein
incorporated by reference in its
entirety). A count of more than 500 mm' in the blood is considered
eosinophilia. Lower than normal
counts of eosinophils occur in some diseases such as alcoholism and
overprotection of cortisol. (Id.) An
aspect of the invention detects or determines the numbers or concentrations of
eosinophils in a subject
suspected of having Parkinson's Disease. If the numbers or concentrations of
eosinophils in the subject is
lower than normal (e.g. 30 mm' in the blood), then the result may be used by a
care-giver to determine the
diagnosis of the disease.
a. Compounds
The methods of the invention further comprise administration to a subject of
the compounds that
follow. In the groups, radicals, or moieties defined in this "Compounds"
section, the number of carbon
atoms is often specified preceding the group, for example, C16 alkyl means an
alkyl group or radical having
1 to 6 carbon atoms. In general, for groups comprising two or more subgroups
which are disclosed in this
"Compounds" section, the last-named group is the radical attachment point, for
example, "thioalkyl" means
a monovalent radical of the formula HS-Alk-. Unless otherwise specified below,
conventional definitions
of terms control and conventional stable atom valences are presumed and
achieved in all formulas and
groups.
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An embodiment of the invention further comprises administration to a subject
of the compounds
of formula 1, wherein
A
ONF\ I
0
R4
I,
1
A is CH2, 0 or N-C1 6-alkyl;
121 is selected from
= NHR11, NMeR1
= NHIV 2, NMeR1 2;
= NHCH2-R1 3;
= NH-C3 6-cycloalkyl, whereas optionally one carbon atom is replaced by a
nitrogen atom,
whereas the ring is optionally substituted with one or two residues selected
from the group
consisting of Cl 6-alkyl, 0-Ci 6-alkyl, NHS02-phenyl, NHCONH-phenyl, halogen,
CN,
S02-C16-alkyl, COO-C16-alkyl;
= a C9 or 10-bicyclic-ring, whereas one or two carbon atoms are replaced by
nitrogen atoms and
the ring system is bound via a nitrogen atom to the basic structure of formula
1 and whereas
the ring system is optionally substituted with one or two residues selected
from the group
consisting of Cl 6-alkyl, COO-C16-alkyl, C1 6-haloalkyl, 0-Ci 6-alkyl, NO2,
halogen, CN,
NHS02-C1 6-alkyl, methoxy-phenyl;
= a group selected
from NHCH(pyridinyl)CH2C00-C1 6-alkyl,
NHCH(CH2O-C1 6-alkyl)-benzoimidazolyl, optionally substituted with halogen or
CN;
= or 1-aminocyclopentyl, optionally substituted with methyl-oxadiazole
R11
is phenyl, optionally substituted with one or two residues selected from the
group
consisting of Cl 6-alkyl, C2 6-alkenyl, C2 6-alkynyl, C1 6-haloalkyl, C1 6-
alkylene-OH,
C2 6-alkenylene-OH, C2 6-alkynylene-OH,
CH2CON(C1 6-alky1)2,
CH2NHCONH-C3 6-cycloalkyl, CN, CO-pyridinyl, CONR11 1R11 2, COO-C1 6-alkyl,
N(S02-C1 6-alkyl)(CH2CON(Ci 4-alky1)2) 0-C1 6-alkyl, 0-pyridinyl, S02-C16-
alkyl,
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S 02-C1_6-alkylen-OH, S 02-C3_6 -cycloalkyl, S 02-
piperidinyl, SO2NH-C1_6-alkyl,
SO2N(C1_6-alkyl)2, halogen, CN, CO-morpholinyl, CH2-pyridinyl or a
heterocyclic ring
optionally substituted with one or two residues selected from the group
consisting of
Ci_6 -alkyl, NHC1_6-alkyl and =0;
R1.1.1 H, Ci_6 -alkyl, C3_6-cycloalkyl, C1_6 -
haloalkyl, CH2CON(C1_6-alkyl,)2,
CH2C0-azetindinyl, C1_6-alkylen-C3_6-cycloalkyl, CH2-pyranyl, CH2-tetrahydro-
furanyl, CH2-furanyl, C1_6-alkylen-0H or thiadiazolyl, optionally substituted
with
Ci_6 -alkyl ;
R1.1.2 H, Ci_6-alkyl, S02C1_6-alkyl;
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one N or 0, replacing a carbon atom of the ring,
optionally
substituted with one or two residues selected from the group consisting of
Ci_6-alkyl,
C14-alkylene-OH, OH, =0;
Or
Ril is phenyl, wherein two adjacent residues are together forming a five-
or six-membered
carbocyclic aromatic or non-aromatic ring, optionally containing independently
from each
other one or two N, S, or SO2, replacing a carbon atom of the ring, wherein
the ring is
optionally substituted with C1_4-alkyl or =0;
R1.2 is selected from
= heteroaryl, optionally substituted with one or two residues selected from
the group
consisting of Ci_6-alkyl, C2_6-alkenyl, C2_6-
alkynyl, C3_6-cycloalkyl,
CH2C00-C1_6-alkyl, CONR1.2.1R1.2.2, coR1.2.3, COO-C1_6-alkyl, CONH2, 0-C1_6-
alkyl,
halogen, CN, 502N(C1_6-alky1)2 or heteroaryl optionally substituted with one
or two
residues selected from the group consisting of Ci_6-alkyl;
= heteroaryl, optionally substituted with a five- or six-membered
carbocyclic non-
aromatic ring containing independently from each other two N, 0, S, or SO2,
replacing
a carbon atom of the ring;
= a aromatic or non-aromatic C9 or 10-bicyclic-ring, whereas one or two
carbon atoms are
replaced by N, 0 or S each optionally substituted with one or two residues
selected
from the group consisting of N(C1_6-alky1)2, CONH-C1_6-alkyl, =0;
= a heterocyclic non-aromatic ring, optionally substituted with pyridinyl;
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= 4,5-dihydro-naphtho[2,1-d]thiazole, optionally substituted with NHCO-C1_6-
alkyl,
R1.2.1 H, C1_6-alkyl, C1_6-alkylene-C3_6-cycloalkyl, C1_4-alkylene-
phenyl, C1_4-alkylene-
furanyl, C3_6-cycloalkyl, C14-alkylene-O-C1_4-alkyl, C1_6-haloalkyl or a five-
or
six-membered carbocyclic non-aromatic ring, optionally containing
independently
from each other one or two N, 0, S, or SO2, replacing a carbon atom of the
ring,
optionally substituted with 4-cyclopropylmethyl-piperazinyl
R1.2.2 H, Ci_6-alkyl;
R1.2.3 a five- or six-membered carbocyclic non-aromatic ring,
optionally containing
independently from each other one or two N, 0, S, or SO2, replacing a carbon
atom
of the ring;
R1.3 is selected from phenyl, heteroaryl or indolyl, each optionally
substituted with one or two
residues selected from the group consisting of Ci_6-alkyl, C3_6-cycloalkyl, 0-
C1_6-alkyl,
0-C1_6-haloalkyl, phenyl, heteroaryl;
R2 is selected from the group consisting of Ci_6-alkylene-phenyl, C1_6-
alkylene-naphthyl, and
C1_6-alkylene-heteroaryl; each optionally substituted with one, two or three
residues selected from
the group consisting of Ci_6-alkyl, C1_6-haloalkyl, 0-C1_6-alkyl, 0-C1_6-
haloalkyl, halogen;
= is H, Ci_6-alkyl;
R4 is H, Ci_6-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1 (above), wherein
A is CH2, 0 or N-C1_4-alkyl;
= is selected from
= NHR", NMeR1.1;
= NHR1'2, NMeR1'2;
= NHCH2-R1.3;
Ril is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Ci_6-alkyl, C2_6-alkenyl, C2_6-alkynyl, C1_6-haloalkyl, C1_6-
alkylene-OH,
C2_6-alkenylene-OH, C2_6-alkynylene-OH,
CH2CON(C1_6-alkyl)2,
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CH2NHCONH-C3_6-cycloalkyl, CN, CO-pyridinyl, CONR1.1.1R1.1.2, COO-C1_6-alkyl,
N(S 02-C1_6 -alkyl)(CH2CON(Ci_4-alkyl)2) 0-C1_6-alkyl, 0-pyridinyl, SO2-C16-
alkyl,
S02-C1_6-alkylen-OH, S 02-C3_6 -cycloalkyl, S 02-
piperidinyl, SO2NH-C1_6-alkyl,
SO2N(C1_6-alky1)2, halogen, CN, CO-morpholinyl, CH2-pyridinyl or a
heterocyclic ring
optionally substituted with one or two residues selected from the group
consisting of
Ci_6 -alkyl, NHC1_6-alkylõ =0;
R1.1.1 H, Ci_6 -alkyl, C3_6-cycloalkyl, Ci_6-
haloalkyl, CH2CON(Ci_6-alkyl,)2,
CH2C0-azetindinyl, Ci_6-alkylen-C3_6-cycloalkyl, CH2-pyranyl, CH2-tetrahydro-
furanyl, CH2-furanyl, Ci_6-alkylen-OH or thiadiazolyl, optionally substituted
with
Ci_6 -alkyl ;
R1.1.2 H, Ci_6-alkyl, S02C1_6-alkyl;
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one N or 0, replacing a carbon atom of the ring,
optionally
substituted with one or two residues selected from the group consisting of
Ci_6-alkyl,
Ci_4-alkylene-OH, OH, =0;
Or
Ril is phenyl, wherein two adjacent residues are together forming a five-
or six-membered
carbocyclic aromatic or non-aromatic ring, optionally containing independently
from each
other one or two N, S, or SO2, replacing a carbon atom of the ring, wherein
the ring is
optionally substituted with Ci_4-alkyl or =0;
R1.2 is selected from
= heteroaryl, optionally substituted with one or two residues selected from
the group
consisting of Ci_6-alkyl, C2_6-alkenyl, C2_6-
alkynyl, C3_6-cycloalkyl,
CH2C00-Ci_6-alkyl, CONR1.2.1R1.2.2, coR1.2.3, COO-Ci_6-alkyl, CONH2, 0-C1_6-
alkyl,
halogen, CN, 502N(Ci_4-alky1)2 or heteroaryl optionally substituted with one
or two
residues selected from the group consisting of Ci_6-alkyl;
= heteroaryl, optionally substituted with a five- or six-membered
carbocyclic non-
aromatic ring containing independently from each other two N, 0, S, or SO2,
replacing
a carbon atom of the ring;
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R1.2.1 H, C1_6-alkyl, C1_6-alkylene-C3_6-cycloalkyl, C1_4-alkylene-
phenyl, C1_4-alkylene-
furanyl, C3_6-cycloalkyl, C14-alkylene-O-C1_4-alkyl, C1_6-haloalkyl or a five-
or
six-membered carbocyclic non-aromatic ring, optionally containing
independently
from each other one or two N, 0, S, or SO2, replacing a carbon atom of the
ring,
optionally substituted with 4-cyclopropylmethyl-piperazinyl
R1.2.2 H, Ci_6-alkyl;
R1.2.3 a five- or six-membered carbocyclic non-aromatic ring,
optionally containing
independently from each other one or two N, 0, S, or SO2, replacing a carbon
atom
of the ring;
R1.3 is selected from phenyl, heteroaryl or indolyl, each optionally
substituted with one or two
residues selected from the group consisting of Ci_6-alkyl, C3_6-cycloalkyl, 0-
C1_6-alkyl,
0-C1_6-haloalkyl, phenyl, heteroaryl; where in some instances R1.3 is selected
from phenyl,
pyrazolyl, isoxazolyl, pyridinyl, pyrimidinyl, indolyl or oxadiazolyl, each
optionally
substituted with one or two residues selected from the group consisting of
Ci_6-alkyl,
C3_6-cycloalkyl, 0-C1_6-alkyl, 0-C1_6-haloalkyl, phenyl, pyrrolidinyl;
R2 is selected from the group consisting of Ci_6-alkylene-phenyl, C1_6-
alkylene-naphthyl, and
C1_6-alkylene-thiophenyl; each optionally substituted with one, two or three
residues selected from
the group consisting of Ci_6-alkyl, C1_6-haloalkyl, 0-C1_6-alkyl, 0-C1_6-
haloalkyl, halogen;
R3 is H, C1_4-alkyl;
R4 is H, C1_4-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1 (above), wherein
A is CH2, 0 or N-C1_4-alkyl;
121 is selected from
= NHR", NMeR1.1;
Ril is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Ci_6-alkyl, C2_6-alkenyl, C2_6-alkynyl, C1_6-haloalkyl, C1_6-
alkylene-OH,
C2_6-alkenylene-OH, C2_6-alkynylene-OH,
CH2CON(C1_6-alky1)2,
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CH2NHCONH-C3_6-cycloalkyl, CN, CO-pyridinyl, CONIV11R1.1.2, c00-c 1_6-alkyl,
N(S02-C1_6-alkyl)(CH2CON(C1_4-alky1)2) 0-C1_6-alkyl, 0-pyridinyl, S02-C1_6-
alkyl,
S02-C1_6-alkylen-OH, S02-C3_6-cycloalkyl,
S02-piperidinyl, SO2NH-C1_6-alkyl,
SO2N(C1_6-alky1)2, halogen, CN, CO-morpholinyl, CH2-pyridinyl or a
heterocyclic ring
optionally substituted with one or two residues selected from the group
consisting of
NHC1_6-alkylõ =0;
H, C3_6-cycloalkyl,
CH2CON(Ci_6-alkyl,)2,
CH2C0-azetindinyl,
CH2-pyranyl, CH2-tetrahydro-
furanyl, CH2-furanyl, Ci_6-alkylen-OH or thiadiazolyl, optionally substituted
with
R1.1.2 H, S02C1_6-alkyl;
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one N or 0, replacing a carbon atom of the ring,
optionally
substituted with one or two residues selected from the group consisting of
Ci_6-alkyl,
Ci_4-alkylene-OH, OH, =0;
Or
Ril
is phenyl, wherein two adjacent residues are together forming a five- or six-
membered
carbocyclic aromatic or non-aromatic ring, optionally containing independently
from each
other one or two N, S, or SO2, replacing a carbon atom of the ring, wherein
the ring is
optionally substituted with Ci_4-alkyl or =0;
R2 is selected from the group consisting of Ci_6-alkylene-phenyl, Ci_6-
alkylene-naphthyl, and
Ci_6-alkylene-thiophenyl; each optionally substituted with one, two or three
residues selected from
the group consisting of Ci_6-alkyl, Ci_6-haloalkyl, 0-Ci_6-haloalkyl,
halogen;
R3 is H,
R4 is H,
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or N-Ci_4-alkyl;
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is selected from
= NHR1'2, NMeR1'2;
R1.2 is selected from
= heteroaryl, optionally substituted with one or two residues selected from
the group
consisting of Ci_6-alkyl, C2_6-alkenyl,
C2_6-alkynyl, C3_6-cycloalkyl,
CH2C00-C1_6-alkyl, CONR1.2.1R1.2.2, coR1.2.3, COO-C1_6-alkyl, CONH2, 0-C1_6-
alkyl,
halogen, CN, SO2N(C1_4-alkyl)2 or heteroaryl optionally substituted with one
or two
residues selected from the group consisting of Ci_6-alkyl;
= heteroaryl, optionally substituted with a five- or six-membered
carbocyclic non-
aromatic ring containing independently from each other two N, 0, S, or SO2,
replacing
a carbon atom of the ring;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
N(C1_6-alky1)2, CONH-C1_6-alkyl, =0;
= piperidinyl, optionally substituted with pyridinyl;
= 4,5-dihydro-naphtho[2,1-d]thiazole, optionally substituted with NHCO-C1_6-
alkyl,
R1.2.1 H, Ci_6-alkyl, C1_6-alkylene-C3_6-cycloalkyl, C1_4-alkylene-
phenyl, C1_4-alkylene-
furanyl, C3_6-cycloalkyl, C14-alkylene-0-C1_4-alkyl, C1_6-haloalkyl or a five-
or
six-membered carbocyclic non-aromatic ring, optionally containing
independently
from each other one or two N, 0, S, or SO2, replacing a carbon atom of the
ring,
optionally substituted with 4-cyclopropylmethyl-piperazinyl
R1.2.2 H, Ci_6-alkyl;
R1.2.3
a five- or six-membered carbocyclic non-aromatic ring, optionally containing
independently from each other one or two N, 0, S, or SO2, replacing a carbon
atom
of the ring;
R2
is selected from the group consisting of Ci_6-alkylene-phenyl, C1_6-alkylene-
naphthyl, and
C1_6-alkylene-thiophenyl; each optionally substituted with one, two or three
residues selected from
the group consisting of Ci_6-alkyl, C1_6-haloalkyl, 0-C1_6-alkyl, 0-C1_6-
haloalkyl, halogen;
R3 is H, C1_4-alkyl;
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R4 is H, C1_4-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1 (above), wherein
A is CH2, 0 or N-C1_4-alkyl;
121 is selected from
= NHR1.2, NMeR1.2;
R1.2 is selected from
= heteroaryl, optionally substituted with one or two residues selected from
the group
consisting of Ci_6-alkyl, C2_6-alkenyl,
C2_6-alkynyl, C3_6-cycloalkyl,
CH2C00-C1_6-alkyl, CONR1.2.1R1.2.2, coR1.2.3, COO-C1_6-alkyl, CONH2, 0-C1_6-
alkyl,
halogen, CN, SO2N(C1_4-alkyl)2 or heteroaryl optionally substituted with one
or two
residues selected from the group consisting of Ci_6-alkyl;
= heteroaryl, optionally substituted with a five- or six-membered
carbocyclic non-
aromatic ring containing independently from each other two N, 0, S, or SO2,
replacing
a carbon atom of the ring;
R1.2.1 H, Ci_6-alkyl, C1_6-alkylene-C3_6-cycloalkyl, C1_4-alkylene-
phenyl, C1_4-alkylene-
furanyl, C3_6-cycloalkyl, C14-alkylene-O-C1_4-alkyl, C1_6-haloalkyl or a five-
or
six-membered carbocyclic non-aromatic ring, optionally containing
independently
from each other one or two N, 0, S, or SO2, replacing a carbon atom of the
ring,
optionally substituted with 4-cyclopropylmethyl-piperazinyl
R1.2.2 H, Ci_6-alkyl;
R1.2.3 a five- or six-membered carbocyclic non-aromatic ring,
optionally containing
independently from each other one or two N, 0, S, or SO2, replacing a carbon
atom
of the ring;
R2 is selected from the group consisting of Ci_6-alkylene-phenyl, C1_6-
alkylene-naphthyl, and
C1_6-alkylene-thiophenyl; each optionally substituted with one, two or three
residues selected from
the group consisting of Ci_6-alkyl, C1_6-haloalkyl, 0-C1_6-alkyl, 0-C1_6-
haloalkyl, halogen;
R3 is H, C1_4-alkyl;
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R4 is H, C14-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or N-C14-alkyl;
121 is selected from
= NHCH2-R13;
R13 is selected from phenyl, pyrazolyl, isoxazolyl, pyridinyl,
pyrimidinyl, indolyl or
oxadiazolyl, each optionally substituted with one or two residues selected
from the group
consisting of Cl 6-alkyl, C36-cycloalkyl, 0-Cl 6-alkyl, 0-C16-haloalkyl,
phenyl,
pyrrolidinyl;
R2 is selected from the group consisting of Ci 6-alkylene-phenyl, C16-
alkylene-naphthyl, and
C16-alkylene-thiophenyl; each optionally substituted with one, two or three
residues selected from
the group consisting of Cl 6-alkyl, C16-haloalkyl, 0-Cl 6-alkyl, 0-C16-
haloalkyl, halogen;
R3 is H, C14-alkyl;
R4 is H, C14-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or N-C14-alkyl;
is selected from
= NHR11, NMeR11;
= NHR12, NMeR12;
= NHCH2-R1 3;
= NH-C3 6-cycloalkyl, whereas optionally one carbon atom is replaced by a
nitrogen atom,
whereas the ring is optionally substituted with one or two residues selected
from the group
CA 03111433 2021-03-02
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consisting of C1_6-alkyl, 0-C1_6-alkyl, NHS02-phenyl, NHCONH-phenyl, halogen,
CN,
S02-C1_6-alkyl, COO-C1_6-alkyl;
= a C9 or 10-bicyclic-ring, whereas one or two carbon atoms are replaced by
nitrogen atoms and
the ring system is bound via a nitrogen atom to the basic structure of formula
1 and whereas
the ring system is optionally substituted with one or two residues selected
from the group
consisting of Ci_6-alkyl, COO-C1_6-alkyl, C1_6-haloalkyl, 0-C1_6-alkyl, NO2,
halogen, CN,
NHS02-C1_6-alkyl, m-methoxyphenyl;
= a group selected from
NHCH(pyridinyl)CH2C00-C1_6-alkyl,
NHCH(CH2O-C1_6-alkyl)-benzoimidazolyl, optionally substituted with Cl;
= or 1-aminocyclopentyl, optionally substituted with methyl-oxadiazolyl;
Ril is phenyl, optionally substituted with one or two residues selected
from the group
consisting of Ci_6-alkyl, C1_6-haloalkyl,
CH2CON(C1_6-alky1)2,
CH2NHCONH-C3_6-cycloalkyl, CN, CONIV11R1.1.2, COO-C1_6-alkyl, 0-C1_6-alkyl,
S02-C1_6-alkyl, S02-C1_6-alkylen-OH, S02-C3_6-cycloalkyl, S02-
piperidinyl,
SO2NH-C1_6-alkyl, SO2N(C1_6-alky1)2, halogen, CN, CO-morpholinyl, CH2-
pyridinyl or a
heterocyclic ring optionally substituted with one or two residues selected
from the group
consisting of Ci_6-alkyl, NHC1_6-alkylõ =0;
H, Ci_6-alkyl, C3_6-cycloalkyl, C1_6-haloalkyl, CH2CON(C1_6-alkyl,)2,
CH2C0-azetindinyl, C1_6-alkylen-C3_6-cycloalkyl, CH2-pyranyl, CH2-tetrahydro-
furanyl, CH2-furanyl, C1_6-alkylen-OH or thiadiazolyl, optionally substituted
with
Ci_6-alkyl;
R1.1.2 H, Ci_6-alkyl, S02C1_6-alkyl;
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH
R1.2 is selected from
= heteroaryl, optionally substituted with one or two residues selected from
the group
consisting of Ci_6-alkyl, C3_6-cycloalkyl, CH2C00-C1_6-alkyl, CONR1.2.1R1.2.2,
COO-C1_6-alkyl, CONH2, 0-C1_6-alkyl, halogen, CN, CO-pyrrolidinyl,
CO-morpholinyl or heteroaryl optionally substituted with one or two residues
selected
from the group consisting of Ci_6-alkyl;
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= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
N(Ci 6-alky1)2, CONH-C1 6-alkyl, =0;
= piperidinyl, optionally substituted with pyridinyl;
= 4,5-dihydro-naphtho 12, 1-d] thiazole, optionally substituted with NHCO-C
1 6-alkyl,
R1 2 1 H, Cl 6-alkyl;
R1 2 2 H, Cl 6-alkyl;
R13 is selected from phenyl, pyrazolyl, isoxazolyl, pyrimidinyl, indolyl or
oxadiazolyl,
each optionally substituted with one or two residues selected from the group
consisting of
Cl 6-alkyl, C3 6-cycloalkyl, 0-Cl 6-alkyl, 0-C1 6-haloalkyl;
R2 is selected from Ci 6-alkylene-phenyl or C1 6-alkylene-naphthyl, both
optionally substituted with
one or two residues selected from the group consisting of Cl 6-alkyl, C1 6-
haloalkyl, 0-Cl 6-alkyl,
0-C1 6-haloalkyl, halogen; or CH2-thiophenyl, optionally substituted with one
or two residues
selected from the group consisting of halogen;
= is H, C14-alkyl;
R4 is H, C14-alkyl;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
= is selected from
= NHR11, NMeR1
= NHR1 2, NMeR1 2;
= NHCH2-R1 3;
= NH-cyclohexyl, optionally substituted with one or two residues selected
from the group
consisting of C14-alkyl, NHS02-phenyl, NHCONH-phenyl, halogen;
= NH-pyrrolidinyl, optionally substituted with one or two residues selected
from the group
consisting of S02-C14-alkyl, COO-C14-alkyl;
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= piperidinyl, optionally substituted with one or two residues selected
from the group consisting
of NHS02-C1 4-alkyl, m-methoxyphenyl;
= dihydro-indolyl, dihydro-isoindolyl, tetrahydro-quinolinyl or tetrahydro-
isoquinolinyl,
optionally substituted with one or two residues selected from the group
consisting of C14-alkyl,
COO-C14-alkyl, C1 4-haloalkyl, 0-C1 4-alkyl, NO2, halogen;
= a group selected from
NHCH(pyridinyl)CH2C00-C1 4-alkyl,
NHCH(CH2O-C1 4-alkyl)-benzoimidazolyl, optionally substituted with Cl;
= or 1-aminocyclopentyl, optionally substituted with methyl-oxadiazolyl;
R11 is phenyl, optionally substituted with one or two residues selected
from the group
consisting of C14-alkyl, C1 4-haloalkyl,
CH2CON(C1 4-alky1)2,
CH2NHCONH-C3 6-cycloalkyl, CN, CONR11 1R112, COO-C14-alkyl, 0-C1 4-alkyl,
S02-C14-alkyl, S02-C1 4-alkylen-OH, S 02-C3 6-cycloalkyl, S02-
piperidinyl,
SO2NH-C1 4-alkyl, SO2N(C1 4-alky1)2, halogen, CO-morpholinyl, CH2-pyridinyl,
or
imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl, triazolyl, tetrazolyl,
oxazolyl,
oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally substituted
with one or two
residues selected from the group consisting of C14-alkyl, NHCi 4-alkylõ =0;
R111 H, Cl 6-alkyl, C3 6-cycloalkyl, C1 4-
haloalkyl, CH2CON(C1 4-alkyl,)2,
CH2C0-azetindinyl, C1 4-alkylen-C3 6-cycloalkyl, CH2-pyranyl, CH2-tetrahydro-
furanyl, CH2-furanyl, C1 4-alkylen-OH or thiadiazolyl, optionally substituted
with
C14-alkyl;
R112 H, C14-alkyl, S02C1 4-alkyl;
or R111 and R1 1 2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH
R12 is selected from
= pyridinyl, pyridazinyl, pyrrolyl, pyrazolyl, isoxazolyl, thiazolyl,
thiadiazolyl,
optionally substituted with one or two residues selected from the group
consisting of
C14-alkyl, C3 6-cycloalkyl, CH2C00-C1 4-alkyl, CONIV 2 11Z1 2 2, COO-C14-
alkyl,
CONH2, 0-C1 4-alkyl, halogen, CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl,
triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, each optionally substituted
with one or
two residues selected from the group consisting of C14-alkyl;
33
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= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
N(C14-alky1)2, CONH-C14-alkyl, =0;
= piperidinyl, optionally substituted with pyridinyl;
= 4,5-dihydro-naphtho12,1-d] thiazole, optionally substituted with NHCO-C14-
alkyl,
R1 2 1 H, C14-alkyl;
R1 2 2 H, C14-alkyl;
R13 is selected from phenyl, pyrazolyl, isoxazolyl, pyrimidinyl,
indolyl or oxadiazolyl, each
optionally substituted with one or two residues selected from the group
consisting of
C14-alkyl, C36-cycloalkyl, 0-C14-alkyl, 0-C14-haloalkyl;
R2 is selected from Ci 6-alkylene-phenyl or C16-alkylene-naphthyl, both
optionally substituted with
one or two residues selected from the group consisting of C14-alkyl, C14-
haloalkyl,
0-C14-haloalkyl, halogen; or CH2-thiophenyl, optionally substituted with one
or two residues
selected from the group consisting of halogen;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
121 is selected from
= NHR11, NMeR11;
= NHIV 2, NMeR12;
= NHCH2-1Z1 3;
= NH-piperidinyl, optionally substituted with pyridinyl;
= NH-cyclohexyl, optionally substituted with one or two residues selected
from the group
consisting of t-Bu, NHS02-phenyl, NHCONH-phenyl, F;
34
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= NH-pyrrolidinyl, optionally substituted with one or two residues selected
from the group
consisting of SO2Me, COO-t-Bu;
= piperidinyl, optionally substituted with one or two residues selected
from the group consisting
of NHS02-n-Bu, m-methoxyphenyl;
= dihydro-indolyl, dihydro-isoindolyl, tetrahydro-quinolinyl or tetrahydro-
isoquinolinyl,
optionally substituted with one or two residues selected from the group
consisting of Me,
COOMe, CF3, OMe, NO2, F, Br;
= a group selected from NHCH(pyridinyl)CH2COOMe, NHCH(CH20Me)-
benzoimidazolyl,
optionally substituted with Cl;
= or 1-aminocyclopentyl, optionally substituted with methyl-oxadiazolyl;
Ril is phenyl, optionally substituted with one or two residues selected
from the group
consisting of Me, Et, t-Bu, CF3, CH2CONMe2, CH2NHCONH-cyclohexyl, CN,
CONR1.1.1R1.1.2, COOMe, COOEt, OMe, SO2Me, SO2CH2CH2OH, SO2Et,
S02-cyclopropyl, S02-piperidinyl, SO2NHEt, SO2NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
H, Me, Et, t-Bu, i-Pr, cyclopropyl, CH2-i-Pr, CH2-t-Bu, CH(CH3)CH2CH3,
CH2CHF2, CH2CONMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R1.1.2 H, Me, Et, SO2Me, SO2Et
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH
R1.2 is selected from
= pyridinyl, pyrrolyl, pyrazolyl, isoxazolyl, thiazolyl, thiadiazolyl,
optionally
substituted with one or two residues selected from the group consisting of Me,
Et, Pr,
Bu, cyclopropyl, CH2COOEt, CONR1.2.1R1.2.2, COOMe, COOEt, CONH2, OMe, Cl,
Br CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl, triazolyl, tetrazolyl,
isoxazolyl,
oxadiazolyl, each optionally substituted Me;
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= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
NMe2, CONHMe, =0;
= 4,5-dihydro-naphtho12,1-d]thiazole, optionally substituted with NHCOMe,
R1.2.1 H, me;
R1.2.2 H, me;
R1.3 is selected from phenyl, pyrazolyl, isoxazolyl, pyrimidinyl,
indolyl or oxadiazolyl, each
optionally substituted with one or two residues selected from the group
consisting of Me,
Et, Pr, cyclopentyl, OMe, OCHF2;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, OCF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
121 is selected from
= NHR"
= NHR1.2,
R1.1 is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Me, Et, Pr, Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN,
CONR1.1.1R1.1.2,
OMe, S02Me, SO2CH2CH2OH, S02Et,
S02-cyclopropyl, S02-piperidinyl, S02NHEt, S02NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
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R1.1.1 H, Me, Et, t-Bu, i-Pr, cyclopropyl, CH2-i-Pr, CH2-t-Bu, CH(CH3)CH2CH3,
CH2CHF2, CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R1.1.2 H, Me, Et, S02Me, S02Et
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH
R1.2 is selected from
= pyridinyl, pyrrolyl, pyrazolyl, isoxazolyl, thiazolyl, thiadiazolyl,
optionally
substituted with one or two residues selected from the group consisting of Me,
Et, Pr,
Bu, cyclopropyl, CH2C00Et, CONR1.2.1R1.2.2, COOMe, COOEt, CONH2, OMe, Cl,
Br CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl, triazolyl, tetrazolyl,
isoxazolyl,
oxadiazolyl, each optionally substituted Me;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
NMe2, CONHMe, =0;
= 4,5-dihydro-naphtho[2,1-d[thiazole, optionally substituted with NHCOMe,
R1.2.1 H, me;
R1.2.2 H, me;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, 0CF3, F, Cl, Br, Et
R3 is H;
R4 is H.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHR", NMeR" ;
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= NHR1.2, NMeR1.2;
= NHCH2-R1.3;
Ril is phenyl, optionally substituted with one or two residues selected
from the group
consisting of Me, Et, Pr, Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN,
CONR1.1.1R1.1.2,
OMe, S02Me, SO2CH2CH2OH, S02Et,
S02-cyclopropyl, S02-piperidinyl, S02NHEt, S02NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
R1.1.1 H, Me, Et, Pr, Bu, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R1.1.2 H, Me, Et, S02Me, S02Et
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH
R1.2 is selected from
= pyridinyl, pyrrolyl, pyrazolyl, isoxazolyl, thiazolyl, thiadiazolyl,
optionally
substituted with one or two residues selected from the group consisting of Me,
Et, Pr,
Bu, cyclopropyl, CH2C00Et, CONR1.2.1R1.2.2, COOMe, COOEt, CONH2, OMe, Cl,
Br CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl, triazolyl, tetrazolyl,
isoxazolyl,
oxadiazolyl, each optionally substituted Me;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
NMe2, CONHMe, =0;
= 4,5-dihydro-naphtho[2,1-d[thiazole, optionally substituted with NHCOMe,
R1.2.1 H, me;
R1.2.2 H, me;
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R1.3 is selected from phenyl, pyrazolyl, isoxazolyl, pyrimidinyl,
indolyl or oxadiazolyl, each
optionally substituted with one or two residues selected from the group
consisting of Me,
Et, Pr, cyclopentyl, OMe, OCHF2;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, OCF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHR", NMeR" ;
Ril is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Me, Et, t-Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN,
CONR1.1.1R1.1.2,
OMe, S02Me, SO2CH2CH2OH, S02Et,
S02-cyclopropyl, S02-piperidinyl, S02NHEt, S02NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
H, Me, Et, Bu, Pr, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R1.1.2 H, Me, Et, S02Me, S02Et
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or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, 0CF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHR", NMeR" ;
Ril is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Me, Et, t-Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN,
CONR1.1 .1 R1.1.2,
COOMe, COOEt, OMe, S02Me, SO2CH2CH2OH, S02Et,
S02-cyclopropyl, S02-piperidinyl, S02NHEt, S02NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
H, Me, Et, Bu, Pr, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R1.1.2 H, Me, Et, S02Me, S02Et
or R1.1.1 and R1.1.2 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH;
CA 03111433 2021-03-02
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R2 is defined as in Table 1 shown below;
R3 is H;
R4 is H;
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
= is selected from
= NHR11, NMeR1
R11 is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Me, Et, t-Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN,
CONIV 11Zi 1 2,
COOMe, COOEt, OMe, S02Me, SO2CH2CH2OH, S02Et,
S02-cyclopropyl, S02-piperidinyl, S02NHEt, S02NMeEt, F, Cl, CO-morpholinyl,
CH2-pyridinyl, or imidazolidinyl, piperidinyl, oxazinanyl, pyrazolyl,
triazolyl, tetrazolyl,
oxazolyl, oxadiazolyl, thiazolyl, pyridinyl, pyrimidinyl, each optionally
substituted with
one or two residues selected from the group consisting of Me, NHMe, =0;
and R111 and R112 together are forming a four-, five- or six-membered
carbocyclic ring,
optionally containing one 0, replacing a carbon atom of the ring, optionally
substituted
with one or two residues selected from the group consisting of CH2OH;
R2 is defined as in Table 1 shown below;
R3 is H;
R4 is H;
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
= is selected from
= NHR11, NMeR1
41
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R11 is phenyl, optionally substituted with one or two residues
selected from the group
consisting of Me, Et, t-Bu, CF3, CH2CONMe2, CH2NHCONH-cyclohexyl, CN,
CONIV 111Z112, COOMe, COOEt, OMe, F, Cl;
R111 H, Me, Et, Bu, Pr, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2CONMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R112 H, Me, Et, SO2Me, SO2Et
R2 is defined as in Table 1 shown below;
R3 is H;
R4 is H;
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
121 is selected from
= NHR11, NMeR11;
Rll is phenyl, optionally substituted with one or two residues
selected from the group
consisting of S02Me, SO2CH2CH2OH, S02Et, S02-cyclopropyl, S02-piperidinyl,
S02NHEt, S02NMeEt;
R111 H, Me, Et, Bu, Pr, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R112 H, Me, Et, S02Me, S02Et
R2 is defined as in Table 1 shown below;
R3 is H;
R4 is H;
42
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Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHR11, NMeR11;
R11 is phenyl, optionally substituted with one residue selected from
the group consisting of Me,
Et, t-Bu, CF3, CH2C0NMe2, CH2NHCONH-cyclohexyl, CN, CONR111R1 1 2, COOMe,
COOEt, OMe, S02Me, SO2CH2CH2OH, S02Et, S02-cyclopropyl, S02-piperidinyl,
S02NHEt, S02NMeEt, F, Cl, and additionally with one residue selected from the
group
consiting of CO-morpholinyl, CH2-pyridinyl, or imidazolidinyl, piperidinyl,
oxazinanyl,
pyrazolyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl, thiazolyl, pyridinyl,
pyrimidinyl,
each optionally substituted with one or two residues selected from the group
consisting of
Me, NHMe, =0;
R111 H, Me, Et, Bu, Pr, cyclopropyl, CH2-Pr, CH2-Bu, CH(CH3)CH2CH3, CH2CHF2,
CH2C0NMe2, CH2C0-azetindinyl, CH2-cyclopropyl, CH2-cyclobutyl,
CH2-pyranyl, CH2-tetrahydrofuranyl, CH2-furanyl, CH2CH2OH or thiadiazolyl,
optionally substituted with Me;
R112 H, Me, Et, S02Me, S02Et
R2 is defined as in Table 1 shown below;
R3 is H;
R4 is H;
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
121 is selected from
= NHR12, NMeR1 2;
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R1.2 is selected from
= pyridinyl, pyridazinyl, pyrrolyl, pyrazolyl, isoxazolyl, thiazolyl,
thiadiazolyl,
optionally substituted with one or two residues selected from the group
consisting of
Me, Et, Pr, Bu, cyclopropyl, CH2C00Et, CONR1.2.11Z1.2.2, COOMe, COOEt, CONH2,
OMe, Cl, Br CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl, triazolyl,
tetrazolyl,
isoxazolyl, oxadiazolyl, each optionally substituted Me;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of
NMe2, CONHMe, =0;
= 4,5-dihydro-naphtho12,1-d]thiazole, optionally substituted with NHCOMe,
R1.2.1 H, me;
R1.2.2 H, me;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, 0CF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHR1'2, NMeR1'2;
R1.2 is selected from pyridinyl, pyridazinyl, pyrrolyl, pyrazolyl,
isoxazolyl, thiazolyl,
thiadiazolyl, optionally substituted with one or two residues selected from
the group
consisting of Me, Et, n-Pr, i-Pr, Bu, cyclopropyl, CH2C00Et, CONR1.2.1R1.2.2,
COOMe,
COOEt, CONH2, OMe, Cl, Br CO-pyrrolidinyl, CO-morpholinyl or pyrazolyl,
triazolyl,
tetrazolyl, isoxazolyl, oxadiazolyl, each optionally substituted Me;
R1.2.1 H, me;
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R1 2 2 H, me;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, OCF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
is selected from
= NHCH2-R1 3 ;
R13 is selected from phenyl, pyrazolyl, isoxazolyl, pyrimidinyl,
indolyl or oxadiazolyl, each
optionally substituted with one or two residues selected from the group
consisting of Me,
Et, Pr, cyclopentyl, OMe, OCHF2;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, 0CF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
A is CH2, 0 or NMe;
121 is selected from
= NH-piperidinyl, optionally substituted with pyridinyl;
CA 03111433 2021-03-02
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= NH-cyclohexyl, optionally substituted with one or two residues selected
from the group
consisting of t-Bu, NHS02-phenyl, NHCONH-phenyl, F;
= NH-pyrrolidinyl, optionally substituted with one or two residues selected
from the group
consisting of SO2Me, COO-t-Bu;
= piperidinyl, optionally substituted with one or two residues selected
from the group consisting
of NHS02-n-Bu, m-methoxyphenyl;
= dihydro-indolyl, dihydro-isoindolyl, tetrahydro-quinolinyl or tetrahydro-
isoquinolinyl,
optionally substituted with one or two residues selected from the group
consisting of Me,
COOMe, CF3, OMe, NO2, F, Br;
= a group selected from NHCH(pyridinyl)CH2COOMe, NHCH(CH20Me)-
benzoimidazolyl,
optionally substituted with Cl;
= or 1-aminocyclopentyl, optionally substituted with Methyl-Oxadiazolyl;
R2 is selected from CH2-phenyl or CH2-naphthyl, both optionally substituted
with one or two residues
selected from the group consisting of CH3, CF3, OCF3, F, Cl, Br, Et; or CH2-
thiophenyl, optionally
substituted with one or two residues selected from the group consisting of Cl,
Br;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is CH2, 0 or NMe, R' is selected from NHR1
2, NMeR1 2; R2 is defined
as in Table 1 shown below; R3 is H; R4 is and R12 is selected from
= pyridinyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
i-Pr, n-Bu, cyclopropyl, CONR1 2 1R1 2 2, COOMe, COOEt, CONH2, OMe, Cl, Br CO-
pyrrolidinyl,
CO-morpholinyl or pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl,
each optionally substituted
Me;
= pyrrolyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
COOMe, COOEt;
= pyrazolyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
cyclopropyl, COOEt, CO-pyrrolidinyl;
= isoxazolyl, optionally substituted with one or two residues selected from
the group consisting of t-Bu,
COOEt;
46
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= thiazolyl, optionally substituted with one or two residues selected from
the group consisting of Me, n-
Pr, i-Pr, Bu, COOMe, COOEt, CH2C00Et, CONR1.2.1R1.2.2;
= thiadiazolyl, optionally substituted with one or two residues selected
from the group consisting of
COOEt;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each optionally substituted
with one or two residues selected from the group consisting of NMe2, CONHMe,
=0;
= 4,5 -dihydro-naphtho [2, 1-d] thiazole, optionally substituted with
NHCOMe,
and
R1.2.1 is H or Me;
R1.2.2 is H or Me.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is CH2, 0 or NMe, R' is selected from
NHR1.2, NMeR1.2; R2 is defined
as in Table 1 shown below; R3 is H; R4 is and R1.2 is selected from
= pyridinyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
i-Pr, n-Bu, CONR1.2.1R1.2.2, COOMe, COOEt, CONH2, OMe, Cl, Br;
= pyrrolyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
COOMe, COOEt;
= pyrazolyl, optionally substituted with one or two residues selected from
the group consisting of Me, Et,
cyclopropyl, COOEt, CO-pyrrolidinyl;
= isoxazolyl, optionally substituted with one or two residues selected from
the group consisting of t-Bu,
COOEt;
= thiazolyl, optionally substituted with one or two residues selected from
the group consisting of Me, n-
Pr, i-Pr, Bu, COOMe, COOEt, CONR1.2.1R1.2.2;
= thiadiazolyl, optionally substituted with one or two residues selected
from the group consisting of
COOEt;
= benzothiazolyl, indazolyl, dihydro-indolyl, indanyl, tetrahydro-
quinolinyl, each optionally substituted
with one or two residues selected from the group consisting of NMe2, CONHMe,
=0;
and
R1.2.1 is H or Me;
R1.2.2 is H or Me.
47
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Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein
= A is CH2, 0 or NMe, 121 is selected from NHR1 2, NMeR1 2; R2 is defined
as in Table 1 shown below;
R3 is H; R4 is H; R12 is pyridinyl, optionally substituted with one or two
residues selected from the
group consisting of Me, Et, i-Pr, n-Bu, CONR1 2 1R1 2 2, COOMe, COOEt, CONH2,
OMe, Cl, Br; R1 2 1
is H or Me and R1 2 2 is H or Me.
= A is CH2, 0 or NMe, R' is selected from NHR1 2, NMeR1 2; R2 is defined as
in Table 1 shown below;
R3 is H; R4 is H; R12 is pyrrolyl, optionally substituted with one or two
residues selected from the group
consisting of Me, Et, COOMe, COOEt; R1 2 1 is H or Me and R1 2 2 is H or Me.
= A is CH2, 0 or NMe, 121 is selected from NHR1 2, NMeR1 2; R2 is defined
as in Table 1 shown below;
R3 is H; R4 is H; R12 is pyrazolyl, optionally substituted with one or two
residues selected from the
group consisting of Me, Et, cyclopropyl, COOEt, CO-pyrrolidinyl; R1 2 1 is H
or Me and R1 2 2 is H or
Me.
= A is CH2, 0 or NMe, R' is selected from NHR1 2, NMeR1 2; R2 is defined as
in Table 1 shown below;
R3 is H; R4 is H; R12 is isoxazolyl, optionally substituted with one or two
residues selected from the
group consisting of t-Bu, COOE; R1 2 1 is H or Me and R1 2 2 is H or Me.
= A is CH2, 0 or NMe, 121 is selected from NHR1 2, NMeR1 2; R2 is defined
as in Table 1 shown below;
R3 is H; R4 is H; R12 is thiazolyl, optionally substituted with one or two
residues selected from the
group consisting of Me, n-Pr, i-Pr, Bu, COOMe, COOEt, CONR1 1R1 2; ; R1 2 1 is
H or Me and R1 2 2
is H or Me.
= A is CH2, 0 or NMe, R' is selected from NHR1 2, NMeR1 2; R2 is defined as
in Table 1 shown below;
R3 is H; R4 is H; R12 is thiadiazolyl, optionally substituted with one or two
residues selected from the
group consisting of COOEt; R1 2 1 is H or Me and R1 2 2 is H or Me.
= A is CH2, 0 or NMe, 121 is selected from NHR1 2, NMeR1 2; R2 is defined
as in Table 1 shown below;
R3 is H; R4 is H; R12 is benzothiazolyl, indazolyl, dihydro-indolyl, indanyl,
tetrahydro-quinolinyl, each
optionally substituted with one or two residues selected from the group
consisting of NMe2, CONHMe,
=0; R1 2 1 is H or Me and R1 2 2 is H or Me.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein all groups are defined as above except R13 is
selected from
= phenyl, optionally substituted with OCHF2;
= pyrazolyl, optionally substituted with Me or Et;
= isoxazolyl, optionally substituted with Pr;
48
CA 03111433 2021-03-02
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= pyrimidinyl, optionally substituted with two OMe;
= indolyl;
= oxadiazolyl, optionally substituted with cyclopentyl.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein all groups are defined as above except A is
CH2.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein all groups are defined as above except A is 0.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein all groups are defined as above except A is
NMe.
Another embodiment of the present invention are compounds of formula 1,
wherein
A is CH2, 0 or NMe;
121 is selected from
/CH3
N
XI XI
1 0
N'
H3 -i XI II
S i 1
N v ,
\ II F 7-0 . 0 V
---CH3
XII /X1
N
N iio,
N-CH3
oJ,I-lb
o Xi
N / 0 -
= S- %
CH3 / -- ,.., 0 N
Ii\lj CH,
N......õ N, N NI
I /I
0 N H3C
4. -- N CH,
-- CH3
/X1 H3Cy0
N
Xi N/X1
/X1 /
0 \
/N......N,CH3 1 I fat \
S"--- 0
N
H3C,-, N N
.--- CH3
49
CA 03111433 2021-03-02
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xi
Iv =
xi 0
xi
ixl \NNJk xi /
N
0
--' C' CH3 N/
1-130"---N
II ,.. ."- N
N /
Nµ 1
CH3 H3C)
,
F13
.-r\f 0 CH3
a-13 H3C
7cH3 0./N--
cH3
CNN ( o
...11 / ,Th
0.,;--L. )
N Q _s 0' N
N 10 N
I
xi xi xi
OH
N___/ CH, 5
/
i 0 N N * 0--11
0.7.-s/0 --S
0.
N
Xi( 0
= N N N *
N
I I
)S N 0 xi CH3
XII
NcIo/
N
0---- I
--- -- CH,
N
I NO N
N\.,..' N Xi N
I
* I N
I. /
N XI\N
H2N ,CH3
H3C 0 0'
HO
H3C
\..........yN * ON
H3C--?L--a
N
H2N \ N 1110 N N
xi I I
0 xi X xi
CA 03111433 2021-03-02
WO 2020/069008
PCT/US2019/052995
p
N-...õ,
1
CH3
N
0 i F 410 HC
is
N )--N
/L-
N FO
N
1 N
1
)(IN Xi Xi
Xil 1.1 )(It 0
11-.-
N \ N 0 0" N)LN H,C,N /
/
CH \ 1
i 3
(N 40 N CH3
. N
/ N-- N 1
-N XI Xi XI
14111
N 0
"11N
N F/ N I, F N
H3 C/...'""`"/"''''' W.'0"."--0
0...,õõ.. N 0 N
1 1 1 1
Xi Xi Xi Xi
N/X1
NN
,0 NX1 0
ON:PN-
, N H3C ---f.........f
1 N i\ 1
0, ___N /N)
y,,--
H3C-iSi N
0-C1-13 H,C 0 1
H3C CH3 Xi
F
IX10'CH3
XI\ N ,õ
XI\ N ^,
N f\l f\l
Xi
F F
B , F F F Br
XII
..õ,x, 0......._\ --- N N
N ' / Na CH3 F
N CH3
1 N 0 N-
Xi
F )(1
51
CA 03111433 2021-03-02
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N/ \
N)(1 ----
/CH3
N ___ N¨X
/ -1
Nµ
CH3
N N
1 \ CI N 0µ
Xi F F
F )S CH3
CI
N
/
H3C,N / N\ /
H3C,0
H3C
N N N 1\I
0 Xi
1 \ \ 1\I 1
Xi )S Xi Xi CH3
is
N
/X1
0
N
= 0 N ,
0 D\¨N ri\iS
r
I, ItCH3
)S\ N F or\I
N)S1
H3C.....NAo
0=s
/\\ 1
H3C 0 CH3
,
/X1
Xi N
N Ill
,p
)S H,C f\I /0
Xi
S/ CH
1
N N¨N ION 3 \NI
o ,c)
ilt \ \\\1 H3µ,, ---N H3C,,NA'0
N \ 1 11 e¨N s,CH
\\ -õ,, 3
CH3 CH3 0
X1 X1
XI µI\J
X1 IN
X1
\NI 0 I 0
N
0
. N¨CH3 * N N __ N
/
* NH, H3C * H3C H3C
i<1 H H
N X.N X.N H
0¨ ) 0 X.N
_______________________________________________ 0¨/ 6
/N ____________________________________________________________________
\ 0
52
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H3Cs
Xi
0
IN,.......õ(0
0
/
H
0
X.N X.N
r...-NN)-....N 4100 000 j
H3C \ /-0
)S HC 41 0-/
CH3
, 1
HN X
0
C)
>---=----N
X 0 Sr\..___/---/ 0
N.......\,y& -) \ 0--CH3
H C V-r\I N H3C,0 Nei
N 3 N \ 0 NI---
CH3 Xi I
,......õ
NI/X1 HN
N i Xi X-z--N 0 X=====:--
N
\xi N
,
N ________________________________________________ e
0 x___N, õc, 0,CH3
0 s
1 0,CH3 CH3 H
CH, S\'`.=,,,."'"-or.---sC 0 N
H3 ----
CH,
NO N
I / I /
XI X X1
1 N
H3C,N *
CH, /N
H3C -1..)
0 NiCH3 0 NI/CH3 0
\ 0 N/CH3 /CH,
Xi CH3 Xi 1 0
t t \ N
N N Xi CH3
t
N
0 0 /CH,
H30_ N\ H3C- NI 0 N
\
0 \
CH3 CH3 H2N CH,
H H
X.N X.N
H H
0 X.N X.N 0
0 0
411 0-/
41 0-/ 4104 0-
0 0-
/ F , Me0
53
CA 03111433 2021-03-02
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,,NI,
IN \ - XI \ N Xi N
H
XI 1 iN XiN
X1 -N
N
. 0 N 404 \CH3
I. H3C-- N \ N 0
0 -
, S
CI CH3 IN
H3C-N
/ N '-- N )--z--- N
X1 N I S N
0\
= .---
N
0 N
f\I I.
H3C
N N N N
0
H30-N
OH
7 N Nr
N,CH3
N
H3C-CH3 S---'''''
...-- N
\\
N
)--z---
0\
N 0
0 C7 N
IN -,
x(N x( N
Xi N
)N
X( N
XIN
CH
y_--N 411 /X1 3
H3C \ /
4,01-13
/ N I \
\\c.:>
N
0 µ
CH3 N
H3C
NI)/ _________________________________________
)- / \
<
______________________________________________ 0
_______________________________________________________ 0 XII
N NI' \
0 CH3
CH3
N)=3 /
CH3
N X1 H3C\ N/.'
XII )/ __ )_µN- CH3 N
N \ XII Xi
I
N 0
H3C)- 0 N 0 N 0 0
cj
H3C-,...\ 0
/X1
N
H3C...., N)---"-- CH3
dxi
NI/xi
H3C\
XII N1)/-)-N . 0 )/- N N- CH3
N
N1\1_ \ _____________________
H3C) __________________________________________ 0
41 \)
0 0
, ,
54
CA 03111433 2021-03-02
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CH3 F
Nl _CH3 F
H3CN
N N N
N-CH,
XII XI XII
I
N 0 N 0 0 N 0
0
H3C
0
N N
/X1 H30....,/--"-1
..s.8
H3R Xi Xi
I I
NO )/ \ N-CH
* 3
NI\ _ N 0 0 N illo 0
O , 0
,
0
Xi 0 OH
1 N 0 N N
\ / CH3 XI
t N H3 I
N 0 0
C
CH3
--N HO
N \\
CH3 \---\ CH3
N XI 0
N- /X1 OH
OH3
N 0 S 0,
\ N N 0 N-
N
\--.13CH3 __
0
CH3 HO
H3C __________ CH3
\--\ N-CH
N 3 cj0
rc)
/X1 N
XII XII N Xi
N,
IN 0 N 0 N 0
Nh
*
0
H:C...)(cH3
H
X.N XI 0
0
)--z--N CH3
S N
NI
L'.../
I NI
N ilo 0 N 0 0
0 '.
I H3)
C
CA 03111433 2021-03-02
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/CH3
\
N
0 /X1 0
N H3C
/X1 \ Xi / CH3
1 N
H3C H3C- N N- CH3 N 1
io -
N-CHCH3
N
1 0
Xi 0 H3C CI
0 0 OH 0 0
XI / CH3 xi
% N 1 N 1 N
Xi N 1 N 1 N 1
% N CH3 CH CH3
N 1
CH3
H3C H3C H3C
CH3 CH3 CH3
CI H3C H3C H3C
0 0
N
Xi /CH3 xi
XI 0
IN N
1\1
1 I
1 / HC
\ /CH,
N
CH3 CH3 I N
N-\_
OH N I
CH,
0
H3C/0
H3C/0
H3C F
0 0 OH 0 0
,CH3 xi ,CH3
N 1 N 1 N 1 N
CH, CH, N
CH3
0 0 0
1 1 1
F CH, CH3 CH3
R i CH3 R CH3 R 0H3
\ ______________________________ N
Ni\ \ __ d
H Xi CH3 Xi CH3 X CH3
X.N 0 IN __ µ / 1\1-µ / N _____ µ /
N N kr-
---- i
N
CH3 /0 N
Br H3C H30 H30
.,..N,
N \ -
õ.. NI,
N \` I N NI \ -N
/1\I N \ i /
\ N CH3 XlµN N \CH3
N 'CH3
H3C R
H3C, N 0 1 N
H,C..... N 0 0 XIIN ----,
1 0
\ CH3
CH, OH N /
56
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CH3
XI N"---4
CH3
o N r\L
1 N ) ) X. N X. N \\)
N
N ----
\ 0
\ ,
N / ) __ ,/ / ) __ e
N ,
Br)- 0 )- 0- N
H3C-N i
\
CH3 Me0 Nr:----
m"
XI
1
N N
I
CH3
f=J / f=1 / A---..:),
H3C N H3C N H3C N
0 0
Xi XI XI Xi
N I N 1 N I N 1
CH3 CH3 CH3 at
0 /NI
X1 XI X1
N H3C"-N /
N \ N 1 H3 N 11110 \cH3
CH3 C N
X(
R CH3
\ ___ Ni\ at
XII
N'-'%\ N N
/
N f=J e N
1 N --- =
N ---- N N
\ XI
N / N ---- N ----
\ \
CH3 N / N / H30
0 CH3
)(It N1\ CH3 Xi NH 0 X.NH
N \ /
N
_c.
12/-)
- 0 N"'<
) ______________________________________________ 0_
?-
= ,
57
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R2 is selected from
X2 X2 F F X2
H3C CI
F
X2 11110 F X2 F
*
CI CI F CI Br
X2 X2 X2
X2
X2 X2
401 10 10
1 10 F CI H3C \
CH3 a
CI F CI F
X2
x2 X2 X2
Br
\ F , Br ;
R3 is H;
R4 is H;
or R3 and R4 together are forming a CH2-CH2 group.
Another embodiment of the present invention are compounds of formula 1,
wherein A is defined
as above; R3 is H; R4 is H; and R2 is defined as in Table 1 shown below; and
R' is selected from
/ CH3 XII
N N
40,
xiIN 0
xi
N H3 xi II N
S, 0
Sz.--.-, 0
1 \\ N N..... ,CH3 N
. N,N N 110 0 \CH3 ......... 4 N
1 / ;
CH3
\ II F -- N
1\1"-N F F
--
/X1
N
CH
N¨CH3
Xi
(3,13, Xi /
0 XI 0 / N
\ I N 0 \
0 N 0 N N
...,..õ,õ".õ.. Nõ. CH, CH , .-- 3
N I / N
/ CH, N--
\
H3C ..--
CH3
58
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H3
CH3 7 CH3
H3C-Th 0i/ N-"" CH3
C r\
C 0 Q 0 0--S....-.
1 õ \
kd N... 1
X N
t
CH3 )
N . 0' N
N N
'- N 10 N * N * N
NI, 1 I
\)--N Xi XXi
0 50H
/NI CH3
Xi r_::::N
N N
0 /
";=S --- S
0'
N
N
0*
N I
* N N N
I I I N XI I
CH3
c (-,
CH3
H C
N
0 3 0 0 N
N * ON
H2N I N * N 10
N
Xi I I
0 X Xi Xi
X
/
N
N"\ 0 /X1
N
P i¨N
H3C rr\IS' CH3 1 \
N
N 0 N-
N
H3C,NA'0 * \
N 0=s
N
X H3C 0 CH3
Xi
/
Xi N
N Ill
*
o
o N--I (N., ,
x, xi
S CH
0 NI N N
0 0
/ /0
H3C-- N 0 H3C,NA'0
\ I 4.S¨ Ns, õ..CH
CH3 CH3
0 4.
NH2
59
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X Xi
Xi N NIC H
N
X. N
4.0 1 0
N 0
N¨ CH3 441 N CH
/
4. x ----< ,
110
0 ¨
H3C H3C \ /N
0 N/CH3
\
Xi CH3
%
H / N
X N X.N X
0
H3C-- N 40
4100 00 j . 00 _/
H3C¨ N\
CH3
,
0 /CH3
N
0
0 N/CH3
/ CH3
Xi 1 0 H
IN Xi \
CH3 N X. N
IN 0
0 r CH3
411 0
¨/
H3C¨ N\0 N
0 \ 0
CH3 H2N CH3 /
H
H H X. N
H
X1 ¨N X.N 0 X.N
. 0 0 410.
O¨
F Me0 CI
,N N s/.:N
N =N XIN Xi
I ----
X \ 1 /N N \
N
N 1110 \CH3
N 0
CH3 , IN 3C
0 X( N x( N
H30¨ N
)
CH---= N --_,
s/.:N N N N3
0 N \\
\ N 0 = .---
N ---- N
ilk . 0
N N N x( N N
XI Xi Xi Xi(
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H3C-N
)--:--- N
0 y_- N 011 CH3
\ H CH3
N 0 3C \ /
N)Iiii\
N--\
XII XII
N 0 N 0
N
0 \
)N
CH3
CH3
H3Cm
CH3
NI/c! N H3G-._ N)---- CH3
/X1
XII Xi XII N H3C
I \
N 0 N 0 0 N 0 0
= N- CH3
0
, '
CH3 F
/X1 CH3 rF
N H3C ,,,,6,
N N /X1
N N - CH3 Is
Xi XII N
I
N 0 N 0 0 N 0 0
0
=
H3C 0
,
0
0
W-1-'6' H3C, N=/--"I'
Xi Xi Xi XII
I I i N
N0 0 N0 0 N I N 0
CH3
N-- N HO
0H CH3
N N S N¨/ /X1
Xi XII XII N ( CH
I N, 0 N 0 N 0
0
CH3 HO
H3C _____ CH3
\-- N N-CH 3
N
N/41*....-- )
XII XII Xi Xi
I I
N 0 0 N 0 N * 0 N ,o
61
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/CH,
H3C CH3
\
N
0 /X1
r \CH3 N
N /X1 H3C
Xi H3C H3C¨N N¨CH3
I
N 0 0 N¨CH3
N 0
1
*
Xi 0 H3C
,
0 OH 0
O ,CH3
X ,CH3
I N I N i N 1
CH, CH3
N 1 N 1
CH3 CH3
H,C H3
CH, C CH3
CI CI H,C H3C
O 0 0
Xi Xi ,CH3
Xi Xi
% N N N /
N 1 N 1 IN \ N HC
CH3 CH3 CH3 N¨\_
OH
H3C CH3 /o /o 0
H3C C H3
H3C H3C
O 0 0 OH 0
Xi ,CH3
,CH3
N \ N
N 1 CH,
N 1 i
N 1 N 1
CH3 CH, CH3
0 0
I I
F F CH, CH3
N \\
NNI\" Xi 71
Nt,-N\\N
% N
1 11 N/' CH3
CH3 Xi% N
t N ,CH3 N \
3
N
N \ CH
0 CH,
Xi
% H3C,....N 0
N
0 H,C,N 0
1 I 0 o
CH3 CH, OH
62
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0 0
Xi XI Xi XI
N 1 N 1 N 1 N 1
CH3 CH3 CH3 CH3
0 0 0 0
Xi XI XI Xi
N 1 N 1 N 1 N 1
CH3 CH3 CH3 CH3
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is defined as above; R3 is H; R4 is H; and
R2 is defined as in Table 1
shown below; and R' is selected from
,CH3
N N 110
Xi
/XI 1
N 0
0 XI
/
N F13 X1 \\
S--.. 0S-1.1:1-0 N
iii
% \\ N
NI, N N 11110 0 L
N
\ II F CH3
NN F F ..---
,CH3 / CH3
N
õ \
N
Xi cNo
C 0 Li ....
µ1\1 11110 0.31 / CH3
-- S
Xi N N Q
,
N
0 \
`=-= N . N 410 N 10 N 10 N
NI---N N \ 1 I
CH3 ....--N )S Xi Xi Xi
H3
CH
N.__ ,
ci.,/ /
0 50H
H3C
/ \\ XII
0.A ) 0
0' N 0..;=-s/ N N . 0..A
0'
N
0 N = N N N 0 40 N --...... I
I I I N XI
XI XI XI CH3 xi \N
63
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N/ \
H2N
0 / 0 0,CH3
H3C
L..../N 1110 ON
N
N H2N \ N 410 N N
I
XI I I
XI o xi xi
xi
)S H3C Xi )S IN
Xi
1 \ IN
N N-N N
,o o
fat \NIN
O
lit 6, ,CH3 11 N-CH3
/
0 . NH2 H30
XI
IS H
IN N X.N H
0 CH3
0 0-
X.N
\ /N lip . 00 j
H3C
0 CH3 0 CH3
cr N N 0 CH3
\
Xi CH3 Xi N
IN IN Xi N / \
/ /
CH3
/
I
X.N X1
to j
H3C'N 40 0 0
00
H3C-N H3C-N 0
CH3 CH3 H2N
0
Xi ,CH3
1 0 H
N X.N H H
0 X (DJ .N X.N
CH3 41104 . 0
N . 0 .
0 \ 0 0-/ 0-
CH3 / F , Me0
H IN ,, ,1\1 \- X1\N Xi N
X.N H
XI 1 iN
0 X.N N
0-
40 0 INSN 404 \at
H3 C
C I CH3
0 -
, IN o
64
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H3C-N
,CH3
N N )--:--- N
I 0 yr- N 011 CH3
0
H3C \ -/ 0 N 0
N- \
XII
CH3
N N 0
N 0 \
x( N x( N
X( CH3
CH
H3C,..õ1
CH3 H /X1
N
3C, N)-11.1- CH3
N /X1
Xi XII N H30 N-
CH3
I \
N 0 0 N 0 0
* N- CH3
0
0 H3C
CH3 F
CH3 rL H3CN ,,,,6,
N N N F /X1 N---
A
XII Xi XII N
I 0 )11
N 0 N 0 0 N 0
* N N
* 0
0
HO
a.,,z
H3C...,7 OH' 's118 0
N XII N-
Xi Xi Xi
1 % N H3
I
N 1110 0 N I N 0 0 N 0
C
OH HO
H3C+ cH3
\---\
cH3 r0
N N- CH3 N
N K CH3 Xi XII Xi
N- I 1
N * 0 N 0 N0 0
* 0
/X1 0 0
N H3C
/X1 \ Xi r CH3
Xi
1 N 1 N
H30-N N- CH3 N I N I
. N - CH3 CH3
CH3
0
0 H30 CI CI
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O OH 0 0 0
,CH3
Xi /
CH,
% N % N % N N
N 1 N 1 N \ IN
I
CH, CH,
H3C
CH, H3C
CH, H3C
CH, /0
H3 C H3C H3C H3C
0
Xi Xi 0 t /
N \ N H3C \
N XI ,CH3 0
XI
CH,
IV N t N
CH,
OH \ N I
CH,
/0
0
H3C H3C F F
O OH 0 0
XI /-----/ Xi ,CH3
Xi / CH, 0
t N \ N 1 N
N 1 N 1 N Xi
CH, CH3 1 N
N 1
0 0 0
1 1 1 CH3
CH, CH3 CH3
O 0
Xi Xi Xi Xi
i N
N 1 N 1 N 1 N 1
CH3 CH, CH,
CH3
X1 XI XI
% N
N 1 N 1 N I
CH, CH3 CH3
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is defined as above; R3 is H; R4 is H; and
R2 is defined as in Table 1
shown below; RI[ is selected from
66
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X
IN N, Xi Xi 0
Xi
CH,
N \N--- A N XII I
S /
¨7---o
H3C
----)____N/
\--CH3 H3 N/ NJ
--':;_/ 'C) CH3
N
I
CH3 H3C--.1\11\1/ (21
N
0
HC H
NI/ b XI Xi X. N
XII (1 i sNly>._.4
), ________________________________________________________________ )
NN 0y N N
I ---, N
I N--
/ N )¨
0 ¨/
Xi HC H3C
CH3
H3C, c
0 Xi' N 0
X
NI,...,f No 0
0J) i CD
H
X N 0 NCH--
C) 3
H3C
¨V) 0
Nh_/ H3C N
\
0 Xi H3C CH3 Xi
X
, 1
/X1
HN
)----:---N 11 HN
N' ------- N Xi
' S 0
0 N X----N
\
)=--- II 11 \\ eo...._\CH3
---- N s O
N
I S \\..?L`= o''''' CH C)CH3 3 CH3 0 N'-'11--
H
CH3
CH,
H3C at
Xi NO N N /
0
Xi IN / Xi
N Xit N 0
I
N(
H3C
CH3 '1\1-0 I-13C-- N \
\ N N N
N = N = / XI
/ __ 0
/X1 /X1 /X1
\ __
N/CH3
I /\N
N ) H3C
H3C
NI)/ _______________ <
)¨ / )
\
____________________ N
_____________________________ 0 N
I-13C/
\
0
NI CH3 N'
N)/--)--
\ _ 0 _____________ N
0 N
NI)
H3C)/¨)
67
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0 X
H
N
X.N i 0
/X
Xi )
NI/X1
N H3S N ---- 1
0
\ / CH3 N)
)/ ___________________________________________ 0
)/¨
N) N¨CH3 CH3
0 0 \ _ \ N\_ II
H3C
0\ i
CH3 0\ CH3 R
CH3
\ ______________________________ N"
N\ \ __ Ni
H Xi CH3 X CH3 X CH3
X.N 0 IN / 1=1¨µ / IN ___ µ /
N N N
N N'
--- i CH3 /0
Br H3C H3C H3C
CH3
N-.4 X CH
H3C 0\ XI H H
, / v m
% N ,...., X.N \ /
), 0
1 \ ,,
)¨) N ¨ )r) __ e
N ---- N , N
\ z CH3
0¨ ) N
0_ H3C¨N i
\ N m , CH3 Br Me0 N----;"
N-5;\ N
L. /
X ¨
N_1 % N
N ----
H3C---N V \
N /
,..N.õ f\l/ N
H3C N H3C N H3C '1\1 CH3
0 /CH3
N
\
X CH3
0 CH3
N\/ N
/
N ,-N N
X1 ¨ \
N CH3
---- =
X
õ...... N
XII ()L1\17 %
N \
N3 /
1
N ---- N ---- N
\ z \ .õ
N , H3C
H
X.N
H
x, I)/ e ,
N ______
0
_/ 0 Ni/ __ )
?_ ______________________ 0¨
, ____________________________ =
68
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H3
CH3 / CH3 0
i/ N."" CH3
C i\ii0 H30
k.-) N.,.
Xi CN
t CI
0 ,
0.,fi / CH3
N . S ' N
N N Q
'44- N AP N 1. N 10 N * N .. * N
N , 1 I
Xi
/NO
;ICH, OH
XII N
_
0'
N
*
0 . I N N
N N N
I I I N \
XI CH3 xi \NI
p NI n
,...,`-'
H2N \
, CH3 N
ON
0H30 0 0
\,.....,..õ/ N .
N
H2N \i
X N * N . N
\ 1
0 Xi X1 Xi
Xi
/
N
N"\ 0 /X1
N
_>
HC
11 'OH 1 \
N
N¨N
N 0
H3C,NA'0 * \
N 0=S
N
H300 CH3
X1
/
Xi N
N Ill
*
0
0 (N¨Ici r N,, ,
x, xi
S CH
0 Ni IN'''. 3 µN \NI
0 0
0 10
H3C--- N H3C,NA'0
\ 1 4. el¨Ns, õ,..CH
CH3 CH3 \\ .,õõ, 3
0 .
NH2
69
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Xi Xi
IV
Xi IV NC H
X. N
4.0 1 0
N 0
N- CH3 441 N CH
/
4. x ----< , 110 0 ¨
H3C H3C \ /N
0 N/CH3
\
X CH3
%
H / N
X N X. N Xi
0
H3C-- N 40
4100 00 j . 00 _/
H3C- N\
CH3
0 /CH3
N
0
0 N/CH3
X / OH3
Xi 1 0 H
IN X \
CH3 N X. N
IN 0
0 ,OH
411 a¨'
H3C- N\0 N
0 \ 0
CH3 H2N CH3 /
H
H H X. N
H
X1 -N X1 -N 0 X. N
. 0 0 410.
O¨
F Me0 CI
1\1 N s/.:N
N =N XN Xi
I ----
X \ 1 /I N
N 1110N \CH3
N 0
CH3 , IN 3C
0 X1( N x( N
H3C- N
)
CH---= N --_,
s/.:N 1\17--' N N3
0 N \
\ N 0 = .---
N ---- \N, . 0
N N N x( N N
Xi X Xi(
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H3C- N
)--:--- N
0 y_- N 011 CH3
\ N 0 H3C
NJ:1
\ /
N-- \
XII CH3 XII
N 0 N 0
N
0 \
N
CH3
CH3
H3Cm
CH3
1\i/c! N H3G-._ CH3
/X1
XII Xi XII N H3C
I \
N 0 N 0 0 N 0 0
* N-
CH3
0
, '
CH3 F
/X1 CH3 rF
N H3C
N N /X1
N N- CH3 )1(i
Xi XII N
I
N 0 N 0 0 N 0 0
0
*
H3C 0
,
0
0
W H30, 7-"I' N.18 0 N
Xi Xi Xi XII
I I i N
N0 0 N0 0 N I N 0
CH3
-- NN HO
\\
OH CH3
N N S N-/ /X1
Xi XII XII N ( OH
I N-
N 40 0 N 0 N 0
* 0
' ,
CH3 HO
H3C _____ CH3
\-- N N-CH 3 r 0 0
N
N/41*....-- )
XII XII Xi Xi
I I
N 0 0 N 0 N * 0 N ,o
71
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/ CH,
H3C cH3
\
N
0 /X'
r \ CH3 N H3C
N /X' \
Xi H3C H3C¨ N N¨ CH3
I
N 0 0 N ¨ CH3
N 0
\
*
Xi 0 H3C
,
0 OH 0
,0H3
X
I N I N i / CH3
N \
CH3 CH3
N 1 N 1
CH3 CH3
H3C H3
OOH
CCH3
CI CI H3C H3C
0 0 0
Xi / CH3
Xi Xi
% N N N /
N \ N 1 N \ N HO
\
CH3 CH3 CH3 N¨ \
_
OH
H3C CH3 /o /o 0
H3C C H3
H3C H3C
0 0 0 OH 0
Xi / CH3
/ CH3
N \ N
N \ CH3
N \ i
N 1 N 1
CH3 CH3 CH3
0 0
I I
F F CH3 CH3
,... N
N \\
7 N
N N\" I,. NN'
N
% N
1 11 N \
CH3 Xi% N
t N ,0H3 N \
3
N
N \ CH
0 CH3
Xi
% H3C,...,. N 0
N
0 H3C, N 0
1 I 0 0
CH3 CH3 OH
72
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0 0
Xi XI Xi XI
N 1 N 1 N 1 N 1
CH3 CH3 CH3
CH3
0 0 0 0
Xi Xi Xi Xi
N 1 N 1 N 1 N 1
CH3 CH3 CH3 CH3
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is defined as above; R3 is H; R4 is H; and
R2 is defined as in Table 1
shown below; and R' is selected from
,CH3
N N 110
Xi
/Xi 1
N 0
0 Xi
/
N H3 X1 \\
S--.. 0S-1.1:1-0 N
iii
% \\ N N N
,CH3
N-.....N N 11110 0 L
N
\ II F CH3 CH3 /
NN F F ...---
2H3 7 CH3
N
õ \
N
Xi cNo
C 0 Li ....
µ1\1 11110 0.31 / CH3
-- S
Xi N N Q
,
N
0
40 \\ S
`=-= N . N 410 N 10 N 10 N
NI---N N \ 1 I
CH3 ....--N Xi Xi Xi Xi
H3
i/ N." CH3 Niiii...../ CH,
0 50H
H3Cm
/ \\ XII
0--1 0 N
; S ) === / N . cxiA
0' N
0'
N
0 N = N N N 0 40 N N --..._ I
I I I
Xi Xi XI CH3 xi \N1
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N3
H2N
0 / 0 0CH3
H3C ,
L..../N 1110 ON
N
N H2N \ N 410 N N
I
XI I I
o xi xi
xi
H,C Xi Xi IN
X
1 \ I
N N-N N
N
o
,o
fik \
O
lit 6",õ ,CH3 11 N-CH3
/
0 . NH2 H30
XI
/(1 H
IN N X.N H
0 i X.N
CH3
\ /N ii 0
0- 4100 00 j
H3C
0 CH3 0 0H3
/ /
N N 0 CH3
\ 1
X OH
Xi N
IN IN Xi \
CH3
/
IN
X.N XI
H3C,N . 0 0
. 00 j
H3C-N H3C-N 0
CH3 CH3 H2N
X /CH3
1 0 H
N X.N H H
0 X.N X.N
/ _/
0 . 0
CH3 . .
N 0 .
0 \ 0 0-/ 0-
CH3 / F , Me0
HIN ,, ,
N \- X1\N XiN
X.N H
XI 1 iN
0 X.N N
0-
. 0 INS
e
CI CH3 404 \CH3
H3 lei
0-
, IN 0
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H3C-N
CH3
N N )--:--- N
I 0 yr- N 011 CH3
0
H3C \ -/ 0 N 0
N- \
XII
CH3
N N 0
N 0 \
x( N x( N
CH3
OH3
H30,1
CH3
H N/X1
CH 3C-..... N)-1111- 3
N /X1
Xi XII N H3C N-
CH3
I \
N 0 0 N 0 0
* N- CH3
0
0 H3C
CH3 F
CH3 rL H3CN ,,,,6,
N N N F /X1 NI---A
XII Xi XII N
I 0 )11
N 0 N, 0 N 0
* N N, 0
0
HO
ON.,,,
H3C..., Nr N.18 0 OH
N XII N¨
Xi Xi Xi
1 % N H3
I
N 1110 0 N I N 0 0 N 0
C
OH
3 HO
H3C cH3
\---\
cH3 r0
/X1 N N¨ OH
N
N K CH3 Xi )1 Xi
N¨ I 1
N * 0 N 0 N 110 0
* 0
/X1 0 0
N H3C
/X1 \ Xi r CH3
Xi
1 N 1 N
H30-N N- CH3 N I N I
. N - CH3 CH3 CH3
0
0 H30 CI CI
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O OH 0 0 0
,CH,
Xi x
CH,
% N % N % N N
N 1 N 1 N \ N I
CH, CH3 CH, CH3
H30
CH H3C
CH3 H,C
CH, r 0
H,C H3 C H,C H3C
0
Xi Xi
t N /
N \ N H3C
\ Xi ,CH,
CH3
H3C/ H3 C N N t N
CH
OH \ 3 N I
CH,
0
0
F F
O OH 0 0
,CH
Xi x CH,
0
t N \ N 1 N
N 1 N 1 N Xi
CH, CH3 1 N
N 1
0 0 0
1 1 1 CH3
CH, CH3 CH3
O 0
Xi Xi Xi Xi
i N
N 1 N 1 N 1 1
CH3 CH3 CH3 NH3
X1 XI XI
N 1 N 1 N 1
CH3 CH3 CH3
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein A is defined as above; R3 is H; R4 is H; and
R2 is defined as in Table 1
shown below; RI[ is selected from
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X
IN N, Xi Xi 0
Xi
CH3
N \N--- A N XII I
S /
¨7---o
c_._ ----)____N/
\--CH3 H3 N/ NJ
--':;_/ .. 'C) CH3
N
I
CH3 H3C--.1\11\1/ (21
N
0
HO H
o/Ntzz:19 el b X X. N
XII (1 sNly>._.4
), ________________________________________________________________________ )¨
/
NN N
1 ---- N
I y, N
N--
Xi H3C H3C 0
CH3
H3C, c
0 Xil N o
X
NI,...,f No 0
0J) C)
H
--
_____________________________________________ L CH
rrk0 3 3C
--
X.N
0
H
N H3C CH3
h H3C N
\
0 Xi X
X
, 1
/X1
HN
)----:---N 11 HN
N' -------N Xi 0 )7=N
)=--- 0 N NII "0
N
\\ eo...._\CH3
'
I S \\..?L`.0'''''CH C)CH3 3 CH3 0 N-.11--
H
CH3
CH3
H3C at
Xi N
N N /
0
Xi Li XI Xi% N 0
I
N--..
H30
IN-, --(--7-i"-N
CH3 'I\I-0 H3C. N \
\ N N N
N = N = / X( X(
/ 0
/X1 /X1 /X1
\ _______________________________________________________________________
xi.N---N OH
I /\N
\\c..> N ) H3C
H3C
1\1)/ <
)¨ / \
____________________ N
_____________________________ 0 N
H30/
\
0
)¨)4 CH3 N'
?-)---
\ _ 0
0 N
NI) I \I
H30
77
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0 X H
N /1 .
XN i 0
/X
Xi )
NX
N H3 C\ N ---- 1
0
\ / N CH3 N)
)/¨) N¨ CH3 N
) _____________________________________ N
\ _ \ \ _ 0 II
0
CH3 H3C
0
0\ i
CH3 0\ CH3 R
CH3
\ ______________________________ 1\1"
___________________________________________________ N\ \ __ Ni
H Xi CH3 Xi CH3 Xi CH3
X.N 0 IN ___ / 1N¨µ / IN ___ µ /
N N N
N N'
--- i CH3 /0
Br H30 H30 H30
CH3
Xi 3
N"---.4 CH
o IN 1\
H30 0 /\ XI
, v H H
m
% N ,.. .., X. N\\)
)/ ______________________________________ 0
1 \ ,,
)¨) N ¨ )r) __ e
N ---- N , N
\ z CH3
0¨ ) N
0_ H3C¨
N i
\ N CH3 Br Me0 N-- "
N-5;\ N
L. /
XI ¨
1\1_1 % N
N ----
H3C---N V \
N /
õNõ N
H3C N H3C N H3C '1\1 CH3
i0 N1CH3
\
XI CH3
1 0 CH3
N / N
N .,.. N N
X1 ¨ \
N CH3
---- =
õ...... N
Xi
XII ()L1\17 %
N \
N3 /
1
N ---- N ---- N
\ z \ .õ
N , H30
H
X.N
H
x, I)/ e ,
N ______
0
_/ 0 Ni/ __ )
?_ ______________________ 0-
, ___________________________ =
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Table 1: R2 is defined as one of the groups shown below in the definitions 1
to 4:
Definition 1 x2 x2 F
H,C 0 CI 0
X2
1110 F F
CI CI
F X2 X2 X2
F
X2 F
S0 10
CI Br CI F
X2 X2 X, X2
01 10 .
H,C
F 0 CI
CI F CH, Br .
Definition 2 x2 X2
X2 X2
H,C CI 0
1. F CI10 CI CI CI F
X2 X2
1$1 40
H,C
CH, Br .
,
Definition 3 X2
X2
\ Br
CI -0S)
, .
Definition 4 X2 X2
F .
,
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein the compounds of formula 1 are present in the
form of the individual
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optical isomers, mixtures of the individual enantiomers or racemates, e.g., in
the form of the
enantiomerically pure compounds.
Another embodiment of the present invention further comprises administration
to a subject of the
compounds of formula 1, wherein the compounds of formula 1 are present in the
form of the acid addition
salts thereof with pharmacologically acceptable acids as well as optionally in
the form of the solvates and/or
hydrates.
b. Co-Crystals and Salts
Additional embodiments of the present invention further comprise
administration to a subject of
the co-crystals of the compounds of formula 2 (below). In general, for groups
comprising two or more
subgroups in this "Co-Crystals and Salts" section, the first named subgroup is
the radical attachment point,
for example, the substituent 'C13-alkyl-aryl' means an aryl group which is
bound to a C1-3-alkyl-group,
the latter of which is bound to the core or to the group to which the
substituent is attached.
R3
0 R2a
* (HX) R2b
2
wherein
is Cl 6-alkyl, C1 6-haloalkyl, 0-C1 6-haloalkyl, halogen;
is 1, 2 or 3; and in some instances 1 or 2;
R2a and R2b are each independently selected from H, Cl 6-alkyl, C1 6-alkenyl,
Ci 6-alkynyl, C3 6-cycloalkyl,
COO-C16-alkyl, 0-Ci 6-alkyl, CONR2b 1R2b 2, halogen;
R2b is H, Cl 6-alkyl, Co 4-alkyl-C3 6-cycloalkyl, C1 6-haloalkyl;
R2b 2 is H, Cl 6-alkyl;
or R2b and R2b 2 are together a C3 6-alkylene group forming with the nitrogen
atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom
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R3 is H, C1_6-alkyl;
X is an anion selected from the group consisting of chloride, bromide,
iodide, sulphate, phosphate,
methanesulphonate, nitrate, maleate, acetate, benzoate, citrate, salicylate,
fumarate, tartrate,
dibenzoyltartrate, oxalate, succinate, benzoate and p-toluenesulphonate; and
in some instances
chloride or dibenzoyltartrate
is 0, 0.5, 1, 1.5 or 2; and in some instances 1 or 2;
with a co-crystal former selected from the group consisting of orotic acid,
hippuric acid, L-pyroglutamic
acid, D-pyroglutamic acid, nicotinic acid, L-(+)-ascorbic acid, saccharin,
piperazine, 3-hydroxy-2-
naphtoic acid, mucic (galactaric) acid, pamoic (embonic) acid, stearic acid,
cholic acid, deoxycholic acid,
nicotinamide, isonicotinamide, succinamide, uracil, L-lysine, L-proline, D-
valine, L-arginine, glycine, in
some instances ascorbic acid, mucic acid, pamoic acid, succinamide, nicotinic
acid, nicotinamide,
isonicotinamide, 1-lysine, 1-proline.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C1_6-alkyl, C1_6-alkenyl, C1_6-alkynyl, C3_6-cycloalkyl, 0-C1_6-
alkyl, CONR2a.1R2a.2 ;
R2a.1 is H, C1_6-alkyl, C1_6-haloalkyl;
R2a.2 is H, Ci_6-alkyl;
R2b is H, C1_6-alkyl, C1_6-alkenyl, C1_6-alkynyl, C3_6-cycloalkyl, COO-Ci_6-
alkyl, 0-C1_6-alkyl,
coNR2b.iR2b.2, halogen;
R2b.i is H, C1_6-alkyl, C0_4-alkyl-C3_6-cycloalkyl, C1_6-haloalkyl;
R2b.2 is H, Ci_6-alkyl;
or R2" and R2b'2 are together a C3_6-alkylene group forming with the nitrogen
atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom and the
remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
Rza is H, Ci_6-alkyl, Ci_6-alkynyl, C3_6-cycloalkyl, 0-C1_6-alkyl,
CONR2a.1R2a.2 ;
R2a.1 is C 1_6 -alkyl ;
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R2a.2 is H;
R2b is H, C1_6-alkyl, 0-C1_6-alkyl, CONR2b.1R2b.2;
R2b.1 is C1_6-alkyl, Co4-alkyl-C3_6-cycloalkyl, C1_6-haloalkyl;
R2b.2 is H, C1_6-alkyl;
or R2" and R2b'2 are together a C3_6-alkylene group forming with the nitrogen
atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom and the
remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C14-alkyl, C14-alkynyl, C3_6-cycloalkyl, 0-C14-alkyl,
CONR2a.1R2a.2;
R2a.1 is C1_4-alkyl;
R2a.2 is H;
R2b is H, C14-alkyl, 0-C14-alkyl, CONR2b.1R2b.2;
R2b.1 is C14-alkyl, C04-alkyl-C3_6-cycloalkyl, C14-haloalkyl;
R2b.2 is H, C14-alkyl;
or R2" and R2b'2 are together a C3_6-alkylene group forming with the nitrogen
atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom and the
remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C14-alkyl,
R2b is H, CONR2b.1R2b.2;
R2b.1 is C14-alkyl, C04-alkyl-C3_6-cycloalkyl, C14-haloalkyl;
R2b.2 is H, C14-alkyl;
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or R2b.1 and R2b.2 are together a C3_6-alkylene group forming with the
nitrogen atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom and the
remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R1 is Ci_6-alkyl, C1_6-haloalkyl, 0-C1_6-haloalkyl, halogen;
is 1 or 2;
R2a is H, C1_4-alkyl;
R2b is H, CONR2"R2b.2;
R2b.1 is C1_4-alkyl, C0_4-alkyl-C3_6-cycloalkyl, C1_4-haloalkyl;
R2b.2 is H, C1_4-alkyl;
or R2" and R2b'2 are together a C3_6-alkylene group forming with the nitrogen
atom a heterocyclic
ring, wherein optionally one carbon atom or the ring is replaced by an oxygen
atom
R3 is H, Ci_6-alkyl;
X is an anion selected from the group consisting of chloride or
dibenzoyltartrate
is 1 or 2.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C1_4-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C1_4-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b.2 is C1_4-alkyl; in some instances Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C1_4-alkyl; in some instances Methyl, Ethyl, Propyl;
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R2b is H, CONR2b.1R2b.2;
R2b.1 is Co_4-alkyl-C3_6-cycloalkyl;
R2b.2 is H, C1_4-alkyl; in some instances H, Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2a is H, C1_4-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C1_4-haloalkyl;
R2b.2 is H, C1_4-alky; in some instances H, Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein
R2b.1 and R2b'2 are together a C3_6-alkylene group forming with the nitrogen
atom a heterocyclic ring,
wherein optionally one carbon atom or the ring is replaced by an oxygen atom
and the remaining residues
are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein IV, m, R2a, R2b, R3, X and j
are defined as above and the
co-crystal former is selected from the group consisting of ascorbic acid,
mucic acid, pamoic acid,
succinamide, nicotinic acid, nicotinamide, isonicotinamide, 1-lysine, 1-
proline, or hydrates or
hydrochlorides of the same.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2a, wherein R2a, R2b, R3, X and j are
defined as above
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H
CI =
* (HX). R2b
2a.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b.2 is C14-alkyl; in some instances Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C04-alkyl-C3_6-cycloalkyl;
R2b.2 is H, C14-alkyl; in some instances H, Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C14-haloalkyl;
R2b.2 is H, C14-alky; in some instances H, Methyl, Ethyl, Propyl;
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and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2a, wherein
R2b and R2b 2 are together a C3 6-alkylene group forming with the nitrogen
atom a heterocyclic ring,
wherein optionally one carbon atom or the ring is replaced by an oxygen atom
and the remaining residues are defined as above.
The free bases of compounds of formula 2 (j = 0) are often amorphous and are
used for a process
of manufacturing co-crystal, nevertheless salts of compounds of formula 2 are
employed in some
instances for a process of manufacturing co-crystal. Thus, another aspect of
the invention are salts of
compounds of formula 2 wherein IV, m, R2a, R2b, tc ¨3
are defined as for the co-crystals above and
X is an anion selected from the group consisting of chloride, bromide,
iodide, sulphate, phosphate,
methanesulphonate, nitrate, maleate, acetate, benzoate, citrate, salicylate,
fumarate, tartrate,
dibenzoyltartrate, oxalate, succinate, benzoate and p-toluenesulphonate; in
some instances
chloride, or dibenzoyltartrate
is 0, 0.5, 1, 1.5 or 2; in some instances 1 or 2.
Another aspect of the present invention further comprises administration to a
subject of the co-
crystals of the compounds of formula 2, wherein IV, m, R2a, R2b, R3 are
defined as for the co-crystals
above and
X is an anion selected from the group consisting of chloride or
dibenzoyltartrate
is 1 or 2.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2, wherein IV, m, R2a, R2b, R3 are defined as for
the salts above and X is
chloride and j is 2.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2, wherein IV, m, R2a, R2b, R3 are defined as for
the salts above and X is
dibenzoyltartrate and j is 1.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein R2a, R2b, R3, X and j are defined as
above
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OO
2a
C I =
* (HX). R2b
2a.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b.2 is C14-alkyl; in some instances Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C04-alkyl-C3_6-cycloalkyl;
R2b.2 is H, C14-alkyl; in some instances H, Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein
R2a is H, C14-alkyl; in some instances Methyl, Ethyl, Propyl;
R2b is H, CONR2b.1R2b.2;
R2b.1 is C14-haloalkyl;
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R2b 2 is H, C1 4-alky; in some instances H, Methyl, Ethyl, Propyl;
and the remaining residues are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein
R2b and R2b 2 are together a C3 6-alkylene group forming with the nitrogen
atom a heterocyclic ring,
wherein optionally one carbon atom or the ring is replaced by an oxygen atom
and the remaining residues
are defined as above.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein IV, m, R2a, R2b, R3 are defined as for
the salts above and X is
chloride and j is 2.
Another aspect of the present invention further comprises administration to a
subject of the salts
of the compounds of formula 2a, wherein IV, m, R2a, R2b, R3 are defined as for
the salts above and X is
dibenzoyltartrate and j is 1. Another aspect of the invention are salts of
compounds of formula 2a,
wherein IV, m, R2a, R2b, R3 are defined as for the salts above and X is (S)-
(S)-(+)-2,3-dibenzoyl-tartrate
and j is 1.
c. Formulations
Additional embodiments of the present invention further comprise
administration to a subject of a
pharmaceutical composition containing compounds of formula 3
o *),
H R1
N¨R
0
CI 0
* (HX)1 N \ 3
wherein
is H, Cl 6-alkyl, Co 4-alkyl-C3 6-cycloalkyl, C1 6-haloalkyl;
R2 is H, Cl 6-alkyl;
X is an anion selected from the group consisting of chloride or 1/2
dibenzoyltartrate
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j is 1 or 2.
An embodiment of the present invention further comprises administration to a
subject of a pharmaceutical
composition containing compounds of formula 3 wherein
R1 is H, C16-alkyl;
R2 is H, C16-alkyl;
X is an anion selected from the group consisting of chloride or 1/2
dibenzoyltartrate
j is 1 or 2.
An embodiment of the present invention further comprises administration to a
subject of a pharmaceutical
composition containing compounds of formula 3 wherein
R1 is H, Methyl, Ethyl, Propyl, Butyl;
R2 is H, Methyl, Ethyl, Propyl, Butyl;
X is an anion selected from the group consisting of chloride or 1/2
dibenzoyltartrate, such as chloride;
j is 1 or 2, in some instances 2.
An embodiment of the present invention further comprises administration to a
subject of a pharmaceutical
composition containing compounds of formula 3 wherein
R1 is H, Methyl, Ethyl, Propyl, Butyl;
R2 is H, Methyl;
X is an anion selected from the group consisting of chloride or 1/2
dibenzoyltartrate, such as chloride;
j is 1 or 2, in some instances 2.
An embodiment of the present invention further comprises administration to a
subject of a pharmaceutical
composition containing compounds of formula 3 wherein
R1 is H, Methyl;
R2 is H, Methyl;
X is an anion selected from the group consisting of chloride or 1/2
dibenzoyltartrate, such as chloride;
j is 1 or 2, in some instances 2.
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An embodiment of the present invention further comprises administration to a
subject of a pharmaceutical
composition containing compounds described in Table 2 as a hydrochloride. An
additional embodiment of
the present invention further comprises administration to a subject of a
pharmaceutical composition
containing compounds describe in Table 2 as a di-hydrochloride.
TABLE 2
Structure
0
NH
HN
N
1
\
101
CI
0
1\
HI\l/¨o
2 ci 4110 N
e¨
\\
)_ 0
0
/¨o
3 ci = N HN
o
)_
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# Structure
,c) N ) 0
H
N
...../.-.õ, HN / \
N*---
4
CI
0
0 ) 0
N H
N
HN / \
N"--- F
N..,...- F
CI0
CIJN ) 0
N if
H
N
HN / \ )
N"---
6 N
C's
OJN ) 0
N if
/
N
HN
N"---
7 N
C's
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Structure
µN-7
o
8 HN
CI 41100
NI)/ ) (
)-
oo
/=0
9 ciHN)__\
N/
0.N)" 0
/
N
H
N
N
CI
Another object of the present invention is administration to a subject of a
pharmaceutical dosage
form of the compounds described above, wherein the dosage is an orally
deliverable dosage form.
Another object of the present invention is administration to a subject of a
pharmaceutical dosage
form of the compounds described above, which is in the form of a tablet,
capsule, pellets, powder or
granules.
Another object of the present invention is administration to a subject of the
pharmaceutical dosage
forms described above for use as medicament.
Another object of the present invention is the use of the above pharmaceutical
dosage forms for the
preparation of a medicament for the treatment of a neurodegenerative disease
or condition selected from
Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington
disease, amyotrophic lateral
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sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, vascular
dementia, and progressive
supranuclear palsy.
Another object of the present invention is a process for the treatment and/or
prevention of a disease
or condition selected from neurodegenerative disease such as Alzheimer's
disease, Parkinson's disease,
frontotemporal dementia, Huntington disease, amyotrophic lateral sclerosis,
multiple sclerosis, glaucoma,
myotonic dystrophy, vascular dementia, and progressive supranuclear palsy,
characterized in that an
effective amount of the above defined pharmaceutical dosage form is
administered orally to a subject or
patient once, twice, thrice or several times daily.
d. Dosage Forms/Ingredients
Solid pharmaceutical compositions ready for use/ingestion made from a compound
of formula 3
comprise powders, granules, pellets, tablets, capsules, chewable tablets,
dispersible tables, troches and
lozenges. In detail:
= Capsule formulations according to the invention comprise the powdery
intermediate of a compound of
formula 3, an intermediate blend comprising the powdery intermediate, pellets
or granules obtained by
conventional wet-, dry or hot-melt granulation or hot-melt extrusion or spray-
drying of a suitable
intermediate blend, filled in conventional capsules, e.g. hard gelatin or HPMC
capsules.
= The Capsule formulations from above may also comprise the powdery
intermediate of a compound of
formula 3 in a compacted form.
= Capsule formulations according to the invention comprise the compound of
formula 3 suspended or
diluted in a liquid or mixture of liquids.
= Tablet formulations according to the invention comprise such tablets
obtained by direct compression
of a suitable final blend or by tableting of pellets or granules obtained by
conventional wet-, dry or hot-
melt granulation or hot-melt extrusion or spray-drying of a suitable
intermediate blend.
Another object of the present invention is a dosage form where a pH-adjusting
or buffering agent
is added for stability improvement of the active ingredient. The pH-adjusting
/ buffering agent may be a
basic amino acid, which has an amino group and alkaline characteristics
(isoelectric point, pI: 7.59-10.76),
such as e.g. L-arginine, L-lysine or L-histidine. A buffering agent within the
meaning of this invention is
L-arginine. L-arginine has a particular suitable stabilizing effect on the
compositions of this invention, e.g.
by suppressing chemical degradation of compounds of formula 3.
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Thus, in an embodiment, the present invention is directed to a pharmaceutical
composition (e.g. an
oral solid dosage form, particularly a tablet) comprising a compound of
formula 3 and L-arginine for
stabilizing the composition, particularly against chemical degradation; as
well as one or more
pharmaceutical excipients.
Suitably the pharmaceutical excipients used within this invention are
conventional materials such
as cellulose and its derivates, D-mannitol, corn starch, pregelatinized starch
as a filler, copovidone as a
binder, crospovidone as disintegrant, magnesium stearate as a lubricant,
colloidal anhydrous silica as a
glidant, hypromellose as a film-coating agent, polyethylene glycol as a
plasticizer, titanium dioxide, iron
oxide red/yellow as a pigment, and talc, etc.
In detail pharmaceutical excipients can be a first and second diluent, a
binder, a disintegrant and a
lubricant; an additional disintegrant and an additional glidant are a further
option.
= Diluents suitable for a pharmaceutical composition according to the
invention are cellulose powder,
microcrystalline cellulose, lactose in various crystalline modifications,
dibasic calcium phosphate
anhydrous, dibasic calcium phosphate dihydrate, erythritol, low substituted
hydroxypropyl cellulose,
mannitol, starch or modified starch (e.g. pregelatinized or partially
hydrolyzed) or xylitol. Among those
diluents mannitol and microcrystalline cellulose are employed in some
instances.
= Diluents that find use as the second diluent are the above-mentioned
diluents mannitol and
microcrystalline cellulose.
= Lubricants suitable for a pharmaceutical composition according to the
invention are talc,
polyethyleneglycol, calcium behenate, calcium stearate, sodium
stearylfumarate, hydrogenated castor
oil or magnesium stearate. The lubricant in some instances is magnesium
stearate.
= Binders suitable for a pharmaceutical composition according to the
invention are copovidone
(copolymerisates of vinylpyrrolidon with other vinylderivates), hydroxypropyl
methylcellulose
(HPMC), hydroxypropylcellulose (HPC), polyvinylpyrrolidon (povidone),
pregelatinized starch,
stearic-palmitic acid, low-substituted hydroxypropylcellulose (L-HPC),
copovidone and pregelatinized
starch being employed in some formulations. The above mentioned binders
pregelatinized starch and
L-HPC show additional diluent and disintegrant properties and can also be used
as the second diluent
or the disintegrant.
= Disintegrants suitable for a pharmaceutical composition according to the
present invention are corn
starch, crospovidone, polacrilin potassium, croscarmellose sodium, low-
substituted
hydroxypropylcellulose (L-HPC) or pregelatinized starch; such as
croscarmellose sodium.
= As an optional glidant colloidal silicon dioxide can be used.
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An exemplary composition according to the present invention comprises the
diluent mannitol,
microcrystalline cellulose as a diluent with additional disintegrating
properties, the binder copovidone, the
disintegrant croscarmellose sodium, and magnesium stearate as the lubricant.
Typical pharmaceutical compositions comprise (% by weight)
10-50 % active ingredient
20-88 % diluent 1,
5-50% diluent 2,
1-5 % binder,
1-15 % disintegrant, and
0.1-5 % lubricant.
Pharmaceutical compositions according to some embodiments comprise (% by
weight)
10-50 % active ingredient
20-75 % diluent 1,
5-30% diluent 2,
2-30 % binder,
1-12 % disintegrant, and
0.1-3 % lubricant
Pharmaceutical compositions according to some embodiments comprise (% by
weight)
10-90 % active ingredient
5-70 % diluent 1,
5-30% diluent 2,
0-30 % binder,
1-12 % disintegrant, and
0.1-3 % lubricant
Pharmaceutical compositions according to some embodiments comprise (% by
weight)
10-50 % active ingredient
20-75 % diluent 1,
5-30% diluent 2,
2-30 % binder,
0,5-20 % buffering agent,
1-12 % disintegrant, and
0.1-3 % lubricant
Pharmaceutical compositions according to some embodiments comprise (% by
weight)
30-70 % active ingredient
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20-75 % diluent 1,
5-30% diluent 2,
2-30 % binder,
0,5-20 % buffering agent,
1-12 % disintegrant, and
0.1-3 % lubricant
Pharmaceutical compositions containing 10-90% of active ingredient, such as 30-
70 % active
ingredient (% by weight) are employed in some instances.
A tablet formulation according to the invention may be uncoated or coated,
e.g. film-coated, using
suitable coatings known not to negatively affect the dissolution properties of
the final formulation. For
instance the tablets can be provided with a seal coat for protection of the
patients environment and clinical
staff as well as for moisture protection purposes by dissolving a high
molecular weight polymer as
polyvinylpyrrolidone or hydroxypropyl-methylcellulose together with
plasticizers, lubricants and
optionally pigments and tensides in water or organic solvent as acetone and
spraying this mixture on the
tablet cores inside a coating equipment as a pan coater or a fluidized bed
coater with wurster insert.
Additionally, agents such as beeswax, shellac, cellulose acetate phthalate,
polyvinyl acetate
phthalate, zein, film forming polymers such as hydroxypropyl cellulose,
ethylcellulose and polymeric
methacrylates can be applied to the tablets, provided that the coating has no
substantial effect on the
disintegration/dissolution of the dosage form and that the coated dosage form
is not affected in its stability.
After the dosage form is film-coated, a sugar coating may be applied onto the
sealed pharmaceutical
dosage form. The sugar coating may comprise sucrose, dextrose, sorbitol and
the like or mixtures thereof.
If desired, colorants or opacifiers may be added to the sugar solution.
Solid formulations of the present invention tend to be hygroscopic. They may
be packaged using
PVC-blisters, PVDC-blisters or a moisture-proof packaging material such as
aluminum foil blister packs,
alu/alu blister, transparent or opaque polymer blister with pouch,
polypropylene tubes, glass bottles and
HDPE bottles optionally containing a child-resistant feature or may be tamper
evident. The primary
packaging material may comprise a desiccant such as molecular sieve or silica
gel to improve chemical
stability of the API. Opaque packaging such as colored blister materials,
tubes, brown glass bottles or the
like can be used to prolong shelf life of the API by reduction of photo
degradation.
e. Dosages
A dosage range of the compound of formula 3 is usually between 100 and 1000
mg, in particular
between 200 and 900 mg, 300 and 900 mg or 350 and 850 mg or 390 and 810 mg. It
is possible to give one
or two tablets, where in some instances two tablets for a daily oral dosage of
100, 200, 300, 350, 400, 450,
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500, 550, 600, 650, 700, 750, 800, 850, 900 mg, and in some instances 350,
400, 450, 750, 800, 850 are
employed.
The dosages range can be achieved by one tablet or by two tablets; in some
instances two tablets
are administered, each containing half of the dosage.
The application of the active ingredient may occur up to three times a day,
such as one or two times
a day. Particular dosage strengths are 400 mg or 800 mg.
f Used Terms and Definitions
Terms not specifically defined herein should be given the meanings that would
be given to them
by one of skill in the art in light of the disclosure and the context. As used
in the specification, however,
unless specified to the contrary, the following terms have the meaning
indicated and the following
conventions are adhered to.
The term, "about" means 5% more or less of the specified value. Thus, about
100 minutes could
also be read as from 95 to 105 minutes.
In case a compound of the present invention is depicted in form of a chemical
name and as a formula
in case of any discrepancy the formula shall prevail. An asterisk is may be
used in sub-formulas to indicate
the bond which is connected to the core molecule as defined.
Unless specifically indicated, throughout the specification and the appended
claims, a given
chemical formula or name shall encompass tautomers and all stereo, optical and
geometrical isomers (e.g.
enantiomers, diastereomers, E/Z isomers etc...) and racemates thereof as well
as mixtures in different
proportions of the separate enantiomers, mixtures of diastereomers, or
mixtures of any of the foregoing
forms where such isomers and enantiomers exist, as well as salts, including
pharmaceutically acceptable
salts thereof and solvates thereof such as for instance hydrates including
solvates of the free compounds or
solvates of a salt of the compound.
The term "substituted" as used herein, means that any one or more hydrogens on
the designated
atom is replaced with a selection from the indicated group, provided that the
designated atom's normal
valence is not exceeded, and that the substitution results in a stable
compound.
By the term "optionally substituted" is meant within the scope of the
invention the above-mentioned
group, optionally substituted by a lower-molecular group. Examples of lower-
molecular groups regarded
as chemically meaningful are groups consisting of 1-200 atoms. Of interest are
such groups that have no
negative effect on the pharmacological efficacy of the compounds. For example
the groups may comprise:
= Straight-chain or branched carbon chains, optionally interrupted by
heteroatoms, optionally substituted
by rings, heteroatoms or other common functional groups.
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= Aromatic or non-aromatic ring systems consisting of carbon atoms and
optionally heteroatoms, which
may in turn be substituted by functional groups.
= A number of aromatic or non-aromatic ring systems consisting of carbon
atoms and optionally
heteroatoms which may be linked by one or more carbon chains, optionally
interrupted by heteroatoms,
optionally substituted by heteroatoms or other common functional groups.
The compounds disclosed herein can exist as therapeutically acceptable salts.
The present invention
includes compounds listed above in the form of salts, including acid addition
salts. Suitable salts include
those formed with both organic and inorganic acids. Such acid addition salts
will normally be
pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable
salts may be of utility in
the preparation and purification of the compound in question. Basic addition
salts may also be formed and
be pharmaceutically acceptable. For a more complete discussion of the
preparation and selection of salts,
refer to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P.
Heinrich. Wiley- VCHA, Zurich,
Switzerland, 2002).
The term "therapeutically acceptable salt," as used herein, represents salts
or zwitterionic forms of
the compounds disclosed herein which are water or oil-soluble or dispersible
and therapeutically acceptable
as defined herein. The salts can be prepared during the final isolation and
purification of the compounds or
separately by reacting the appropriate compound in the form of the free base
with a suitable acid.
Representative acid addition salts include acetate, adipate, alginate, L-
ascorbate, aspartate, benzoate,
benzenesulfonate (besylate), bisulfate, butyrate, camphorate,
camphorsulfonate, citrate, digluconate,
formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,
hemisulfate, heptanoate, hexanoate,
hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate
(isethionate), lactate,
maleate, malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate,
naphthylenesulfonate,
nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-
phenylproprionate,
phosphonate, picrate, pivalate, propionate, pyroglutamate, Succinate,
Sulfonate, tartrate, L-tartrate,
trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-
toluenesulfonate (p-tosylate),
and undecanoate. Also, basic groups in the compounds disclosed herein can be
quaternized with methyl,
ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,
dibutyl, and diamyl Sulfates;
decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and
benzyl and phenethyl bromides.
Examples of acids which can be employed to form therapeutically acceptable
addition salts include
inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric,
and organic acids such as
oxalic, maleic, succinic, and citric. Salts can also be formed by coordination
of the compounds with an
alkali metal or alkaline earth ion. Hence, the present invention contemplates
sodium, potassium,
magnesium, and calcium salts of the compounds disclosed herein, and the like.
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Basic addition salts can be prepared during the final isolation and
purification of the compounds
by reacting a carboxy group with a suitable base such as the hydroxide,
carbonate, or bicarbonate of a metal
cation or with ammonia or an organic primary, secondary, or tertiary amine.
The cations of therapeutically
acceptable salts include lithium, sodium, potassium, calcium, magnesium, and
aluminum, as well as
nontoxic quaternary amine cations such as ammonium, tetramethylammonium,
tetraethylammonium,
methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine,
ethylamine, tributylamine,
pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,
dicyclohexylamine, procaine,
dibenzylamine, N, N-dibenzylphenethylamine, 1-ephenamine, and N,P-
dibenzylethylenediamine. Other
representative organic amines useful for the formation of base addition salts
include ethylenediamine,
ethanolamine, diethanolamine, piperidine, and piperazine.
While it may be possible for the compounds of the subject invention to be
administered as the raw
chemical, it is also possible to present them as a pharmaceutical formulation.
Accordingly, provided herein
are pharmaceutical formulations which comprise one or more of certain
compounds disclosed herein, or
one or more pharmaceutically acceptable salts, esters, prodrugs, amides, or
solvates thereof, together with
one or more pharmaceutically acceptable carriers thereof and optionally one or
more other therapeutic
ingredients. The carrier(s) must be "acceptable" in the sense of being
compatible with the other ingredients
of the formulation and not deleterious to the recipient thereof. Proper
formulation is dependent upon the
route of administration chosen. Any of the well-known techniques, carriers,
and excipients may be used as
suitable and as understood in the art; e.g., in Remington's Pharmaceutical
Sciences. The pharmaceutical
compositions disclosed herein may be manufactured in any manner known in the
art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating,
entrapping or compression processes.
"Heterocyclic rings" ("het") include five-, six- or seven-membered, saturated
or unsaturated
heterocyclic rings or 5-10 membered, bicyclic hetero rings which may contain
one, two or three
heteroatoms, selected from among oxygen, sulphur and nitrogen; the ring may be
linked to the molecule by
a carbon atom or, if present, by a nitrogen atom. The following are examples
of five-, six- or seven-
membered, saturated or unsaturated heterocyclic rings:
NO 0 N¨v 0
cv\S
0
0
Unless stated otherwise, a heterocyclic ring may be provided with a keto
group. Examples include:
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NQ
,
S N
N6 CS) N--li
\N
so2
0
Examples of 5-10-membered bicyclic hetero rings are pyrrolizine, indole,
indolizine, isoindole,
indazole, purine, quinoline, isoquinoline, benzimidazole, benzofurane,
benzopyrane, benzothiazole,
benzoisothiazole, pyridopyrimidine, pteridine, pyrimidopyrimidine,
NO[N NJN ,
Although the term heterocyclic rings includes heterocyclic aromatic groups,
the term heterocyclic
aromatic groups ("hetaryl") denotes five- or six-membered heterocyclic
aromatic groups or 5-10 membered,
bicyclic hetaryl rings which may contain one, two or three heteroatoms,
selected from among oxygen,
sulphur and nitrogen, which contain sufficient conjugated double bonds that an
aromatic system is formed.
The ring may be linked to the molecule through a carbon atom or if present
through a nitrogen atom. The
following are examples of five- or six-membered heterocyclic aromatic groups:
O z S zN, N,
SA 00 n \\ k\k N
\N=i N
N-N
r\ir
0 -N_IN N7N
Examples of 5-10-membered bicyclic hetaryl rings include pyrrolizine, indole,
indolizine,
isoindole, indazole, purine, quinoline, isoquinoline, benzimidazole,
benzofuran, benzopyrane,
benzothiazole, benzoisothiazole, pyridopyrimidine, pteridine,
pyrimidopyrimidine.
The term "halogen" as used herein means a halogen substituent selected from
fluoro, chloro, bromo
or iodo.
By the term "C16-alkyl" (including those which are part of other groups) are
meant branched and
unbranched alkyl groups with 1 to 6 carbon atoms, and by the term 'C14 -alkyl"
are meant branched and
unbranched alkyl groups with 1 to 4 carbon atoms. Alkyl groups with 1 to 4
carbon atoms are present in
some instances. Examples of these include: methyl, ethyl, n-propyl, iso-
propyl, n-butyl, iso-butyl, sec-butyl,
tert-butyl, n-pentyl, iso-pentyl, neo-pentyl or hexyl. The abbreviations Me,
Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu,
etc. may optionally also be used for the above-mentioned groups. Unless stated
otherwise, the definitions
propyl, butyl, pentyl and hexyl include all the possible isomeric forms of the
groups in question. Thus, for
example, propyl includes n-propyl and iso-propyl, butyl includes iso-butyl,
sec-butyl and tert-butyl etc.
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By the term 'C16-alkylene' (including those which are part of other groups)
are meant branched
and unbranched alkylene groups with 1 to 6 carbon atoms and by the term 'C14-
alkylene' are meant
branched and unbranched alkylene groups with 1 to 4 carbon atoms. Alkylene
groups with 1 to 4 carbon
atoms are present in some instances. Examples include: methylene, ethylene,
propylene, 1-methylethylene,
butylene, 1-methylpropylene, 1,1-dimethylethylene, 1,2-dimethylethylene,
pentylene, 1,1-
dimethylpropylene, 2,2-dimethylpropylene, 1,2-dimethylpropylene, 1,3-
dimethylpropylene or hexylene.
Unless stated otherwise, the definitions propylene, butylene, pentylene and
hexylene also include all the
possible isomeric forms of the relevant groups with the same number of
carbons. Thus for example propyl
also includes 1-methylethylene and butylene includes 1-methylpropylene, 1,1-
dimethylethylene, 1,2-
dimethylethylene.
The term " C26-alkenyl " (including those which are part of other groups)
denotes branched and
unbranched alkenyl groups with 2 to 6 carbon atoms and the term 'C24-alkenyl'
denotes branched and
unbranched alkenyl groups with 2 to 4 carbon atoms, provided that they have at
least one double bond.
Employed in some instances are alkenyl groups with 2 to 4 carbon atoms.
Examples include: ethenyl or
vinyl, propenyl, butenyl, pentenyl, or hexenyl. Unless otherwise stated, the
definitions propenyl, butenyl,
pentenyl and hexenyl include all possible isomeric forms of the groups in
question. Thus, for example,
propenyl includes 1-propenyl and 2-propenyl, butenyl includes 1-, 2- and 3-
butenyl, 1-methyl-1 -propenyl,
1-methyl-2-propenyl etc.
By the term "C2 6-alkenylene" (including those which are part of other groups)
are meant branched
and unbranched alkenylene groups with 2 to 6 carbon atoms and by the term "C2
4-alkenylene" are meant
branched and unbranched alkylene groups with 2 to 4 carbon atoms. Alkenylene
groups with 2 to 4 carbon
atoms are present in some instances. Examples include: ethenylene,
propenylene, 1-methylethenylene,
butenylene, 1-methylpropenylene, 1,1-dimethylethenylene, 1,2-
dimethylethenylene, pentenylene, 1,1-
dimethylpropenylene, 2,2-dimethylpropenylene, 1,2-dimethylpropenylene, 1,3-
dimethylpropenylene or
hexenylene. Unless stated otherwise, the definitions propenylene, butenylene,
pentenylene and hexenylene
include all the possible isomeric forms of the respective groups with the same
number of carbons. Thus, for
example, propenyl also includes 1-methylethenylene and butenylene includes 1-
methylpropenylene, 1,1-
dimethylethenylene, 1,2-dimethylethenylene.
By the term 'C26-alkynyl' (including those which are part of other groups) are
meant branched and
unbranched alkynyl groups with 2 to 6 carbon atoms and by the term 'C24-
alkynyl' are meant branched
and unbranched alkynyl groups with 2 to 4 carbon atoms, provided that they
have at least one triple bond.
Alkynyl groups with 2 to 4 carbon atoms are present in some instances.
Examples include: ethynyl,
propynyl, butynyl, pentynyl, or hexynyl. Unless stated otherwise, the
definitions propynyl, butynyl,
pentynyl and hexynyl include all the possible isomeric forms of the respective
groups. Thus, for example,
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propynyl includes 1-propynyl and 2-propynyl, butynyl includes 1-, 2- and 3-
butynyl, 1-methyl-1 -propynyl,
1-methyl-2-propynyl etc.
By the term "C2 6-alkynylene" (including those which are part of other groups)
are meant branched
and unbranched alkynylene groups with 2 to 6 carbon atoms and by the term "C2
4-alkynylene" are meant
branched and unbranched alkylene groups with 2 to 4 carbon atoms. Alkynylene
groups with 2 to 4 carbon
atoms are present in some instances. Examples include: ethynylene,
propynylene, 1-methylethynylene,
butynylene, 1-methylpropynylene, 1,1-dimethylethynylene, 1,2-
dimethylethynylene, pentynylene, 1,1-
dimethylpropynylene, 2,2-dimethylpropynylene, 1,2-dimethylpropynylene, 1,3-
dimethylpropynylene or
hexynylene. Unless stated otherwise, the definitions propynylene, butynylene,
pentynylene and hexynylene
include all the possible isomeric forms of the respective groups with the same
number of carbons. Thus for
example propynyl also includes 1-methylethynylene and butynylene includes 1-
methylpropynylene, 1,1-
dimethylethynylene, 1,2-dimethylethynylene.
The term "C3 6-cycloalkyl" (including those which are part of other groups) as
used herein means
cyclic alkyl groups with 3 to 8 carbon atoms, where in some instances such
groups are cyclic alkyl groups
with 5 to 6 carbon atoms. Examples include: cyclopropyl, cyclobutyl,
cyclopentyl or cyclohexyl.
By the term "C1 6-haloalkyl" (including those which are part of other groups)
are meant branched
and unbranched alkyl groups with 1 to 6 carbon atoms wherein one or more
hydrogen atoms are replaced
by a halogen atom selected from among fluorine, chlorine or bromine, such as
fluorine and chlorine, e.g.,
fluorine. By the term "C1 4-haloalkyl" are meant correspondingly branched and
unbranched alkyl groups
with 1 to 4 carbon atoms, wherein one or more hydrogen atoms are replaced
analogously to what was stated
above. C1 4-haloalkyl is presentin some instances. Examples include: CH2F,
CHF2, CF3.
The term 'C111-alkyl", wherein n is an integer from 2 to n, either alone or in
combination with
another radical denotes an acyclic, saturated, branched or linear hydrocarbon
radical with 1 to n C atoms.
For example the term C15-alkyl embraces the radicals H3C-, H3C-CH2-, H3C-CH2-
CH2-, H3C-CH(CH3)-,
H3C-CH2-CH2-CH2-, H3C-CH2-CH(CH3)-, H3C-CH(CH3)-CH2-, H3C-C(CH3)2-, H3C-CH2-
CH2-CH2-CH2-,
H3C-CH2-CH2-CH(CH3)-, H3C-CH2-CH(CH3)-CH2-, H3C-CH(CH3)-CH2-CH2-, H3C-CH2-
C(CH3)2-,
H3C-C(CH3)2-CH2-, H3C-CH(CH3)-CH(CH3)- and H3C-CH2-CH(CH2CH3)-.
The term "C1 n-haloalkyl", wherein n is an integer from 2 to n, either alone
or in combination with
another radical denotes an acyclic, saturated, branched or linear hydrocarbon
radical with 1 to n C atoms
wherein one or more hydrogen atoms are replaced by a halogen atom selected
from among fluorine, chlorine
or bromine, such as fluorine and chlorine, e.g., fluorine. Examples include:
CH2F, CHF2, CF3.
The term "Ci n-alkylene" wherein n is an integer 2 to n, either alone or in
combination with another
radical, denotes an acyclic, straight or branched chain divalent alkyl radical
containing from 1 to n carbon
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atoms. For example the term
C14-alkylene
includes -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -C(CH3)2-,
-CH(CH2CH3)-, -CH(CH3)-CH2-, -CH2-CH(CH3)-, -CH2-CH2-CH2-CH2-, -CH2-CH2-
CH(CH3)-, -CH(CH3
)-CH2-CH2-, -CH2-CH(CH3)-CH2-, -CH2-C(CH3)2-, -C(CH3)2-CH2-, -CH(CH3)-CH(CH3)-
, -CH2-CH(CH2
CH3)-, -CH(CH2CH3)-CH2-, -CH(CH2CH2CH3)- , -CH(CH(CH3))2- and -C(CH3)(CH2CH3)-
.
The term "C211-alkenyl", is used for a group as defined in the definition for
"C111-alkyl" with at least
two carbon atoms, if at least two of those carbon atoms of said group are
bonded to each other by a double
bond.
The term "C211-alkynyl", is used for a group as defined in the definition for
'C111-alkyl' with at least
two carbon atoms, if at least two of those carbon atoms of said group are
bonded to each other by a triple
bond.
The term "C311-cycloalkyl", wherein n is an integer from 4 to n, either alone
or in combination with
another radical denotes a cyclic, saturated, unbranched hydrocarbon radical
with 3 to n C atoms. For
example the term C37-cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and
cycloheptyl.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an," and
"the" include plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to
"a cell" includes a plurality of such cells and reference to "the peptide"
includes reference to one or more
peptides and equivalents thereof, e.g. polypeptides, known to those having
skill in the art, and so forth.
By "an individual suffering from or at risk of suffering from an aging-
associated cognitive
impairment" is meant an individual that is about more than 50% through its
expected lifespan, such as more
than 60%, e.g., more than 70%, such as more than 75%, 80%, 85%, 90%, 95% or
even 99% through its
expected lifespan. The age of the individual will depend on the species in
question. Thus, this percentage
is based on the predicted life-expectancy of the species in question. For
example, in humans, such an
individual is 50 year old or older, e.g., 60 years old or older, 70 years old
or older, 80 years old or older, 90
years old or older, and usually no older than 100 years old, such as 90 years
old., i.e., between the ages of
about 50 and 100, e.g., 50. . . 55 . . . 60. . . 65 . . . 70. . . 75 . . . 80.
. . 85 . . . 90. . . 95 . . . 100 years old
or older, or any age between 50 ¨ 1000, that suffers from an aging-associated
condition as further described
below, e.g., cognitive impairment associated with the natural aging process;
an individual that is about 50
years old or older, e.g., 60 years old or older, 70 years old or older, 80
years old or older, 90 years old or
older, and usually no older than 100 years old, i.e., between the ages of
about 50 and 100, e.g., 50 . . . 55.
. . 60 . . . 65. . . 70 . . . 75 . . . 80. . . 85 . . . 90. . . 95. . . 100
years old, that has not yet begun to show
symptoms of an aging-associated condition e.g., cognitive impairment; an
individual of any age that is
suffering from a cognitive impairment due to an aging-associated disease, as
described further below, and
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an individual of any age that has been diagnosed with an aging-associated
disease that is typically
accompanied by cognitive impairment, where the individual has not yet begun to
show symptoms of
cognitive impairment. The corresponding ages for non-human subjects are known
and are intended to apply
herein.
As summarized elsewhere, in some instances the subject is a mammal. Mammalian
species that
may be treated with the present methods include canines and felines; equines;
bovines; ovines; etc., and
primates, including humans. The subject methods, compositions, and reagents
may also be applied to
animal models, including small mammals, e.g., murine, lagomorpha, etc., for
example, in experimental
investigations.
As used herein and as described above, "treatment" refers to any of (i) the
prevention of the disease
or disorder, or (ii) the reduction or elimination of symptoms of the disease
or disorder. Treatment may be
effected prophylactically (prior to the onset of disease) or therapeutically
(following the onset of the
disease). The effect may be prophylactic in terms of completely or partially
preventing a disease or
symptom thereof and/or may be therapeutic in terms of a partial or complete
cure for a disease and/or
adverse effect attributable to the disease. Thus, the term "treatment" as used
herein covers any treatment of
an aging-related disease or disorder in a mammal, and includes: (a) preventing
the disease from occurring
in a subject which may be predisposed to the disease but has not yet been
diagnosed as having it; (b)
inhibiting the disease, i.e., arresting its development; or (c) relieving the
disease, i.e., causing regression of
the disease. Treatment may result in a variety of different physical
manifestations, e.g., modulation in gene
expression, rejuvenation of tissue or organs, etc. The therapeutic agent may
be administered before, during
or after the onset of disease. The treatment of ongoing disease, where the
treatment stabilizes or reduces
the undesirable clinical symptoms of the patient, is of particular interest.
Such treatment may be performed
prior to complete loss of function in the affected tissues. The subject
therapy may be administered during
the symptomatic stage of the disease, and in some cases after the symptomatic
stage of the disease.
In some embodiments, the condition that is treated is an aging-associated
impairment in cognitive
ability in an individual. By cognitive ability, or "cognition," it is meant
the mental processes that include
attention and concentration, learning complex tasks and concepts, memory
(acquiring, retaining, and
retrieving new information in the short and/or long term), information
processing (dealing with information
gathered by the five senses), visuospatial function (visual perception, depth
perception, using mental
imagery, copying drawings, constructing objects or shapes), producing and
understanding language, verbal
fluency (word-finding), solving problems, making decisions, and executive
functions (planning and
prioritizing). By "cognitive decline", it is meant a progressive decrease in
one or more of these abilities,
e.g., a decline in memory, language, thinking, judgment, etc. By "an
impairment in cognitive ability" and
"cognitive impairment", it is meant a reduction in cognitive ability relative
to a healthy individual, e.g., an
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age-matched healthy individual, or relative to the ability of the individual
at an earlier point in time, e.g., 2
weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10
years or more previously.
By "aging-associated cognitive impairment," it is meant an impairment in
cognitive ability that is typically
associated with aging, including, for example, cognitive impairment associated
with the natural aging
process, e.g., mild cognitive impairment (M.C.I.); and cognitive impairment
associated with an aging-
associated disorder, that is, a disorder that is seen with increasing
frequency with increasing senescence,
e.g., a neurodegenerative condition such as Alzheimer's disease, Parkinson's
disease, frontotemporal
dementia, Huntington disease, amyotrophic lateral sclerosis, multiple
sclerosis, glaucoma, myotonic
dystrophy, vascular dementia, progressive supranuclear palsy, ataxia,
associated frailty, and the like.
g. Combinations
The compounds of general formula 1 may be used on their own or combined with
other active
substances of formula 1 according to the invention. The compounds of general
formula 1 may optionally
also be combined with other pharmacologically active substances. These
include, B2-adrenoceptor-agonists
(short and long-acting), anti-cholinergics (short and long-acting), anti-
inflammatory steroids (oral and
topical corticosteroids), cromoglycate, methylxanthine, dissociated-
glucocorticoidmimetics, PDE3
inhibitors, PDE4- inhibitors, PDE7- inhibitors, LTD4 antagonists, EGFR-
inhibitors, Dopamine agonists,
PAF antagonists, Lipoxin A4 derivatives, FPRL1 modulators, LTB4-receptor
(BLT1, BLT2) antagonists,
Histamine H1 receptor antagonists, Histamine H4 receptor antagonists, dual
Histamine Hl/H3-receptor
antagonists, P13-kinase inhibitors, inhibitors of non-receptor tyrosine
kinases as for example LYN, LCK,
SYK, ZAP-70, FYN, BTK or ITK, inhibitors of MAP kinases as for example p38,
ERK1, ERK2, JNK1,
JNK2, JNK3 or SAP, inhibitors of the NF-KB signalling pathway as for example
IKK2 kinase inhibitors,
iNOS inhibitors, MRP4 inhibitors, leukotriene biosynthese inhibitors as for
example 5-Lipoxygenase (5-
LO) inhibitors, cPLA2 inhibitors, Leukotriene A4 Hydrolase inhibitors or FLAP
inhibitors, Non-steroidal
anti-inflammatory agents (NSAIDs), CRTH2 antagonists, DP 1-receptor
modulators, Thromboxane
receptor antagonists, additional CCR3 antagonists, CCR4 antagonists, CCR1
antagonists, CCR5
antagonists, CCR6 antagonists, CCR7 antagonists, CCR8 antagonists, CCR9
antagonists, CCR30
antagonistsõ CXCR3 antagonists, CXCR4 antagonists, CXCR2 antagonists, CXCR1
antagonists, CXCR5
antagonists, CXCR6 antagonists, CX3CR3 antagonists, Neurokinin (NK1, NK2)
antagonists, Sphingosine
1-Phosphate receptor modulators, Sphingosine 1 phosphate lyase inhibitors,
Adenosine receptor modulators
as for example A2a-agonists, modulators of purinergic receptors as for example
P2X7 inhibitors, Histone
Deacetylase (HDAC) activators, Bradykinin (BK1, BK2) antagonists, TACE
inhibitors, PPAR gamma
modulators, Rho-kinase inhibitors, interleukin 1-beta converting enzyme (ICE)
inhibitors, Toll-Like
receptor (TLR) modulators, HMG-CoA reductase inhibitors, VLA-4 antagonists,
ICAM-1 inhibitors, SHIP
agonists, GABAa receptor antagonist, ENaC-inhibitors, Melanocortin receptor
(MC1R, MC2R, MC3R,
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MC4R, MC5R) modulators, CGRP antagonists, Endothelin antagonists, TNFa
antagonists, anti-TNF
antibodies, anti-GM-CSF antibodies, anti-CD46 antibodies, anti-IL-1
antibodies, anti-IL-2 antibodies, anti-
IL-4 antibodies, anti-IL-5 antibodies, anti-IL-13 antibodies, anti-IL-4/IL-13
antibodies, anti-TSLP
antibodies, anti-0X40 antibodies, mucoregulators, immunotherapeutic agents,
compounds against swelling
of the airways, compounds against cough, VEGF inhibitors, but also
combinations of two or three active
substances.
In some embodiments, the other active substances are betamimetics,
anticholinergics,
corticosteroids, PDE4-inhibitors, LTD4-antagonists, EGFR-inhibitors, CRTH2
inhibitors, 5-LO-inhibitors,
Histamine receptor antagonists and SYK-inhibitors, but also combinations of
two or three active substances,
i.e.:
= Betamimetics with corticosteroids, PDE4-inhibitors, CRTH2-inhibitors or
LTD4-antagonists,
= Anticholinergics with betamimetics, corticosteroids, PDE4-inhibitors,
CRTH2-inhibitors or LTD4-
antagonists,
= Corticosteroids with PDE4-inhibitors, CRTH2-inhibitors or LTD4-
antagonists
= PDE4-inhibitors with CRTH2-inhibitors or LTD4-antagonists
= CRTH2-inhibitors with LTD4-antagonists.
In these embodiments, the compounds that make up the combination are co-
administered to a subject. The
terms "co-administration" and "in combination with" include the administration
of two or more therapeutic
agents either simultaneously, concurrently or sequentially within no specific
time limits. In one
embodiment, the agents are present in the cell or in the subject's body at the
same time or exert their
biological or therapeutic effect at the same time. In one embodiment, the
therapeutic agents are in the same
composition or unit dosage form. In other embodiments, the therapeutic agents
are in separate compositions
or unit dosage forms. In certain embodiments, a first agent can be
administered prior to (e.g., minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours,
96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes,
45 minutes, 1 hour, 2 hours,
4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2
weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second
therapeutic agent. "Concomitant
administration" of a known therapeutic drug with a pharmaceutical composition
of the present disclosure
means administration of the compound and second agent at such time that both
the known drug and the
composition of the present invention will have a therapeutic effect. Such
concomitant administration may
involve concurrent (i.e. at the same time), prior, or subsequent
administration of the drug with respect to
the administration of a subject compound. Routes of administration of the two
agents may vary, where
representative routes of administration are described in greater detail below.
A person of ordinary skill in
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the art would have no difficulty determining the appropriate timing, sequence
and dosages of administration
for particular drugs and compounds of the present disclosure. In some
embodiments, the compounds (e.g.,
a subject compound and the at least one additional compound) are administered
to the subject within twenty-
four hours of each other, such as within 12 hours of each other, within 6
hours of each other, within 3 hours
of each other, or within 1 hour of each other. In certain embodiments, the
compounds are administered
within 1 hour of each other. In certain embodiments, the compounds are
administered substantially
simultaneously. By administered substantially simultaneously is meant that the
compounds are
administered to the subject within about 10 minutes or less of each other,
such as 5 minutes or less, or 1
minute or less of each other.
A "companion diagnostic" or "companion diagnostic device" means an in vitro
diagnostic device
or an imaging tool that provides information that is essential for the safe
and effective use of a corresponding
therapeutic product. The use of an in vitro diagnostic companion device with a
particular therapeutic
product is stipulated in the instructions for use in the labeling of both the
device and the corresponding
therapeutic product, as well as in the labeling of any generic equivalents and
biosimilar equivalents of the
therapeutic product.
Companion diagnostic testing can be in several forms, including by way of
example and not
limitation: test that screen for familial genetic patterns and difficult to
diagnose conditions; prognosis tests
predicting the future course of a disease; theranostic test to indicate a
patient's response to a prescribed
therapy; monitoring tests that evaluate the effectiveness and appropriate
dosing of a prescribed therapy; and
recurrence tests analyzing the patient's risk for a recurrence of the disease.
See Agarwal A, et al.,
Pharmgenomics Pers Med. 8:99-110 (2015) which is herein incorporated by
reference in its entirety.
h. Pharmaceutical Forms
Suitable preparations for administering the compounds of formula 1 and the co-
crystal or salt forms
of formulae 2 and 2a include for example tablets, capsules, suppositories,
solutions and powders etc. The
content of the pharmaceutically active compound(s) should be in the range from
0.05 to 90 wt.-%, such as
0.1 to 50 wt.-% of the composition as a whole. Suitable tablets may be
obtained, for example, by mixing
the active substance(s) with known excipients, for example inert diluents such
as calcium carbonate,
calcium phosphate or lactose, disintegrants such as corn starch or alginic
acid, binders such as starch or
gelatine, lubricants such as magnesium stearate or talc and/or agents for
delaying release, such as
carboxymethyl cellulose, cellulose acetate phthalate, or polyvinyl acetate.
The tablets may also comprise
several layers.
Coated tablets may be prepared accordingly by coating cores produced
analogously to the tablets
with substances normally used for tablet coatings, for example collidone or
shellac, gum arabic, talc,
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titanium dioxide or sugar. To achieve delayed release or prevent
incompatibilities the core may also consist
of a number of layers. Similarly the tablet coating may consist of a number or
layers to achieve delayed
release, possibly using the excipients mentioned above for the tablets.
Syrups or elixirs containing the active substances or combinations thereof
according to the
invention may additionally contain a sweetener such as saccharine, cyclamate,
glycerol or sugar and a
flavour enhancer, e.g. a flavouring such as vanillin or orange extract. They
may also contain suspension
adjuvants or thickeners such as sodium carboxymethyl cellulose, wetting agents
such as, for example,
condensation products of fatty alcohols with ethylene oxide, or preservatives
such as p-hydroxybenzoates.
Solutions are prepared in the usual way, e.g. with the addition of isotonic
agents, preservatives such
as p-hydroxybenzoates or stabilisers such as alkali metal salts of
ethylenediaminetetraacetic acid, optionally
using emulsifiers and/or dispersants, while if water is used as diluent, for
example, organic solvents may
optionally be used as solubilisers or dissolving aids, and the solutions may
be transferred into injection vials
or ampoules or infusion bottles.
Capsules containing one or more active substances or combinations of active
substances may for
example be prepared by mixing the active substances with inert carriers such
as lactose or sorbitol and
packing them into gelatine capsules.
Suitable suppositories may be made for example by mixing with carriers
provided for this purpose,
such as neutral fats or polyethyleneglycol or the derivatives thereof.
Excipients which may be used include, for example, water, pharmaceutically
acceptable organic
solvents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g.
groundnut or sesame oil), mono-
or polyfunctional alcohols (e.g. ethanol or glycerol), carriers such as e.g.
natural mineral powders (e.g.
kaolins, clays, talc, chalk), synthetic mineral powders (e.g. highly dispersed
silicic acid and silicates), sugars
(e.g. cane sugar, lactose and glucose), emulsifiers (e.g. lignin, spent
sulphite liquors, methylcellulose, starch
and polyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc,
stearic acid and sodium lauryl
sulphate).
For oral use the tablets may obviously contain, in addition to the carriers
specified, additives such
as sodium citrate, calcium carbonate and dicalcium phosphate together with
various additional substances
such as starch, e.g., potato starch, gelatine and the like. Lubricants such as
magnesium stearate, sodium
laurylsulphate and talc may also be used to produce the tablets. In the case
of aqueous suspensions the
active substances may be combined with various flavor enhancers or colorings
in addition to the
abovementioned excipients.
For administering the compounds of formula 1 or the co-crystal or salt forms
of formulae 2 and 2a
preparations or pharmaceutical formulations which are suitable for inhalation
may be employed. Inhalable
preparations include inhalable powders, propellant-containing metered-dose
aerosols or propellant-free
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inhalable solutions. Within the scope of the present invention, the term
propellant-free inhalable solutions
also include concentrates or sterile inhalable solutions ready for use. The
formulations which may be used
within the scope of the present invention are described in more detail in the
next part of the specification.
The inhalable powders which may be used according to the invention may contain
a compound of
formula 1 or a co-crystal or salt form of formulae 2 and 2a either on their
own or in admixture with suitable
physiologically acceptable excipients.
If the active substances of the compounds of formula 1 or the co-crystal or
salt forms of formulae
2 and 2a are present in admixture with physiologically acceptable excipients,
the following physiologically
acceptable excipients may be used to prepare these inhalable powders according
to the invention:
monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose,
saccharose, maltose), oligo- and
polysaccharides (e.g. dextrans), polyalcohols (e.g. sorbitol, mannitol,
xylitol), salts (e.g. sodium chloride,
calcium carbonate) or mixtures of these excipients. In some instances, mono-
or disaccharides are used,
such as lactose or glucose, e.g., in the form of their hydrates, e.g.,
lactose, such as lactose monohydrate.
Within the scope of the inhalable powders according to the invention the
excipients have a
maximum average particle size of up to 250 tim, such as between 10 and 150
tim, and including between
15 and 80 tim. It may sometimes seem appropriate to add finer excipient
fractions with an average particle
size of 1 to 9 tim to the excipient mentioned above. These finer excipients
are also selected from the group
of possible excipients listed hereinbefore. Finally, in order to prepare the
inhalable powders according to
the invention, micronized active substance of the compounds of formula 1 or
the co-crystal or salt forms of
formulae 2 and 2a, such as with an average particle size of 0.5 to 10 inn,
including from 1 to 5 inn, is added
to the excipient mixture. Processes for producing the inhalable powders
according to the invention by
grinding and micronising and finally mixing the ingredients together are known
from the prior art.
The inhalable powders according to the invention may be administered using
inhalers known from
the prior art.
The inhalation aerosols containing propellant gas according to the invention
may contain a
compound of formula 1 or a co-crystal or salt form of formulae 2 and 2a
dissolved in the propellant gas or
in dispersed form. The compounds of formula 1 or the co-crystal or salt forms
of formulae 2 and 2a may
be contained in separate formulations or in a common formulation, in which
they are either both dissolved,
both dispersed or in each case only one component is dissolved and the other
is dispersed. The propellant
gases which may be used to prepare the inhalation aerosols are known from the
prior art. Suitable propellant
gases are selected from among hydrocarbons such as n-propane, n-butane or
isobutane and
halohydrocarbons such as fluorinated derivatives of methane, ethane, propane,
butane, cyclopropane or
cyclobutane. The abovementioned propellant gases may be used on their own or
mixed together. In some
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instances, propellant gases are halogenated alkane derivatives selected from
TG134a and TG227 and
mixtures thereof.
The propellant-driven inhalation aerosols may also contain other ingredients
such as co-solvents,
stabilisers, surfactants, antioxidants, lubricants and pH adjusters. All these
ingredients are known in the art.
The propellant-driven inhalation aerosols according to the invention mentioned
above may be
administered using inhalers known in the art (MDIs = metered dose inhalers).
Moreover, the active substances of the compounds of formula 1 or the co-
crystal or salt forms of formulae
2 and 2a according to the invention may be administered in the form of
propellant-free inhalable solutions
and suspensions. The solvent used may be an aqueous or alcoholic, such as an
ethanolic solution. The
solvent may be water on its own or a mixture of water and ethanol. The
relative proportion of ethanol
compared with water is not limited but the maximum is in some instances up to
70 percent by volume, such
as up to 60 percent by volume and including up to 30 percent by volume. The
remainder of the volume is
made up of water. The solutions or suspensions containing a compound of
formula 1 or a co-crystal or salt
form of formulae 2 and 2a are adjusted to a pH of 2 to 7, such as 2 to 5,
using suitable acids. The pH may
be adjusted using acids selected from inorganic or organic acids. Examples of
particularly suitable inorganic
acids include hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid
and/or phosphoric acid.
Examples of particularly suitable organic acids include ascorbic acid, citric
acid, malic acid, tartaric acid,
maleic acid, succinic acid, fumaric acid, acetic acid, formic acid and/or
propionic acid etc. In some
instances, the inorganic acids are hydrochloric and sulphuric acids. It is
also possible to use the acids which
have already formed an acid addition salt with one of the active substances.
Of the organic acids, ascorbic
acid, fumaric acid and citric acid are employed in some instances. If desired,
mixtures of the above acids
may be used, particularly in the case of acids which have other properties in
addition to their acidifying
qualities, e.g. as flavourings, antioxidants or complexing agents, such as
citric acid or ascorbic acid, for
example. According to the invention, in some instances hydrochloric acid is
employed to adjust the pH.
If desired, the addition of edetic acid (EDTA) or one of the known salts
thereof, sodium edetate, as
stabilizer or complexing agent may be omitted in these formulations. Other
embodiments may contain this
compound or these compounds. In an embodiment the content based on sodium
edetate is less than 100
mg/100m1, such as less than 50mg/100m1, and including less than 20mg/100m1.
Inhalable solutions in
which the content of sodium edetate is from 0 to 10mg/100m1 are employed in
some instances. Co-solvents
and/or other excipients may be added to the propellant-free inhalable
solutions, such as those which contain
hydroxyl groups or other polar groups, e.g. alcohols - particularly isopropyl
alcohol, glycols - particularly
propyleneglycol, polyethyleneglycol, polypropyleneglycol, glycolether,
glycerol, polyoxyethylene
alcohols and polyoxyethylene fatty acid esters. The terms excipients and
additives in this context denote
any pharmacologically acceptable substance which is not an active substance
but which can be formulated
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with the active substance or substances in the physiologically suitable
solvent in order to improve the
qualitative properties of the active substance formulation. In some
embodiments, these substances have no
pharmacological effect or, in connection with the desired therapy, no
appreciable or at least no undesirable
pharmacological effect. The excipients and additives include, for example,
surfactants such as soya lecithin,
oleic acid, sorbitan esters, such as polysorbates, polyvinylpyrrolidone, other
stabilizers, complexing agents,
antioxidants and/or preservatives which guarantee or prolong the shelf life of
the finished pharmaceutical
formulation, flavourings, vitamins and/or other additives known in the art.
The additives also include
pharmacologically acceptable salts such as sodium chloride as isotonic agents.
In some embodiments excipients include antioxidants such as ascorbic acid, for
example, provided
that it has not already been used to adjust the pH, vitamin A, vitamin E,
tocopherols and similar vitamins
and provitamins occurring in the human body.
Preservatives may be used to protect the formulation from contamination with
pathogens. Suitable
preservatives are those which are known in the art, particularly acetyl
pyridinium chloride, benzalkonium
chloride or benzoic acid or benzoates such as sodium benzoate in the
concentration known from the prior
art. The preservatives mentioned above may be present in concentrations of up
to 50 mg/100 ml, such as
between 5 and 20 mg/100 ml.
In some embodiments, the formulations contain, in addition to the solvent
water and the compounds of
formula 1 or the co-crystal or salt forms of formulae 2 and 2a, only
benzalkonium chloride and sodium
edetate. In an embodiment, no sodium edetate is present.
The dosage of the compounds according to the invention is naturally highly
dependent on the
method of administration and the complaint which is being treated. When
administered by inhalation the
compounds of formula 1 or the co-crystal or salt forms of formulae 2 and 2a
are characterized by a high
potency even at doses in the tig range. The compounds of formula 1 or the co-
crystal or salt forms of
formulae 2 and 2a may also be used effectively above the tig range. The dosage
may then be in the gram
range, for example.
In another aspect the present invention relates to the above-mentioned
pharmaceutical formulations
as such which are characterized in that they contain a compound of formula 1
or a co-crystal or salt form
of formulae 2 and 2a, particularly the above-mentioned pharmaceutical
formulations which can be
administered by inhalation.
The following examples of formulations illustrate the present invention
without restricting its
scope:
i. Examples of Pharmaceutical Formulations
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A) Tablets per tablet
active substance 1, 2, or 2a 100 mg
lactose 140 mg
maize starch 240 mg
polyvinylpyrrolidone 15 mg
magnesium stearate 5 mg
500 mg
The finely ground active substance, lactose and some of the maize starch are
mixed together. The
mixture is screened, then moistened with a solution of polyvinylpyrrolidone in
water, kneaded, wet
granulated and dried. The granules, the remaining maize starch and the
magnesium stearate are screened
and mixed together. The mixture is pressed into tablets of suitable shape and
size.
B) Tablets per tablet
active substance 1, 2, or 2a 80 mg
lactose 55 mg
maize starch 190 mg
microcrystalline cellulose 35 mg
polyvinylpyrrolidone 15 mg
sodium carboxymethyl starch 23 mg
magnesium stearate 2 mg
400 mg
The finely ground active substance, some of the corn starch, lactose,
microcrystalline cellulose and
polyvinylpyrrolidone are mixed together, the mixture is screened and worked
with the remaining corn starch
and water to form a granulate which is dried and screened. The sodium
carboxymethyl starch and the
magnesium stearate are added and mixed in and the mixture is compressed to
form tablets of a suitable size.
C) Ampoule solution
active substance 1, 2, or 2a 50 mg
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sodium chloride 50 mg
water for inj. 5 ml
The active substance is dissolved in water at its own pH or optionally at pH
5.5 to 6.5 and sodium
chloride is added to make the solution isotonic. The resulting solution is
filtered to remove pyrogens and
the filtrate is transferred under aseptic conditions into ampoules which are
then sterilized and heat-sealed.
The ampoules contain 5 mg, 25 mg and 50 mg of active substance.
D) Metering aerosol
active substance 1, 2, or 2a 0.005
sorbitan trioleate 0.1
monofluorotrichloromethane and
TG134a : TG227 2:1 ad 100
The suspension is transferred into a conventional aerosol container with
metering valve. Preferably
50 ial suspension are released on each actuation. The active substance may
also be released in higher doses
if desired (e.g. 0.02 wt.-%).
E) Solutions (in mg/100m1)
active substance 1, 2, or 2a 333.3 mg
benzalkonium chloride 10.0 mg
EDTA 50.0 mg
HC1 (1N) ad pH 2.4
This solution can be prepared in the usual way.
F) Inhalable powder
active substance 1, 2, or 2a 12 tig
lactose monohydrate ad 25 mg
The inhalable powder is prepared in the usual way by mixing the individual
ingredients.
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j. Indications
Methods of improving cognition or other symptoms of cognitive disease through
treating a
subject/patient diagnosed with cognitive-associated disease are provided.
Aspects of the methods include
modulating CCR3, e.g. with a CCR3 modulating agent, in a manner sufficient to
treat the patient for the
cognitive-associated disease. The methods include treating the cognitive-
associated disease with an orally
administrable and bioavailable composition, including a composition of
compound of formula 1, a co-
crystal or salt of formulae 2 or 2a, or a formulation of formula 3, described
above. As summarized above
and described below in greater detail, a variety of aging associated
impairments, e.g., cognitive-associated
diseases may be treated by embodiments of the invention. In some instances,
the target condition is a
cognitive-associated disease condition that is associated with
neurodegeneration, e.g., as evidenced by
neural compromise, such as one of more of, reduced neurogeneration, e.g., as
manifested by decreased
numbers of BrdU or EdU positive cells, Ki67 positive cells, and Dcx positive
cells when compared to non-
diseased tissue. The composition, which modulates CCR3, can be administered to
a patient/subject
diagnosed with the cognitive-associated disease, such as (by way of example
and not limitation): mild
cognitive impairment (MCI); Alzheimer' s disease; Parkinson's disease;
frontotemporal dementia (FTD);
Huntington's disease; amyotrophic lateral sclerosis (ALS); multiple sclerosis
(MS); glaucoma; myotonic
dystrophy; dementia; progressive supranuclear palsy (PSP); ataxia; multiple-
system atrophy; and frailty;
which are further described below. The methods of the invention can further
comprise monitoring
improvement in the progression of the neurodegenerative disease through
measuring cognitive or physical
improvement.
Methods of improving motor coordination, function, or other symptoms of motor
disorders through
treating a subject/patient diagnosed with motor disorders are provided.
Aspects of the methods include
modulating CCR3, e.g. with a CCR3 modulating agent, in a manner sufficient to
treat the patient for the
motor disorder. The methods include treating the motor disorder with an orally
administrable and
bioavailable composition, including a composition of compound of formula 1, a
co-crystal or salt of
formulae 2 or 2a, or a formulation of formula 3, described above. As
summarized above and described
below in greater detail, a variety of aging associated impairments, e.g.,
motor disorders may be treated by
embodiments of the invention. In some instances, the target condition is a
motor disorder that is associated
with neurodegeneration, e.g., as evidenced by neural compromise, such as one
of more of, reduced
neurogeneration, e.g., as manifested by decreased numbers of BrdU or EdU
positive cells, Ki67 positive
cells, and Dcx positive cells when compared to non-diseased tissue. The
composition, which modulates
CCR3, can be administered to a patient/subject diagnosed with the motor
disorder, such as (by way of
example and not limitation): Parkinson's disease; Parkinsonism; Dementia with
Lewy Bodies; ataxia;
dystonia; cervical dystonia; chorea; Huntington' s disease, multiple system
atrophy; spasticity; progressive
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supranuclear palsy; Tardive dyskinesia; Tourette syndrome; and tremor; which
are further described below.
The methods of the invention can further comprise monitoring improvement in
the progression of the
neurodegenerative disease through measuring cognitive or physical improvement.
Mild cognitive impairment (M.C.I.) is a modest disruption of cognition that
manifests as problems
with memory or other mental functions such as planning, following
instructions, or making decisions that
have worsened over time while overall mental function and daily activities are
not impaired. Thus, although
significant neuronal death does not typically occur, neurons in the aging
brain are vulnerable to sub-lethal
age-related alterations in structure, synaptic integrity, and molecular
processing at the synapse, all of which
impair cognitive function. Individuals suffering from or at risk of developing
an aging-associated cognitive
impairment who will benefit from treatment with the subject compounds of the
invention, e.g., by the
methods disclosed herein, also include individuals of any age that are
suffering from a cognitive impairment
due to an aging-associated disorder; and individuals of any age that have been
diagnosed with an aging-
associated disorder that is typically accompanied by cognitive impairment,
where the individual has not yet
begun to present with symptoms of cognitive impairment. Examples of such aging-
associated disorders
include by way of not of limitation, those listed below.
Alzheimer's disease. Alzheimer's disease is characterized by a progressive,
inexorable loss of
cognitive function associated with an excessive number of senile plaques in
the cerebral cortex and
subcortical gray matter, in addition to excessive 13-amyloid and
neurofibrillary tangles consisting of tau
protein. The common form affects persons >60 years old, and its incidence
increases as age advances. It
accounts for more than 65% of the dementias in the elderly.
The cause of Alzheimer's disease is not known. The disease runs in families in
about 15 to 20% of
cases. The remaining, so-called sporadic cases have some genetic associations.
The disease has an
autosomal dominant genetic pattern in most early-onset and some late-onset
cases but a variable late-life
penetrance. Environmental factors are the focus of active investigation.
In the course of the disease, synapses, and ultimately neurons are lost within
the cerebral cortex,
hippocampus, and subcortical structures (including selective cell loss in the
nucleus basalis of Meynert),
locus coeruleus, and nucleus raphae dorsalis. Cerebral glucose use and
perfusion is reduced in some areas
of the brain (parietal lobe and temporal cortices in early-stage disease,
prefrontal cortex in late-stage
disease). Neuritic or senile plaques (composed of neurites, astrocytes, and
glial cells around an amyloid
core) and neurofibrillary tangles (composed of paired helical filaments) play
a role in the pathogenesis of
Alzheimer's disease. Senile plaques and neurofibrillary tangles occur with
normal aging, but they are much
more prevalent in persons with Alzheimer's disease.
Parkinson's Disease. Parkinson's Disease (PD) is an idiopathic, slowly
progressive, degenerative
CNS disorder characterized by slow and decreased movement (bradykinesia),
muscular rigidity, resting
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tremor (dystonia), muscle freezing, and postural instability. Originally
considered primarily a motor
disorder, PD is now recognized to also cause depression and emotional changes.
PD also can affect
cognition, behavior, sleep, autonomic function, and sensory function. The most
common cognitive
impairments include an impairment in attention and concentration, working
memory, executive function,
producing language, and visuospatial function. A characteristic of PD is
symptoms related to reduced motor
function usually precede those related to cognitive impairment, which aids in
diagnosis of the disease.
In primary Parkinson's disease, the pigmented neurons of the substantia nigra,
locus coeruleus, and
other brain stem dopaminergic cell groups degenerate. The cause is not known.
The loss of substantia nigra
neurons, which project to the caudate nucleus and putamen, results in
depletion of the neurotransmitter
dopamine in these areas. Onset is generally after age 40, with increasing
incidence in older age groups.
Parkinson's disease is newly diagnosed in about 60,000 Americans each year and
currently affects
approximately one million Americans. Even though PD is not fatal in itself,
its complications are the
fourteenth leading cause of death in the United States. At present, PD cannot
be cured, and treatment is
generally prescribed to control symptoms, with surgery prescribed in later,
severe cases.
Treatment options for PD include administration of pharmaceuticals to help
manage motor deficits.
These options increase or substitute for the neurotransmitter, dopamine, of
which PD patients have low
brain concentrations. Such medications include: carbidopa/levodopa (which
create more dopamine in the
brain); apomorphine, pramipexolole, ropinirole, and rotingotine (dopamine
agonists); selegiline and
rasagiline (MAO-B inhibitors which prevent breakdown of dopamine); entacapone
and tolcapone
(Catechol-O-methyltransferase [COW] inhibitors which make more levodopa
available in the brain);
benztropine and trihexyphenidyl (anticholinergics); and amantadine (controls
tremor and stiffness).
Exercise/physical therapy is also commonly prescribed to help maintain
physical and mental function.
Current treatment options, however treat the symptoms of PD, are not curative,
and fail to prevent
disease progression. Additionally, current medications tend to lose efficacy
in late stage PD. The most
prescribed drug, levodopa, commonly results in adverse effects within 5 to 10
years after commencing the
medication. These adverse effects can be severe and can result in motor
fluctuations and unpredictable
swings in motor control between doses as well as jerking/twitching
(dyskinesia) which are difficult to
manage and are even as disabling as PD' s own symptoms. Thus, there remains a
need for new therapies
with new mechanisms of action which can either be administrated along or in
combination with current PD
medications.
Parkinsonism. Secondary parkinsonism (also referred to as atypical Parkinson's
disease or
Parkinson' s plus) results from loss of or interference with the action of
dopamine in the basal ganglia due
to other idiopathic degenerative diseases, drugs, or exogenous toxins. The
most common cause of secondary
parkinsonism is ingestion of antipsychotic drugs or reserpine, which produce
parkinsonism by blocking
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dopamine receptors. Less common causes include carbon monoxide or manganese
poisoning,
hydrocephalus, structural lesions (tumors, infarcts affecting the midbrain or
basal ganglia), subdural
hematoma, and degenerative disorders, including nigrostriatal degeneration.
Certain disorders like
Progressive Supranuclear Palsy (PSP), Multiple System Atrophy (MSA),
Corticobasal degeneration (CBD)
and Dementia with Lewy Bodies (DLB) can exhibit Parkinsonism symptoms before
the cardinal symptoms
necessary to the specific diagnosis can be made, and thus may be labeled as
"Parkinsonism."
Assessing Progression of PD
Several rating scales have been utilized for evaluating the progression of PD.
The most widely-
used scales include the Unified Parkinson's Disease Rating Scale (UPDRS, which
was introduced in 1987)
(J. Rehabil Res. Dev., 2012 49(8): 1269-76), and the Hoehn and Yahr scale
(Neruology, 1967 17(5): 427-
42). Additional scales include the Movement Disorder Society (MDS)'s updated
UPDRS scale (MDS-
UPDRS) as well as the Schwab and England Activities of Daily Living (ADL)
Scale.
The UPDRS scale evaluates 31 items that contributed to three subscales: (1)
mentation, behavior,
and mood; (2) activities of daily living; and (3) motor examination. The Hoehn
and Yahr scale classifies
PD into five stages with discreet substages: 0 ¨ no signs of disease; 1 ¨
symptoms on one side only; 1.5 ¨
symptoms on one side but also involving neck and spine; 2 ¨ symptoms on both
sides with no balance
impairment; 2.5 ¨ mild symptoms on both sides, with recovery when the 'pull'
test is given; 3 ¨ balance
impairment with mild to moderate disease; 4 ¨ severe disability, but ability
to walk or stand unassisted; and
¨ need a wheelchair or bedridden without assistance. The Schwab and England
scale classifies PD into
several percentages (from 100% - complete independent to 10% - total
dependent).
Frontotemporal dementia. Frontotemporal dementia (FTD) is a condition
resulting from the
progressive deterioration of the frontal lobe of the brain. Over time, the
degeneration may advance to the
temporal lobe. Second only to Alzheimer's disease (AD) in prevalence, FTD
accounts for 20% of pre-senile
dementia cases. Symptoms are classified into three groups based on the
functions of the frontal and temporal
lobes affected:
Behavioral variant FTD (bvFTD), with symptoms including lethargy and
aspontaneity on the one
hand, and disinhibition on the other; progressive nonfluent aphasia (PNFA), in
which a breakdown in
speech fluency due to articulation difficulty, phonological and/or syntactic
errors is observed but word
comprehension is preserved; and semantic dementia (SD), in which patients
remain fluent with normal
phonology and syntax but have increasing difficulty with naming and word
comprehension. Other cognitive
symptoms common to all FTD patients include an impairment in executive
function and ability to focus.
Other cognitive abilities, including perception, spatial skills, memory and
praxis typically remain intact.
FTD can be diagnosed by observation of frontal lobe and/or anterior temporal
lobe atrophy in structural
MRI scans.
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A number of forms of FTD exist, any of which may be treated or prevented using
the subject
methods and compositions. For example, one form of frontotemporal dementia is
Semantic Dementia (SD).
SD is characterized by a loss of semantic memory in both the verbal and non-
verbal domains. SD patients
often present with the complaint of word-finding difficulties. Clinical signs
include fluent aphasia, anomia,
impaired comprehension of word meaning, and associative visual agnosia (the
inability to match
semantically related pictures or objects). As the disease progresses,
behavioral and personality changes are
often seen similar to those seen in frontotemporal dementia although cases
have been described of 'pure'
semantic dementia with few late behavioral symptoms. Structural MRI imaging
shows a characteristic
pattern of atrophy in the temporal lobes (predominantly on the left), with
inferior greater than superior
involvement and anterior temporal lobe atrophy greater than posterior.
As another example, another form of frontotemporal dementia is Pick's disease
(PiD, also PcD). A
defining characteristic of the disease is build-up of tau proteins in neurons,
accumulating into silver-
staining, spherical aggregations known as "Pick bodies." Symptoms include loss
of speech (aphasia) and
dementia. Patients with orbitofrontal dysfunction can become aggressive and
socially inappropriate.
They may steal or demonstrate obsessive or repetitive stereotyped behaviors.
Patients with
dorsomedial or dorsolateral frontal dysfunction may demonstrate a lack of
concern, apathy, or decreased
spontaneity. Patients can demonstrate an absence of self-monitoring, abnormal
self-awareness, and an
inability to appreciate meaning.
Patients with gray matter loss in the bilateral posterolateral orbitofrontal
cortex and right anterior
insula may demonstrate changes in eating behaviors, such as a pathologic sweet
tooth. Patients with more
focal gray matter loss in the anterolateral orbitofrontal cortex may develop
hyperphagia. While some of the
symptoms can initially be alleviated, the disease progresses and patients
often die within two to ten years.
Huntington's disease. Huntington's disease (HD) is a hereditary progressive
neurodegenerative
disorder characterized by the development of emotional, behavioral, and
psychiatric abnormalities; loss of
intellectual or cognitive functioning; and movement abnormalities (motor
disturbances). The classic signs
of HD include the development of chorea - involuntary, rapid, irregular, jerky
movements that may affect
the face, arms, legs, or trunk - as well as cognitive decline including the
gradual loss of thought processing
and acquired intellectual abilities. There may be impairment of memory,
abstract thinking, and judgment;
improper perceptions of time, place, or identity (disorientation); increased
agitation; and personality
changes (personality disintegration). Although symptoms typically become
evident during the fourth or
fifth decades of life, the age at onset is variable and ranges from early
childhood to late adulthood (e.g., 70s
or 80s).
HD is transmitted within families as an autosomal dominant trait. The disorder
occurs as the result
of abnormally long sequences or "repeats" of coded instructions within a gene
on chromosome 4 (4p16.3).
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The progressive loss of nervous system function associated with HD results
from loss of neurons in certain
areas of the brain, including the basal ganglia and cerebral cortex.
Amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a
rapidly progressive,
invariably fatal, neurological disease that attacks motor neurons. Muscular
weakness and atrophy and signs
of anterior horn cell dysfunction are initially noted most often in the hands
and less often in the feet. The
site of onset is random, and progression is asymmetric. Cramps are common and
may precede weakness.
Rarely, a patient survives 30 years; 50% die within 3 years of onset, 20% live
5 years, and 10% live 10
years.
Diagnostic features include onset during middle or late adult life and
progressive, generalized
motor involvement without sensory abnormalities. Nerve conduction velocities
are normal until late in the
disease. Recent studies have documented the presentation of cognitive
impairments as well, particularly a
reduction in immediate verbal memory, visual memory, language, and executive
function.
A decrease in cell body area, number of synapses and total synaptic length has
been reported in
even normal-appearing neurons of the ALS patients. It has been suggested that
when the plasticity of the
active zone reaches its limit, a continuing loss of synapses can lead to
functional impairment. Promoting
the formation or new synapses or preventing synapse loss may maintain neuron
function in these patients.
Multiple Sclerosis. Multiple Sclerosis (MS) is characterized by various
symptoms and signs of
CNS dysfunction, with remissions and recurring exacerbations. The most common
presenting symptoms
are paresthesias in one or more extremities, in the trunk, or on one side of
the face; weakness or clumsiness
of a leg or hand; or visual disturbances, e.g., partial blindness and pain in
one eye (retrobulbar optic
neuritis), dimness of vision, or scotomas. Common cognitive impairments
include impairments in memory
(acquiring, retaining, and retrieving new information), attention and
concentration (particularly divided
attention), information processing, executive functions, visuospatial
functions, and verbal fluency.
Common early symptoms are ocular palsy resulting in double vision (diplopia),
transient weakness of one
or more extremities, slight stiffness or unusual fatigability of a limb, minor
gait disturbances, difficulty with
bladder control, vertigo, and mild emotional disturbances; all indicate
scattered CNS involvement and often
occur months or years before the disease is recognized. Excess heat may
accentuate symptoms and signs.
The course is highly varied, unpredictable, and, in most patients, remittent.
At first, months or years
of remission may separate episodes, especially when the disease begins with
retrobulbar optic neuritis.
However, some patients have frequent attacks and are rapidly incapacitated;
for a few the course can be
rapidly progressive.
Glaucoma. Glaucoma is a common neurodegenerative disease that affects retinal
ganglion cells
(RGCs). Evidence supports the existence of compartmentalized degeneration
programs in synapses and
dendrites, including in RGCs. Recent evidence also indicates a correlation
between cognitive impairment
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in older adults and glaucoma (Yochim BP, et al. Prevalence of cognitive
impairment, depression, and
anxiety symptoms among older adults with glaucoma. J Glaucoma. 2012;21(4):250-
254).
Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominant
multisystem disorder
characterized by dystrophic muscle weakness and myotonia. The molecular defect
is an expanded
trinucleotide (CTG) repeat in the 3' untranslated region of the myotonin
protein kinase gene on chromosome
19q. Symptoms can occur at any age, and the range of clinical severity is
broad. Myotonia is prominent in
the hand muscles, and ptosis is common even in mild cases. In severe cases,
marked peripheral muscular
weakness occurs, often with cataracts, premature balding, hatchet facies,
cardiac arrhythmias, testicular
atrophy, and endocrine abnormalities (e.g., diabetes mellitus). Mental
retardation is common in severe
congenital forms, while an aging-related decline of frontal and temporal
cognitive functions, particularly
language and executive functions, is observed in milder adult forms of the
disorder. Severely affected
persons die by their early 50s.
Dementia. Dementia describes a class of disorders having symptoms affecting
thinking and social
abilities severely enough to interfere with daily functioning. Other instances
of dementia in addition to the
dementia observed in later stages of the aging-associated disorders discussed
above include vascular
dementia, and dementia with Lewy bodies, described below.
In vascular dementia, or "multi-infarct dementia", cognitive impairment is
caused by problems in
supply of blood to the brain, typically by a series of minor strokes, or
sometimes, one large stroke preceded
or followed by other smaller strokes. Vascular lesions can be the result of
diffuse cerebrovascular disease,
such as small vessel disease, or focal lesions, or both. Patients suffering
from vascular dementia present
with cognitive impairment, acutely or subacutely, after an acute
cerebrovascular event, after which
progressive cognitive decline is observed. Cognitive impairments are similar
to those observed in
Alzheimer's disease, including impairments in language, memory, complex visual
processing, or executive
function, although the related changes in the brain are not due to AD
pathology but to chronic reduced
blood flow in the brain, eventually resulting in dementia. Single photon
emission computed tomography
(SPECT) and positron emission tomography (PET) neuroimaging may be used to
confirm a diagnosis of
multi-infarct dementia in conjunction with evaluations involving mental status
examination.
Dementia with Lewy bodies.
Dementia with Lewy bodies (DLB), also known under a variety of other names
including Lewy
body dementia, diffuse Lewy body disease, cortical Lewy body disease, and
senile dementia of Lewy type,
is a type of dementia characterized anatomically by the presence of Lewy
bodies (clumps of alpha-synuclein
and ubiquitin protein) in neurons, detectable in post mortem brain histology.
Its primary feature is cognitive
decline, particularly of executive functioning. Alertness and short-term
memory will rise and fall.
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Persistent or recurring visual hallucinations with vivid and detailed pictures
are often an early
diagnostic symptom. DLB it is often confused in its early stages with
Alzheimer's disease and/or vascular
dementia, although, where Alzheimer's disease usually begins quite gradually,
DLB often has a rapid or
acute onset. DLB symptoms also include motor symptoms similar to those of
Parkinson's. DLB is
distinguished from the dementia that sometimes occurs in Parkinson's disease
by the time frame in which
dementia symptoms appear relative to Parkinson symptoms. Parkinson's disease
with dementia (PDD)
would be the diagnosis when dementia onset is more than a year after the onset
of Parkinson's. DLB is
diagnosed when cognitive symptoms begin at the same time or within a year of
Parkinson symptoms.
Treating DLB is a complex process and requires a multifaceted approach.
(Neurology, 2017 89:1-
13). Typical Parkinsonism therapies like dopaminergic and anticholinergic
drugs can exacerbate cognition
and behavior symptoms. Optimal treatment commonly utilizes both pharmacologic
(exercise, cognitive
training, and caregiver-oriented training) and non-pharmacologic approaches.
For cognitive symptoms,
acetylcholinesterase inhibitors can be administered (e.g. rivastigmine,
donepezil) as can the NMDA
receptor antagonist, memantine. For neuropsychiatric symptoms,
acetylcholinesterase inhibitors can
improve apathy and hallucinations. Antipsychotics unfortunately increase
mortality risk in DLB patients.
Motor symptoms are less responsive to dopaminergic treatments in DLB patients
and can exacerbate the
risk of psychosis. Levodopa can be used, but only a low threshold doses, hence
a distinct need in the field
for new agents to treat DLB..
Progressive supranuclear palsy. Progressive supranuclear palsy (PSP) is a
brain disorder that causes
serious and progressive problems with control of gait and balance, along with
complex eye movement and
thinking problems. One of the classic signs of the disease is an inability to
aim the eyes properly, which
occurs because of lesions in the area of the brain that coordinates eye
movements. Some individuals
describe this effect as a blurring. Affected individuals often show
alterations of mood and behavior,
including depression and apathy as well as progressive mild dementia. The
disorder's long name indicates
that the disease begins slowly and continues to get worse (progressive) and
causes weakness (palsy) by
damaging certain parts of the brain above pea-sized structures called nuclei
that control eye movements
(supranuclear). PSP was first described as a distinct disorder in 1964, when
three scientists published a
paper that distinguished the condition from Parkinson's disease. It is
sometimes referred to as Steele-
Richardson-Olszewski syndrome, reflecting the combined names of the scientists
who defined the disorder.
Although PSP gets progressively worse, no one dies from PSP itself.
Ataxia. People with ataxia have problems with coordination because parts of
the nervous system
that control movement and balance are affected. Ataxia may affect the fingers,
hands, arms, legs, body,
speech, and eye movements. The word ataxia is often used to describe a symptom
of incoordination which
can be associated with infections, injuries, other diseases, or degenerative
changes in the central nervous
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system. Ataxia is also used to denote a group of specific degenerative
diseases of the nervous system called
the hereditary and sporadic ataxias which are the National Ataxia Foundation's
primary emphases.
Multiple-system atrophy. Multiple-system atrophy (MSA) is a degenerative
neurological disorder.
MSA is associated with the degeneration of nerve cells in specific areas of
the brain. This cell degeneration
causes problems with movement, balance, and other autonomic functions of the
body such as bladder
control or blood-pressure regulation.
The cause of MSA is unknown and no specific risk factors have been identified.
Around 55% of
cases occur in men, with typical age of onset in the late 50s to early 60s.
MSA often presents with some of
the same symptoms as Parkinson's disease. However, MSA patients generally show
minimal if any response
to the dopamine medications used for Parkinson's.
Dystonia. Dystonia is a condition which involves sustained involuntary muscle
contractions. Such
contracts can exhibit twisting, repetitive movements. This disorder may affect
the entire body or specific
parts of the body, referred to as generalized dystonia or focal dystonia
(respectively). Cervical dystonia
can cause long-lasting or intermittent contractions in the neck muscles. There
is no cure for dystonia.
Current therapies include carbidopa-levodopa, trihexyphenidyl, benztropine,
tetrabenazine, diazepam,
clonazepam, baclofen, physical therapy, speech therapy, stretching, massage,
and invasive surgery.
Frailty. Frailty Syndrome ("Frailty") is a geriatric syndrome characterized by
functional and
physical decline including decreased mobility, muscle weakness, physical
slowness, poor endurance, low
physical activity, malnourishment, and involuntary weight loss. Such decline
is often accompanied and a
consequence of diseases such as cognitive dysfunction and cancer. However,
Frailty can occur even without
disease. Individuals suffering from Frailty have an increased risk of negative
prognosis from fractures,
accidental falls, disability, comorbidity, and premature mortality. (C.
Buigues, et al. Effect of a Prebiotic
Formulation on Frailty Syndrome: A Randomized, Double-Blind Clinical Trial,
Int. J. Mol. Sci. 2016, 17,
932). Additionally, individuals suffering from Frailty have an increased
incidence of higher health care
expenditure. (Id.)
Common symptoms of frailty can be determined by certain types of tests. For
example,
unintentional weight loss involves a loss of at least 10 lbs. or greater than
5% of body weight in the
preceding year; muscle weakness can be determined by reduced grip strength in
the lowest 20% at baseline
(adjusted for gender and BMI); physical slowness can be based on the time
needed to walk a distance of 15
feet; poor endurance can be determined by the individual's self-reporting of
exhaustion; and low physical
activity can be measured using a standardized questionnaire. (Z. Palace et
al., The Frailty Syndrome,
Today's Geriatric Medicine 7(1), at 18 (2014)).
In some embodiments, the subject methods and compositions find use in slowing
the progression
of aging-associated cognitive, motor or other age-related impairment. In other
words, cognitive, motor, or
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other abilities in the individual will decline more slowly following treatment
by the disclosed methods than
prior to or in the absence of treatment by the disclosed methods. In some such
instances, the subject methods
of treatment include measuring the progression of cognitive, motor or other
age-related ability decline after
treatment, and determining that the progression of decline is reduced. In some
such instances, the
determination is made by comparing to a reference, e.g., the rate of decline
in the individual prior to
treatment, e.g., as determined by measuring cognitive, motor, or other age-
related abilities prior at two or
more time points prior to administration of the subject blood product.
The subject methods and compositions also find use in stabilizing the
cognitive, motor or other
abilities of an individual, e.g., an individual suffering from aging-
associated cognitive decline or an
individual at risk of suffering from aging-associated cognitive decline. For
example, the individual may
demonstrate some aging-associated cognitive impairment, and progression of
cognitive impairment
observed prior to treatment with the disclosed methods will be halted
following treatment by the disclosed
methods. As another example, the individual may be at risk for developing an
aging-associated cognitive
decline (e.g., the individual may be aged 50 years old or older, or may have
been diagnosed with an aging-
associated disorder), and the cognitive abilities of the individual are
substantially unchanged, i.e., no
cognitive decline can be detected, following treatment by the disclosed
methods as compared to prior to
treatment with the disclosed methods.
The subject methods and compositions also find use in reducing cognitive,
motor, or other age-
related impairment in an individual suffering from an aging-associated
impairment. In other words, the
affected ability is improved in the individual following treatment by the
subject methods. For example, the
cognitive ability in the individual is increased, e.g., by 2-fold or more, 5-
fold or more, 10-fold or more, 15-
fold or more, 20-fold or more, 30-fold or more, or 40-fold or more, including
50-fold or more, 60-fold or
more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-old or more,
following treatment by the
subject methods relative to the cognitive ability that is observed in the
individual prior to treatment by the
subject methods.
In some instances, treatment by the subject methods and compositions restores
the cognitive, motor,
or other ability in the individual suffering from aging-associated cognitive
or motor decline, e.g., to their
level when the individual was about 40 years old or less. In other words,
cognitive or motor impairment is
abrogated.
k. Methods of Diagnosing and Monitoring for Improvement of Neurodegenerative-
Associated Disease
One having ordinary skill in the art would recognize that among the variety of
methods to diagnose
and monitor disease progression and improvement in neurodegenerative-
associated disease, the following
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types of assessments could be used alone or in combination with subjects
suffering from neurodegenerative
disease. The following types of methods are presented as examples and are not
limited to the recited
methods. One having ordinary skill in the art would recognize that other
methods to monitor disease would
be useful in practicing the invention. Those methods are also contemplated by
the methods of the invention.
i. General Cognition
The methods of the invention further comprise methods of monitoring the effect
of a medication or
treatment on a subject for treating cognitive impairment and/or age-related
dementia, the method
comprising comparing cognitive function before and after treatment. Those
having ordinary skill in the art
recognize that there are well-known methods of evaluating cognitive function.
For example, and not by
way of limitation, the method may comprise evaluation of cognitive function
based on medical history,
family history, physical and neurological examinations by clinicians who
specialize dementia and cognitive
function, laboratory tests, and neuropsychological assessment. Additional
embodiments which are
contemplated by the invention include: the assessment of consciousness, such
as using the Glasgow Coma
Scale (EMV); mental status examination, including the abbreviated mental test
score (AMTS) or mini-
mental state examination (MMSE) (Folstein et al., J. Psychiatr. Res 1975;
12:1289-198); global assessment
of higher functions; estimation of intracranial pressure such as by
fundoscopy.
In one embodiment, examinations of peripheral nervous system may be used to
evaluate cognitive
function, including any one of the followings: sense of smell, visual fields
and acuity, eye movements and
pupils (sympathetic and parasympathetic), sensory function of face, strength
of facial and shoulder girdle
muscles, hearing, taste, pharyngeal movement and reflex, tongue movements,
which can be tested
individually (e.g. the visual acuity can be tested by a Snellen chart; a
reflex hammer used testing reflexes
including masseter, biceps and triceps tendon, knee tendon, ankle jerk and
plantar (i.e. Babinski sign);
Muscle strength often on the MRC scale 1 to 5; Muscle tone and signs of
rigidity.
ii. Multiple Sclerosis
In addition to monitoring improvement for symptoms associated with cognition,
the progression or
improvement of neurodegeneration associated with multiple sclerosis (MS) can
be monitored using
techniques well-known to those having ordinary skill in the art. By way of
example, and not limitation,
monitoring can be performed through techniques such as: cerebrospinal fluid
(CSF) monitoring; magnetic
resonance imaging (MRI) to detect lesions and development of demyelinating
plaques; evoked potential
studies; and gait monitoring.
CSF analysis may be performed, for example, through lumbar puncture to obtain
pressure,
appearance, and CSF content. Normal values typically range as follows:
pressure (70-180 mm H20);
appearance is clear and colorless; total protein (15 ¨ 60 mg/100mL); IgG is 3-
12% of the total protein;
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glucose is 50 ¨ 80 mg/100 mL; cell count is 0-5 white blood cells and no red
blood cells; chloride (110 ¨
125 mEq/L). Abnormal results may indicate the presence or progression of MS.
MRI is another technique that may be performed to monitor disease progression
and improvement.
Typical criteria for monitoring MS with MRI include the appearance of patchy
areas of abnormal white
matter in cerebral hemisphere and in paraventricular areas, lesions present in
the cerebellum and/or brain
stem as well as in the cervical or thoracic regions of the spinal cord.
Evoked potentials may be used to monitor the progression and improvement of MS
in subjects.
Evoked potentials measure slowing of electrical impulses such as in Visual
Evoked Response (VER), Brain
Stem Auditory Evoked Responses (BAER), and Somatosensory Evoked Responses
(SSER). Abnormal
responses help to indicate that there is a decrease in the speed of conduction
in central sensory pathways.
Gait monitoring can also be used to monitor disease progression and
improvement in MS subjects.
MS is often accompanied by an impairment in mobility and an abnormal gait due
in part to fatigue.
Monitoring may be performed, for example, with the use of mobile monitoring
devices worn by subjects.
(Moon, Y., et al., Monitoring gait in multiple sclerosis with novel wearable
motion sensors, PLOS One,
12(2):e0171346 (2017)).
iii. Huntington's
In addition to monitoring improvement for symptoms associated with cognition,
the progression or
improvement of neurodegeneration associated with Huntington's Disease (HD) can
be monitored using
techniques well-known to those having ordinary skill in the art. By way of
example, and not limitation,
monitoring can be performed through techniques such as: motor function;
behavior; functional assessment;
and imaging.
Examples of motor function that may be monitored as an indication of disease
progression or
improvement include chorea and dystonia, rigidity, bradykinesia, oculomotor
dysfunction, and gait/balance
changes. Techniques for performing the monitoring of these metrics are well-
known to those having
ordinary skill in the art. (See Tang C, et al., Monitoring Huntington's
disease progression through
preclinical and early stages, Neurodegener Dis Manag 2(4):421-35 (2012)).
The psychiatric effects of HD present opportunities to monitor disease
progression and
improvement. For example, psychiatric diagnoses may be performed in order to
determine whether the
subject suffers from depression, irritability, agitation, anxiety, apathy and
psychosis with paranoia. (Id.)
Functional assessment may also be employed to monitor disease progression or
improvement.
Total functional score techniques have been reported (Id.), and often declines
by one point per year in some
HD groups.
MRI or PET may be employed also to monitor disease progression or improvement.
For example,
there is a loss of striatal projection neurons in HD, and change in number of
these neurons may be monitored
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in subjects. Techniques to determine neuronal change in HD subjects include
imaging Dopamine D2
receptor binding. (Id.)
iv. ALS
In addition to monitoring improvement for symptoms associated with cognition,
the progression or
improvement of neurodegeneration associated with Amyotrophic Lateral Sclerosis
(ALS) can be monitored
using techniques well-known to those having ordinary skill in the art. By way
of example, and not
limitation, monitoring can be performed through techniques such as: functional
assessment; determining
muscle strength; measuring respiratory function; measuring lower motor neuron
(LMN) loss; and
measuring upper motor neuron (UMN) dysfunction.
Functional assessment can be performed using a functional scale well-known to
those having
ordinary skill in the art, such as the ALS Functional Rating Scale (ALSFRS-R),
which evaluates symptoms
related to bulbar, limb, and respiratory function. The rate of change is
useful in predicting survival as well
as disease progression or improvement. Another measure includes the Combined
Assessment of Function
and Survival (CAFS), ranking subjects' clinical outcomes by combining survival
time with change in
ALSFRS-R. (Simon NG, et al., Quantifying Disease Progression in Amyotrophic
Lateral Sclerosis, Ann
Neurol 76:643-57 (2014)).
Muscle strength may be tested and quantified through use of composite Manual
Muscle Testing
(MMT) scoring. This entails averaging measures acquired from several muscle
groups using the Medical
Research Council (MRC) muscle strength grading scale. (Id.) Hand-held
dynamometry (HHD) may also
be used, among other techniques. (Id.)
Respiratory function can be performed using portable spirometry units, used to
obtain Forced Vital
Capacity (FVC) at baseline to predict the progression or improvement of the
disease. Additionally,
maximal inspiratory pressure, sniff nasal inspiratory pressure (SNIP), and
supping FVC may be determined
and used to monitor disease progression/improvement. (Id.)
Loss in lower motor neurons is another metric which can be utilized to monitor
disease progression
or improvement in ALS. The Neurophysiological Index may be determined by
measuring compound
muscle action potentials (CMAPs) on motor nerve conduction studies, of which
parameters include CMAP
amplitude and F-wave frequency. (Id. and de Carvalho M, et al., Nerve
conduction studies in amyotrophic
lateral sclerosis. Muscle Nerve 23:344-352, (2000)). Lower motor neuron unit
numbers (MUNE) may be
estimated as well. In MUNE, the number of residual motor axons supplying a
muscle through estimation
of the contribution of individual motor units to the maximal CMAP response is
estimated, and used to
determine disease progression or improvement. (Simon NG, et al., supra).
Additional techniques for
determining loss of LMN include testing nerve excitability, electrical
impedance myography, and using
muscle ultrasound to detect changes in thickness in muscles. (Id.)
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Dysfunction of upper motor neurons is another metric which can be utilized to
monitor disease
progression or improvement in ALS. Techniques for determining dysfunction
include performing MRI or
PET scans on the brain and spinal cord, transcranial magnetic stimulation; and
determining levels of
biomarkers in the cerebrospinal fluid (CSF).
v. Glaucoma
In addition to monitoring improvement for symptoms associated with cognition,
the progression or
improvement of neurodegeneration associated with glaucoma can be monitored
using techniques well-
known to those having ordinary skill in the art. By way of example, and not
limitation, monitoring can be
performed through techniques such as: determining intraocular pressure;
assessment of the optic disc or
optic nerve head for damage; visual field testing for peripheral vision loss;
and imaging of the optic disc
and retina for topographic analysis.
vi. Progressive Supranuclear Palsy (PSP)
In addition to monitoring improvement for symptoms associated with cognition,
the progression or
improvement of neurodegeneration associated with Progressive Supranuclear
Palsy (PSP) can be monitored
using techniques well-known to those having ordinary skill in the art. By way
of example, and not
limitation, monitoring can be performed through techniques such as: functional
assessment (activities of
daily living, or ADL); motor assessment; determination of psychiatric
symptoms; and volumetric and
functional magnetic resonance imaging (MRI).
The level of function of a subject in terms of independence, partial
dependence upon others, or
complete dependence can be useful for determining the progression or
improvement in the disease. (See
Duff, K, et al., Functional impairment in progressive supranuclear palsy,
Neurology 80:380-84, (2013)).
The Progressive Supranuclear Palsy Rating Scale (PSPRS) is a rating scale that
comprises twenty-eight
metrics in six categories: daily activities (by history); behavior; bulbar,
ocular motor, limb motor and
gait/midline. The result is a score ranging from 0 ¨ 100. Six items are graded
0 ¨ 2 and twenty-two items
graded 0-4 for a possible total of 100. The PSPRS scores are practical
measures, and robust predictors of
patient survival. They are also sensitive to disease progression and useful in
monitoring disease progression
or improvement. (Golbe LI, et al., A clinical rating scale for progressive
supranuclear palsy, Brain
130:1552-65, (2007)).
The ADL section from the UPDRS (Unified Parkinson's Disease Rating Scale) can
also be used to
quantify functional activity in subjects with PSP. (Duff K, et al., supra).
Similarly, the Schwab & England
Activities Daily Living Score (SE-ADL) can be used for evaluate independence.
(Id.) Additionally, the
motor function sections of the UPDRS are useful as a reliable measure for
assessing disease progression in
PSP patients. The motor section may contain, for example, 27 different
measures for quantifying motor
function in PSP patients. Examples of these include resting tremor, rigidity,
finger tapping, posture, and
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gait). A subject's disease progression or improvement may also be assessed by
performing a baseline
neuropsychological evaluation completed by trained medical personnel, the
assessment using the
Neuropsychiatric Inventory (NPI) to determine the frequency and severity of
behavior abnormalities (e.g.
delusions, hallucinations, agitation, depression, anxiety, euphoria, apathy,
disinhibition, irritability, and
aberrant motor behavior). (Id.)
Functional MRI (fMRI) can be employed to monitor disease progression and
improvement as well.
fMRI is a technique using MRI to measure changes in brain activity in certain
regions of the brain, usually
based on blood flow to those regions. Blood flow is considered to correlate
with brain region activation.
Patients with neurodegenerative disorders like PSP can be subjected to
physical or mental tests before or
during being scanned in an MRI scanner. By way of example, and not limitation,
tests can be a well-
established force control paradigm where patients as asked to produce force
with the hand most affected by
PSP and maximum voluntary contraction (MVC) is measured by fMRI immediately
after the test takes
place. Burciu, RG, et al., Distinct patterns of brain activity in progressive
supranuclear palsy and
Parkinson's disease, Mov. Disord. 30(9):1248-58 (2015)).
Volumetric MRI is a technique where MRI scanners determine volume differences
in regional brain
volume. This may be done, for example, by contrasting different disorders, or
by determining differences
in volume of a brain region in a patient over time. Volumetric MRI may be
employed to determine disease
progression or improvement in neurodegenerative disorders like PSP. The
technique is well-known to those
having ordinary skill in the art. (Messina D, et al., Patterns of brain
atrophy in Parkinson's disease,
progressive supranuclear palsy and multiple system atrophy, Parkinsonism and
Related Disorders,
17(3):172-76 (2011)). Examples of cerebral regions which may be measured
include, but are not limited
to, intracranial volume, cerebral cortex, cerebellar cortex, thalamus,
caudate, putamen, pallidum,
hippocampus, amygdala, lateral ventricles, third ventricle, fourth ventricle,
and brain stem.
vii. Neuro genesis
Noninvasive techniques for evaluating neurogenesis have been reported. (Tamura
Y. et al., J.
Neurosci. (2016) 36(31):8123-31). Positron emission tomography (PET) used with
the tracer, [18F]FLT, in
combinations with the BBB transporter inhibitor probenecid, allows for
accumulation of the tracer in
neurogenic regions of the brain. Such imaging allows for an evaluation of
neurogenesis in patients being
treated for neurodegenerative disease.
viii. Parkinson's Disease and Motor Function
Several rating scales have been utilized for evaluating the progression of PD.
The most widely-
used scales include the Unified Parkinson's Disease Rating Scale (UPDRS, which
was introduced in 1987)
(J. Rehabil. Res. Dev., 2012 49(8): 1269-76), and the Hoehn and Yahr scale
(Neurology, 1967 17(5): 427-
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42). Additional scales include the Movement Disorder Society (MDS)'s updated
UPDRS scale (MDS-
UPDRS) as well as the Schwab and England Activities of Daily Living (ADL)
Scale.
The UPDRS scale evaluates 31 items that contributed to three subscales: (1)
mentation, behavior,
and mood; (2) activities of daily living; and (3) motor examination. The Hoehn
and Yahr scale classifies
PD into five stages with discreet substages: 0 ¨ no signs of disease; 1 ¨
symptoms on one side only; 1.5 ¨
symptoms on one side but also involving neck and spine; 2 ¨ symptoms on both
sides with no balance
impairment; 2.5 ¨ mild symptoms on both sides, with recovery when the 'pull'
test is given; 3 ¨ balance
impairment with mild to moderate disease; 4 ¨ severe disability, but ability
to walk or stand unassisted; and
¨ need a wheelchair or bedridden without assistance. The Schwab and England
scale classifies PD into
several percentages (from 100% - complete independent to 10% - total
dependent).
General motor function can be evaluated using widely-used scales including the
General Motor
Function Scale (GMF). This tests three components: dependence, pain, and
insecurity. (Aberg A.C., et
al. (2003) Disabil. Rehabil. 2003 May 6;25(9):462-72.). Motor function can
also be assessed using home-
monitoring or wearable sensors. For example: gait (speed of locomotion,
variability, leg rigidity) can be
sensed with an accelerometer; posture (trunk inclination) by a gyroscope; leg
movement by an
accelerometer; hand movement by an accelerometer and gyroscope; tremor
(amplitude, frequency,
duration, asymmetry) by an accelerometer; falling by an accelerometer; gait
freezing by an accelerometer;
dyskinesia by an accelerometer, gyroscope, and inertial sensors; bradykinesia
(duration and frequency) by
an accelerometer plus gyroscope, and aphasia (pitch) using a microphone.
(Pastorino M, et al., Journal of
Physics: Conference Series 450 (2013) 012055).
1. Reagents, Devices, and Kits
Also provided are reagents, devices, and kits thereof for practicing one or
more of the above-
described methods. The subject reagents, devices, and kits thereof may vary
greatly. Reagents and devices
of interest include those mentioned above with respect to the methods of
administering the compounds for
formula 1 in the subject.
In addition to the above components, the subject kits will further include
instructions for practicing
the subject methods. These instructions may be present in the subject kits in
a variety of forms, one or more
of which may be present in the kit. One form in which these instructions may
be present is as printed
information on a suitable medium or substrate, e.g., a piece or pieces of
paper on which the information is
printed, in the packaging of the kit, in a package insert, etc. Yet another
means would be a computer
readable medium, e.g. diskette, CD, portable flash drive, etc., on which the
information has been recorded.
Yet another means that may be present is a website address which may be used
via the internet to access
the information at a remote site. Any convenient means may be present in the
kits.
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VII. Examples
The following examples are provided by way of illustration and not by way of
limitation.
a. Pharmaceutical Preparation
The pharmaceutical compositions that are administered to subjects with
cognitive or
neurodegenerative disease that are comprised of the compounds, co-crystals,
and salts described above can
be synthesized, made, and formulated using the examples disclosed in U.S.
Patent Application Publication
Nos. 2013/0266646, 2016/0081998, U.S. Patent Nos. 8,278,302, 8,653,075, RE
45323, 8,742,115,
9,233,950, and 8,680,280, which are herein incorporated by reference in their
entirety. Further, the
pharmaceutical compositions may be prepared as described in the examples
below:
1. Tablet formulation - wet granulation
Copovidone is dissolved in ethanol at ambient temperature to produce a
granulation liquid. An
active CCR3 antagonist ingredient, lactose and part of the crospovidone are
blended in a suitable mixer, to
produce a pre-mix. The pre-mix is moistened with the granulation liquid and
subsequently granulated. The
moist granulate is optionally sieved through a sieve with a mesh size of 1.6-
3.0 mm. The granulate is dried
at 45 C in a suitable dryer to a residual moisture content corresponding to 1-
3 % loss on drying. The dried
granulate is sieved through a sieve with a mesh size of 1.0 mm. The granulate
is blended with part of the
crospovidone and microcrystalline cellulose in a suitable mixer. Magnesium
stearate is added to this blend
after passing through a 1.0 mm sieve for delumping. Subsequently the final
blend is produced by final
blending in a suitable mixer and compressed into tablets. The following tablet
composition can be obtained:
Component mg/tablet %/tablet
Active ingredient 28.500 30.0
Crospovidone 1.500 1.6
Lactose 28.000 29.5
Copovidone 3.000 3.2
Total (granulate) 61.000 64.3
Microcrystalline cellulose 31.000 32.6
Crospovidone 2.500 2.6
Magnesium stearate 0.500 0.5
Total 95.000 100.000
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2. Tablet formulation - melt granulation
An active CCR3 antagonist ingredient, lactose, part of the mcc, polyethylene
glycole, lactose and
part of the crospovidone are blended in a suitable mixer, to produce a pre-
mix. The pre-mix is heated in a
high shear mixer and subsequently granulated. The hot granulate is cooled down
to room temperature and
sieved through a sieve with a mesh size of 1.0 mm. The granulate is blended
with part of the crospovidone
and microcrystalline cellulose in a suitable mixer. Magnesium stearate is
added to this blend after passing
through a 1.0 mm sieve for delumping. Subsequently the final blend is produced
by final blending in a
suitable mixer and compressed into tablets. The following tablet composition
can be obtained:
Component mg/tablet %/tablet
Active ingredient 28.500 30.0
Crospovidone 1.500 1.6
Lactose 11.000 11.6
Polyethylene glycole 14.300 15.1
MCC 5.700 6.0
Total (granulate) 61.000 64.3
Microcrystalline cellulose 31.000 32.6
Crospovidone 2.500 2.6
Magnesium stearate 0.500 0.5
Total 95.000 100.000
3. Tablet formulation - hot melt granulation
An active CCR3 antagonist ingredient, mannit, polyethylene glycole and part of
the crospovidone
are blended in a suitable mixer, to produce a pre-mix. The pre-mix is heated
in a high shear mixer and
subsequently granulated. The hot granulate is cooled down to room temperature
and sieved through a sieve
with a mesh size of 1.0 mm. The granulate is blended with part of the
crospovidone and mannit in a suitable
mixer. Magnesium stearate is added to this blend after passing through a 1.0
mm sieve for delumping.
Subsequently the final blend is produced by final blending in a suitable mixer
and compressed into tablets.
The following tablet composition can be obtained:
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Component mg/tablet %/tablet
Active ingredient 28.500 30.0
Crospovidone 1.500 1.6
Mannit 16.700 17.6
Polyethylene glycole 14.300 15.1
Total (granulate) 61.000 64.3
Mannit 31.000 32.6
Crospovidone 2.500 2.6
Magnesium stearate 0.500 0.5
Total 95.000 100.000
4. Tablet formulation - hot melt extrusion
An active CCR3 antagonist ingredient and stearic-palmitic acid are blended in
a suitable mixer, to
produce a pre-mix. The pre-mix is extruded in a twin-screw-extruder and
subsequently granulated. The
granulate is sieved through a sieve with a mesh size of 1.0 mm. The granulate
is blended with mannit and
crospovidone in a suitable mixer. Magnesium stearate is added to this blend
after passing through a 1.0 mm
sieve for delumping. Subsequently the final blend is produced by final
blending in a suitable mixer and
compressed into tablets. The following tablet composition can be obtained:
Component mg/tablet %/tablet
Active ingredient 28.500 30.0
Stearic-palmitic acid 27.500 28.9
Total (granulate) 56.000 58.9
Mannit 32.600 34.3
Crospovidone 5.600 5.9
Magnesium stearate 0.800 0.9
Total 95.000 100.000
5. Tablet formulation - hot melt extrusion
An active CCR3 antagonist ingredient and stearic-palmitic acid are blended in
a suitable mixer, to
produce a pre-mix. The pre-mix is extruded in a twin-screw-extruder and
subsequently granulated. The
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granulate is sieved through a sieve with a mesh size of 1.0 mm. The granulate
is directly filled into hard
capsules. The following capsule composition can be obtained:
Component mg/tablet %/tablet
Active ingredient 70.000 70.0
Stearic-palmitic acid 30.000 30.0
Total (granulate) 100.000 100.0
Capsule 90.000
Total 190.000 100.000
6. Tablet formulation - roller compaction
An active CCR3 antagonist ingredient, part of mannit and crospovidone and
magnesium stearate
are blended in a suitable mixer, to produce a pre-mix. The pre-mix is
compacted with a roller compactor
and subsequently granulated. Optionally, the granulate is sieved through a
sieve with a mesh size of 0.8
mm. The granulate is blended with part of mannit and crospovidone in a
suitable mixer. Magnesium stearate
is added to this blend after passing through a 1.0 mm sieve for delumping.
Subsequently the final blend is
produced by final blending in a suitable mixer and compressed into tablets.
The following tablet
composition can be obtained:
Component mg/tablet %/tablet
Active ingredient 28.500 30.0
Crospovidone 1.400 1.5
Mannit 34.600 36.4
Magnesium stearate 0.500 0.5
Total (granulate) 65.000 68.4
Mannit 27.000 28.4
Copovidone 1.600 1.7
Crospovidone 0.950 1.0
Magnesium stearate 0.450 0.5
Total 95.000 100.000
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7. Tablet formulation - roller compaction
An active CCR3 antagonist ingredient and magnesium stearate are blended in a
suitable mixer, to
produce a pre-mix. The pre-mix is compacted with a roller compactor and
subsequently granulated.
Optionally, the granulate is sieved through a sieve with a mesh size of 0.8
mm. The granulate is blended
with mannit and croscarmellose sodium in a suitable mixer. Magnesium stearate
is added to this blend after
passing through a 1.0 mm sieve for delumping. Subsequently the final blend is
produced by final blending
in a suitable mixer and compressed into tablets. The following tablet
composition can be obtained:
Component mg/tablet %/tablet
Active ingredient 114.200 66.0
Magnesium stearate 1.800 1.0
Total (granulate) 116.000 67.0
Mannit 51.000 29.5
Croscarmellose sodium 3.500 2.0
Magnesium stearate 2.500 1.5
Total 173.000 100.000
8. Tablet formulation - roller compaction
An active CCR3 antagonist ingredient and magnesium stearate are blended in a
suitable mixer, to
produce a pre-mix. The pre-mix is compacted with a roller compactor and
subsequently granulated.
Optionally, the granulate is sieved through a sieve with a mesh size of 0.8
mm. The granulate is blended
with microcrystalline cellulose and crospovidone in a suitable mixer.
Magnesium stearate is added to this
blend after passing through a 1.0 mm sieve for de-lumping. Subsequently the
final blend is produced by
final blending in a suitable mixer and compressed into tablets. The following
tablet composition can be
obtained:
Component mg/tablet %/tablet
Active ingredient 114.200 66.0
Magnesium stearate 1.800 1.0
Total (granulate) 116.000 67.0
MCC 51.000 29.5
Crospovidone 3.500 2.0
Magnesium stearate 2.500 1.5
Total 173.000 100.000
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9. Coated tablet formulation
Tablet cores according above mentioned formulations can be used to produce
film-coated tablets.
Hydroxypropyl methylcellulose, polyethylene glycol, talc, titanium dioxide and
iron oxide are suspended
in purified water in a suitable mixer at ambient temperature to produce a
coating suspension. The tablet
cores are coated with the coating suspension to a weight gain of about 3 % to
produce film-coated tablets.
The following film coating composition can be obtained:
Component mg/tablet %/tablet
Hypromellose 2.40 48.0
Polyethylene glycol 6000 0.70 14.0
Titanium dioxide 0.90 18.0
Talcum 0.90 18.0
Iron oxide red 0.10 2.0
Purified water __ --
(volatile component)
Total 5.00 100.0
b. Drug Formulation and Administration
The investigational product of the invention (Compound 1) conformed to the
following
chemical structure:
0 N
okr."), 0 0
*if ...(0 It7/LN e
N
a14 i /
N
to (HC1)2
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COMPOUND 1
Those having ordinary skill in the relevant art would recognize that the
compounds, co-crystals,
salts, and formulations described previously in the sections above could also
be used in these examples.
Compound 1 was made available as 100 mg, 200 mg, and 400 mg film-coated
tablets with a
biconvex, round or oval shape and a dull red color. The tablets were produced
by a dry granulation process
and contained microcrystalline cellulose, hydrogen phosphate, croscarmellose
sodium, magnesium stearate,
polyvinyl alcohol, titanium dioxide, polyethylene glycol, talc, iron oxide red
and iron oxide yellow as
inactive ingredients. Placebo tablets matching the investigational product
were produced by a direct
compression process and contained the same inactive ingredients.
c. Pre-Clinical Examples
1. Materials and Methods
(a) Subcutaneous Osmotic Pump Implantation
Alzet mini-pumps were filled, prepared,p and numbered by mouse ID the day
previous to
implantation to allow for priming at 37 C and to allow for blindedness of
treatment. Pumps were implanted
on the back, slightly posterior to the scapulae and slightly lateral to the
midline. Mice were anesthetized
with 3-5% isoflurane using a vaporizer and regulator in an induction chamber,
then moved to the procedure
area and fitted with a nose cone to maintain anesthesia at 1-3% isoflurane. An
ophthalmic ointment was
applied to the eyes to prevent drying. Mice were injected with meloxicam
5mg/kg subcutaneously. Fur was
removed from the incision area using small sharp scissors, and the area was
cleaned with alternating
applications of 70% isopropanol and betadine. An incision 0.5-1cm was made,
and a hemostat was inserted
to spread the subcutaneous tissue to create a pocket for the pump. The pump
was inserted into the pocket
and the wound was closed with wound clips. All surgical tools were autoclaved
prior to first use on a surgery
day. Subsequently, instruments were sterilized with a glass bead sterilizer
between animals. Mice were
placed in a clean recovery cage placed partially atop a warming pad until full
recovery and ambulation.
Mice were tested for anesthesia induction by toe pinch method and monitoring
of respiration. Mice were
monitored post-operatively every 15 minutes until recovery. Mice were
administered a second dose of
meloxicam the following day. If signs of infection were observed, mice
received 5mg/kg Baytril
subcutaneously per day until infection cleared.
(b) Open Field Test
Open Field is used to evaluate general locomotor activity and exploratory
behavior in a novel
environment. Mice are brought to the experimental room for at least 30 min for
acclimation to the
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experimental room conditions (dim lighting) prior to testing. The testing
arena consists of a 50cm x 50cm
square arena. Mice are placed in the center of an arena and tracked for 15
minutes. Time spent in the
peripheral and centers zones is analyzed, along with rearing behavior. 70%
ethanol is used to clean all
surfaces between trials.
(c) Y-Maze
A large Y-maze test assesses short-term memory of the familiarity of a
specific context. Mice are
brought to the experimental room for at least 30 min of acclimation to the
experimental room conditions
(dim lighting) prior to testing. For the initial training trial, the mouse is
placed at the end of one arm of a
large Y-maze designated "start arm" (arm length: 15 inches). The third arm of
the maze is blocked off,
allowing the mouse to explore two of the three arms freely ("start arm" and
"familiar arm") for 5 min. Each
arm contains spatial cues. One hour later, the mouse is placed back into the
maze in the "start arm" and
allowed to explore all three arms with the third arm unblocked ("novel arm").
Movements in and out of
each arm are tracked using automated tracking software (CleverSys). Testing is
performed under dim
lighting, and the apparatus is cleaned with 70% ethanol between trials.
(d) Barnes Maze
A modified Barnes maze was used to assess spatial working/episodic like
learning and memory.
The Barnes maze apparatus consists of a 122cm diameter circular platform with
40 escape holes, each with
a diameter of 5cm placed along three rings of varying distances from the
center of the platform. An escape
box is attached to one of the holes and all holes are left uncovered. Bright
lights and a fan are trained on
the maze to provide adverse stimuli to encourage escape. Visual cues are
placed on all four sides of the
maze. Mice are given a series of 4 or 5 trials with inter-trial intervals of
approximately 10 min, and the
maximum duration of each trial is 90 or 120 sec. For each trial, mice are
placed in the center of the maze.
After 10 seconds, mice are allowed to explore, and the trial is ended if the
mouse has found and entered an
escape box before the end of the trial. Mice that cannot find the escape box
are led to it and allowed to enter
and given 30 sec to remain before being returned to its home cage. Training is
done for 4 days. Data that is
recorded and analyzed include velocity, escape latency, and distance moved.
Mice are divided into groups of 4-5 mice each, with balanced treatment groups.
For example, Group
1 mice are run for 4 trials, then Group 2 mice are run for 4 trials, and so on
until all groups finish testing.
70% ethanol is used to clean the arenas between trials.
(e) Rotarod
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The mice were trained in the rotarod for 3 trials up to 100 seconds each, the
rotarod being a test
of motor coordination. Success or failure was recorded on the last trial, with
success defined by a latency
to fall of > 90 seconds. A binomial test was conducted to compare success
rates between control and
compound-treated mice.
(f) T maze
The Water Maze was filled with water at least 24 hours prior to the test to
allow it to reach room
temperature. The water was dyed with white latex paint to make the animals
visible for tracking and to
allow for the use of a hidden platform. Two distinct visual cues were placed
at the end of both T-arms of
the T-Maze insert. On day 1 animals were given 4 trials each with a visible
platform and a 30 min inter-
trial interval. Animals were given 60 seconds to reach the platform. If they
did not reach the platform in
that time they were guided to it and allowed to remain for 5 seconds before
being removed from the tank.
The goal arm was switched after every third mouse and both treatment groups
had equal numbers of right
and left turn goal arms. After each trial the mice were placed in an empty
cage with blue pads and allowed
to dry off under a red light lamp before being placed back into their home
cage. Day 2 is the testing day,
where animals are subjected to the same test of 4 trials each and a 30 min
inter trial interval, but with a
hidden platform. Animals were scored for right or wrong choice and for latency
to reach the platform, and
a binomial test was conducted to compare success rates between control and
compound-treated mice. All
trials were recorded using TopScan.
(g) Protein quantification
CCL11 protein levels were measured from mouse plasma by sandwich ELISA Plasma
was diluted
1:10 for the assay. (Mouse CCL11/Eotaxin Duo Set ELISA kit, R&D Systems,
Minneapolis, MN).
Human CCL11 from human plasma was measured by a SomaLogic aptamer-based assay
(SOMAscan from SomaLogic, Inc., Boulder, CO). (Gold L, et al. (2010). Aptamer-
Based Multiplexed
Proteomic Technology for Biomarker Discovery. PLOS ONE 5(12): el 5004.
Panels of circulating cytokines from mouse plasma were analyzed by Luminex.
Luminex Assay
Service performed by Eve Technologies (Calgary, Alberta, Canada).
(h) mRNA quantification
Snap-frozen hemibrains were dissected out into cortex, hippocampus, striatum,
and thalamus. The
cortex was split into three homogeneous parts and one part was used for RNA
isolation. RNA was isolated
using the Prolink RNA Mini Kit (Thermo Fisher Scientific #12183025). cDNA was
made using Taqman
RT Kit (Thermo Fisher Sci #N8080234). qPCR was run on a Quant Studio 6 using
custom made Taqman
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Multiplex Primers for IL-lbeta and GAPDH. All samples were run together on a
single plate and analyzed
for ddCT relative to their endogenous control first and then relative to the
control group.
(i) HPLC/MS quantification of Compound 1
Compound levels were measured by MS/MS by Quintara (Hayward, CA).
(j) Flow analyses
Eosinophil Count
200uL of whole blood was collected during perfusion and shipped to Charles
River Clinical
Pathology Services (Shrewsbury, MA) for hematology analysis of eosinophil
count by FACS (fluorescence
activated cell sorter).
Eosinophil Shape Change (ESC)
The ESC determines the shape change of human eosinophils activated by human
eotaxin-1
(PreProTech, Rocky Hill, New Jersey) compared to native eosinophils. The
change is detected as the
change in the forward-scatter measured by FACS (fluorescence activated cell
sorter). The mean forward
scatter of the autofluorescence (eosinophil) population for each sample was
determined in conjunction
with the mean of each set of sample triplicates. Methods of determining ESC
have been previously
described and are known in the art.
CCR3 Receptor Internalization
The CCR3 receptor internalization assay is FACS-based and uses human Eotaxin-1
(PreProTech,
Rocky Hill, New Jersey) and an anti-human CCR3 antibody labeled with APC (R&D
Systems,
Minneapolis, Minnesota) or isotype control IgG2A antibody to monitor CCR3
receptor internalization in
human eosinophils induced by human eotaxin-1 compared to naive eosinophils.
The median APC
fluorescent intensity units for each sample were determined along with the
mean of each set of sample
triplicates. Methods of monitoring CCR3 receptor internalization have been
previously described and are
known in the art.
(k) Histology
Mice were taken down on the day following the end of behavior testing.
Anesthesia was induced
by 2,2,2-tribromoethanol and mice were subsequently perfused with 0.9% saline
trans-cardially. The brains
were dissected and cut sagitally in two even halves. One half was snap frozen
for later use in dry ice, and
the other was fixed in 4% paraformaldehyde in PBS for use in
immunohistochemistry. After two days of
fixation, the hemibrains were transferred to a 30% sucrose in PBS solution and
changed after two days.
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Hemibrains were sectioned at 30um on a microtome at -22 C. Brain sections were
stored in cryoprotectant
media at -20 C until needed for staining. Blocking was done on free floating
sections in the appropriate
serum at 10% serum in PBST 0.5%. Primary antibodies were incubated overnight
at 4C. For light
microscopy, the following antibodies were used in the given concentrations:
DCX, 1:200, Santa Cruz
BioTech, CD68, 1:1000. AbD Serotec. Secondary biotinylated antibodies were
applied the next day at a
concentration of 1:300. Staining visualization was achieved by reaction with
the ABC kit (Vector) and
diaminobenzidine (Sigma). Dehydration of the mounted slides was achieved using
ethanol and xylene dips.
Images were acquired on a Leica light microscope at 5x magnification.
For fluorescent microscopy, the following antibodies were used in the given
concentrations: GFAP,
1:500, DAKO; Ibal, 1:1000, Wako; and BrdU, 1:500, AbCam. Antigen retrieval
protocol was required for
BrdU (2N HC1, 37C, 30min) prior to blocking. The appropriate fluorescent
secondary antibodies were
applied the next day at a concentration of 1:300 for one hour at room
temperature. Prolong Gold Mounting
Media was used to coverslip the slides. Images were acquired on a light
microscope at 5x magnification.
2. Experimental Groups
First Experimental Group (See Figures 1-2): Two-month-old or 18-month-old
C57B1/6 mice were
dosed with either IgG antibody control by IP injection or Compound 1
subcutaneously by Alzet osmotic
pump for either 2 or 4 weeks. During the last week of treatment, mice were
subjected to behavior testing
prior to perfusion on the last day of treatment. All mice received 5
consecutive days of BrdU injection at
150mg/kg IP immediately prior to treatment start.
Drug Formulation. Control rat IgG2A clone 54447 (MAB006, R&D Systems) was
administered
at 5Oug/kg in sterile saline. Compound 1 was formulated in 40% HP-13-
cyclodextrin, and adjusted to pH
6.5 with NaOH (1M). Solutions were prepared fresh weekly and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Young Controls: Young C57BL/6 mice (n=18), aged 1-2
months,
received 5 injections of control IgG by intraperitoneal (IP) injection, one
injection every
3 days, over a period of 14 days.
= Treatment Group 2, Old Controls: Aged C57BL/6 mice (n=18), aged 18
months, received
injections of control IgG by IP injection, one injection every 3 days, over a
period of 14
days.
= Treatment Group 3, Compound 1 (dose 1) in Old: Aged C57BL/6 mice (n=16),
aged 18
months, received infusion of ¨50mg/m1 Compound 1 by Alzet mini-pump, model
2001
(luL/hr), for two weeks with one replacement.
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= Treatment Group 4, Compound 1 (dose 2) in Old: Aged C57BL/6 mice (n=16),
aged 18
months, received infusion of ¨50mg/m1 Compound 1 by Alzet mini-pump, model
2002
(0.5uL/hr), for two weeks.
= Treatment Group 5, Compound 1 (dose 2) in Old: Aged C57BL/6 mice (n=16),
aged 18
months, received infusion of ¨50mg/m1 Compound 1 by Alzet mini-pump, model
2002
(0.5uL/hr), for four weeks with one replacement.
Four-week subcutaneous dosing of Compound 1 in 18-month-old mice increased the
numbers of
doublecortin-positive cells in the hippocampus, an indicator of neurogenesis
(see Figure 1A). Higher
dosing of Compound 1 for 2 weeks led to a trend in increased BrdU-positive
cells in the hippocampus, a
marker for cell proliferation (see Figure 1B). Low or high infusion of
Compound 1 for 2 or 4 weeks led to
improvement in the cued Y-maze, a test for memory (see Figure 2 reporting
percent of time normalized to
total interaction time and number of visits--similar effects were also seen
when evaluating total time spent).
Comparisons are made to control groups (Treatment groups 1 & 2) which were
treated with IgG antibody
with no compound, and dosing and behavior were performed in parallel.
Thus, administration of Compound 1 increased the number of Dcx and BrdU
positive cells, which
indicates that Compound 1 increased neurogenesis and cell survival,
respectively. Compound 1 was able
to improve memory (cognition) as evidenced by performance in the Y-Maze test.
Second Experimental Group (See Figures 3-6): Three-month-old or 16.5-month-old
C57B1/6 mice
were dosed with either vehicle control or Compound 1 subcutaneously by Alzet
osmotic pump for 4 weeks.
During the last week of treatment, mice were subjected to behavior testing
prior to perfusion on the last day
of treatment. All mice received 5 consecutive days of BrdU injection at
150mg/kg IP immediately prior to
treatment start.
Drug Formulation. Compound 1 was formulated in 40% HP-I3-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Solutions were
prepared fresh weekly and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Young Controls: Young C57BL/6 mice (n=19), aged 3
months,
received infusion of vehicle by Alzet mini-pump, model 2002 (0.5uL/hr), for
four weeks
with one replacement.
= Treatment Group 2, Old Controls: Aged C57BL/6 mice (n=19), aged 16
months, received
infusion of vehicle by Alzet mini-pump, model 2002 (0.5uL/hr), for four weeks
with one
replacement.
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= Treatment Group 3, Compound 1 (dose 2) in Old: Aged C57BL/6 mice (n=19),
aged 16
months, received infusion of ¨50mg/m1 Compound 1 by Alzet mini-pump, model
2002
(0.5uL/hr), for four weeks with one replacement.
Four-week subcutaneous dosing of Compound 1 in 16.5-month-old mice improved
cognitive
performance in the cued Y-maze test for memory (see Figure 3). 4-week dosing
of Compound 1 also
trended towards improved performance in the Barnes maze, a test for
hippocampus-dependent spatial
memory (see Figure 4). Trends for improved memory were also observed upon
analysis of the average of
the latency for day 4 trials as well as in the difference between the
latencies of trials 13 and 16. Four-week
dosing of Compound 1 also significantly increased the number of BrdU-positive
cells in the hippocampus,
an indicator of neurogenesis (see Figure 5). Thus, Compound 1 was able to both
improve cell survival and
improve memory (cognition) as evidenced by the results obtained using the Y-
Maze and Barnes Maze tests.
Compound Cerebrospinal Fluid Levels in Mice
Cerebrospinal fluid (CSF) from both the 2-month-old "young" and 16.5-month-old
"old" groups
was collected and levels of Compound 1 were determined by mass spectroscopy.
Figure 6 depicts the
levels of the compound of the invention that were detected in mouse CSF for
both the young and old
groups (both below 10 nM). These CSF levels do not approach the Ki for the
compound in mice (124
nM, determined by cell line receptor binding), and therefore do not cross the
blood-brain barrier (BBB) in
significant concentrations. For comparison, the levels of the compound of the
invention that were
measured in the plasma of young (2-month-old) and old (18-month-mice) perfused
for 2 and 4 weeks
respectively at 0.5 it/hr of 50mg/mL solution were significantly higher (352
31 nM and 355 43 nM,
respectively; values are mean s.e.m.), further indicating that the compound
of the invention fails to
cross the BBB in significant amounts.
This data shows that Compound 1 does not act directly in the CNS, and
therefore acts
peripherally. Additionally, what does cross the BBB is insufficient in
concentration to be effective.
Further, there is no difference in BBB penetration between young and old mice,
indicating that the effects
Compound 1 displays on cognition and neurodegeneration are not due to
differences in the BBB between
the two groups.
Compound Tissue Distribution Levels
The distribution of Compound 1 in mouse tissues was determined after oral
administration of which
was [14C] -radiolabeled ("Labeled Compound") to male pigmented C57BL/6J01aHsd
mice (Harlan Labs,
BV). The Labeled Compound was administered at 10 mg/kg of body weight,
corresponding to 17 timol/kg.
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One animal was sacrificed after 1, 24, and 168 hours post-administration.
Blood, plasma, and eye
radioactivity concentrations were measured using liquid scintillation counting
(LSC). Tissue and organ
concentrations were determined by whole body autoradiography technique (QWBA).
Preparation of the
whole-body animal sections was performed according to known techniques (see S.
Ullberg, et al.,
Autoradiography in Pharmacology in: The Int. Encyclopedia of Pharmacology, J.
Cohen (Ed.), 1(78):221-
39 (1971)), using a crystat microtome Reichert-Jung CRY() MACROCUT or a CRY()
MACROCUT
LEICA CM 3600g.
The following sections were taken at different levels through the embedded
animal, and whole
body sections selected at 5 ¨ 7 levels in order to allow for quantitative
evaluation of radioactivity: adrenal
glands; blood; bone marrow; brain; eye (lens); epididymis; fat (white and
brown); Harderian gland; heart;
kidneys; liver; lung; muscle; pituitary; pancreas; prostate, spinal cord;
spleen salivary gland; skin; testis;
thyroid; thymus; uveal tract. Two sections of each chosen level were taken per
animal, and those sections
lyophilized in the microtome at -20 to -25 C for a minimum of 48 hours.
Figure 7 depicts a chart of values reporting the area under the curve (AUC)
for all three time points,
and quantifies each tissue's exposure to the Labeled POI after administration
of the compound. Figure 7
shows that Compound 1 does not cross the blood-brain barrier (BBB) at
significant levels.
Again, these results show that, because Compound 1 cannot cross the BBB at
appreciable levels, it
acts in a peripheral fashion. As such, Compound l's effects do not directly
act on the central nervous
system, and it overcomes difficulties that have caused the failure of many CNS
disease-targeting
pharmaceutical candidates.
Pharmacokinetic Profiles in P.O. Dosing
Plasma from male 2-month-old C57B1/6 mice was measured for concentrations of
Compound 1 at
time points of 20 minutes, 2 hours, 8 hours, and 12 hours after oral gavage at
2 doses: 30 mg/kg and 150
mg/kg. A dose of 30 mg/kg was found to be sufficient to achieve a greater than
100 nM concentration of
Compound 1 for 8 hours (Figure 8).
Third Experimental Group (See Figures 9-11): Two-month-old C57B1/6 mice were
dosed with
either vehicle control or Compound 1 by oral gavage twice daily for 18 days.
During the last week of
treatment, mice were subjected to behavior testing prior to perfusion on the
last day of treatment. All mice
received 5 consecutive days of BrdU injection at 150mg/kg IP immediately prior
to treatment start.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Solutions were
prepared fresh weekly and stored at 4 C.
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Treatment Groups:
= Treatment Group la, Vehicle treatment: Young C57BL/6 mice (n=15), aged 7
weeks,
received twice daily (BID) oral (PO) treatment of vehicle solution for 18
days, with only 1
injection on the last day, for a total of 35 injections.
= Treatment Group lb, Compound 1 treatment: Young C57BL/6 mice (n=15), aged
7 weeks,
received twice daily (BID) oral (PO) treatment of Compound 1, 30mg/kg, for 18
days, with
only 1 injection on the last day for a total of 35 injections.
= Treatment Group 2a, Vehicle treatment with rmCCL11: Young C57BL/6 mice
(n=15),
aged 7 weeks, received twice daily (BID) oral (PO) treatment of vehicle
solution for 18
days, with only 1 injection on the last day for a total of 35 injections. Mice
concurrently
received peripheral injections (IP) of rmCCL11 starting on day 1 of treatment,
every 3
days, for a total of 5 injections.
= Treatment Group 2b, Compound 1 treatment with rmCCL11: Young C57BL/6 mice
(n=15), aged 7 weeks, received twice daily (BID) oral (PO) treatment of
Compound 1,
30mg/kg, for 18 days, with only 1 injection on the last day for a total of 35
injections. Mice
concurrently received peripheral injections (IP) of rmCCL11 starting on day 1
of treatment,
every 3 days, for a total of 5 injections.
Recombinant mouse CCL11 ("rmE") treatment significantly worsened anxiety in
the Open Field,
but 2 weeks of treatment with Compound 1 twice daily orally improved anxiety
(see Figure 9). rmCCL11
impaired memory in the Y-maze; however, mice treated with Compound 1 were no
longer significantly
different from control mice (see Figure 10). rmCCL11 also impaired memory in
the Barnes maze, and
treatment with Compound 1 significantly improved memory performance (see
Figure 11). Thus, rmE
worsened both anxiety via the Open Field test and memory as evidenced by
performance measured in the
Y-Maze and Barnes Maze tests. However, Compound 1 treatment was able to
attenuate these effects.
Fourth Experimental Group (See Figure 12): 23-month-old C57B1/6 mice were
dosed with either
vehicle control or Compound 1 by oral gavage twice daily for 19 days. After 11
days of treatment, mice
were subjected to Y maze testing.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Vehicle treatment: Aged C57B1/6 mice (n=8), aged 23
months,
received twice daily (BID) oral gavage (PO) treatment of vehicle solution for
19 days.
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= Treatment Group 2, Compound 1 treatment: Aged C57B1/6 mice (n=11), aged
23 months,
received twice daily (BID) oral gavage (PO) treatment of Compound 1, 30 mg/kg,
for 19
days.
Compound 1 treatment significantly improved memory in the Y-maze. Mice treated
with
Compound 1 exhibited intact memory for the novel arm in the number of visits
(Figures 12A). Treatment
with Compound 1 also significantly increased the distance travelled of mice
during the test (Figure 12B).
Fifth Experimental Group (See Figures 13-17): 23-month-old C57B1/6 mice were
dosed with
either vehicle control or Compound 1 subcutaneously twice daily for 21 days.
Three weeks later, mice
were subjected to behavior testing, and sacrificed the day following the last
behavior test.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group lb, Vehicle treatment: Aged C57B1/6 mice (n=9), aged 23
months,
received twice daily (BID) subcutaneous (SQ) treatment of vehicle solution for
21 days.
= Treatment Group 4, Compound 1 treatment: Aged C57B1/6 mice (n=17), aged
23 months,
received twice daily (BID) subcutaneous (SQ) treatment of Compound 1, 30
mg/kg, for 21
days.
Compound 1 treatment significantly improved memory in the Y-maze and the
Barnes Maze. Mice
treated with Compound 1 exhibited intact memory for the novel arm in both the
number of visits (Figures
13A-B) and in the duration of time spent in the novel arm (Figures 13C-D).
Treatment with Compound 1
also significantly increased the velocity of mice during the test (Figure
13E). Mice treated with Compound
1 performed significantly better on the Barnes Maze for spatial memory (Figure
14A) and also exhibited
increased velocity during the test (Figure 14B). Locomotor activity was also
improved in the Open Field,
where there were strong trends towards improvement in both distance travelled
and velocity (Figures 15A
and 15B, respectively).
Levels of inflammatory cytokines were measured by Luminex assay from the
plasma of mice
(Figure 16). There were strong trends in decrease in several inflammatory
markers, including TNFa, IL6,
ILlbeta, IL5, and IL17. Activated microglia was also quantified by IHC
staining in the hippocampus of
mice (Figure 17). Compound 1 treatment resulted in a strong trend towards
decreased microgliosis.
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Sixth Experimental Group (See Figure 18): 23-month-old C57B1/6 mice were dosed
with either
vehicle control or Compound 1 subcutaneously twice daily for 30 days, and then
sacrificed on the day of
the last injection.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group la, Vehicle treatment: Aged C57B1/6 mice (n=9), aged 23
months,
received twice daily (BID) subcutaneous (SQ) treatment of vehicle solution for
30 days.
= Treatment Group 3, Compound 1 treatment: Aged C57B1/6 mice (n=18), aged
23 months,
received twice daily (BID) subcutaneous (SQ) treatment of Compound 1, 30
mg/kg, for 30
days.
Compound 1 treatment resulted in a strong trend towards decreased blood
eosinophils in the blood
by FACS analysis in a subset of animals (n = 2, 5) (Figure 18).
Seventh Experimental Group (See Figure 19): 3-month-old hairless mice were
treated with
oxazolone (Ox) every other day and treated with control saline or 30 mg/kg
Compound 1 for 2 weeks
BID, PO.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Vehicle treatment: 3-month-old Hairless mice (n=3)
received twice
daily (BID) oral gavage (PO) treatment of vehicle solution for 2 weeks.
= Treatment Group 2, Vehicle treatment and Oxazolone treatment: 3-month-old
Hairless
mice (n=6) received twice daily (BID) oral gavage (PO) treatment of vehicle
solution for
2 weeks.
= Treatment Group 3, Compound 1 treatment and Oxazolone treatment: 3-month-
old
Hairless mice (n=6) received twice daily (BID) oral gavage (PO) treatment of
Compound
1, 30 mg/kg, for 2 weeks.
Compound 1 decreased the oxazolone-induced increase in blood eosinophils by
complete blood
count (CBC) analysis. N = 3, 6, 8. *p < 0.05, **p <0.01. Compound 1
dramatically reduced numbers of
blood eosinophils in an oxazolone-induced model of eosinophilia (Figure 19).
This shows that inhibition
of CCR3 can be sufficient to normalize eosinophil levels and function
especially in diseased states,
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demonstrating a second mechanism (in addition to decreased brain inflammation)
by which Compound 1
can be effective in treating neuronal loss and associated motor and cognitive
deficits.
Eighth Experimental Group (See Figures 20-22): Twenty-four-month-old C57 mice
were treated
for 4 weeks with continuous infusion of Compound 1 or vehicle by Alzet osmotic
pump, which were
implanted to continuously deliver the two treatments.
Drug Formulation: Compound 1 was formulated in 40% HP-I3-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Old Controls: Aged C57BL/6 mice (n=15), aged 23
months, received
infusion of vehicle by Alzet mini-pump, model 2002 (0.5uL/hr), for four weeks
with one
replacement.
= Treatment Group 2, Compound 1 (dose 2) in Old: Aged C57BL/6 mice (n=15),
aged 23
months, received infusion of ¨50mg/m1 Compound 1 by Alzet mini-pump, model
2002
(0.5uL/hr), for four weeks with one replacement.
The mice were tested in the Rotarod for motor coordination. Success or failure
was recorded on
the last trial, with success defined by a latency to fall of > 90 seconds.
Compound 1-treated mice succeeded
more than vehicle-treated mice by binomial test. N = 15 each, * p < 0.05. A
greater proportion of mice
treated with Compound 1, 47%, were able to stay on the rod for longer than 90
seconds, a threshold where
only 20% of control-treated mice could stay on after 3 successive trials
(Figure 20). These results suggest
that there is a consistent effect of Compound 1 on motor function in models of
eotaxin elevation.
Mice were tested in the T maze for cognitive function (Figure 21). The number
of successes or
failures to turn down the correct arm was recorded. Compound 1-treated mice
succeeded more than vehicle-
treated mice by binomial test. * p < 0.05.
Fecal output was measured by weighing the dry weight of fecal pellets
overnight (Figure 22).
Gastric function is a well-known symptom of Parkinson's disease. Water and
food intake were measured
over the same time period. Mice treated with Compound 1 had significantly
lower fecal output compared
to control mice. They also had significantly higher water intake overnight
compared to control mice. * p <
0.05. These results indicate that deficits in gastric function may be altered
by Compound 1 treatment.
Ninth Experimental Group (See Figure 23): Three-month-old C57 mice were given
one dose of
lipopolysaccharide (LPS) 10mg/kg IP to induce inflammation and treated with
Compound 1 for 18 days.
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Drug Formulation: Compound 1 was formulated in 40% HP-I3-cyclodextrin, and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Vehicle treatment: Young C57BL/6 mice (n=15), aged 3
months,
received a single injection of LPS and were treated with vehicle, BID, for 18
days by oral
gavage.
= Treatment Group 2, Compound 1 treatment: Young C57BL/6 mice (n=15), aged
3 months,
received a single injection of LPS and were treated with Compound 1, 30 mg/kg,
BID, for
18 days by oral gavage.
Brain sections were immunostained for detection of CD68+ activated microglia.
Compound 1-
treated mice exhibited significantly decreased CD68+ immunoreactivity
(decreased activated microglia) in
contrast to vehicle (saline)-treated mice (Figure 23). N = 9, 10, 8. *p < 0.05
by one-way ANOVA.
These data indicate a potent anti-neuroinflammatory effect of Compound 1, with
therapeutic
potential for reducing neuroinflammation-induced toxicity to neurons in
diseases exhibiting
neurodegeneration or cognitive or motor decline.
Tenth Experimental Group (See Figure 24-29): Three-month-old C57 mice were
given daily
doses of lipopolysaccharide (LPS) 0.5 mg/kg IP to induce inflammation and
treated with Compound 1
chronically for up to 4 weeks.
Drug Formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin and
adjusted to pH
6.5 with NaOH (1M). Vehicle solution was formulated and adjusted for pH
similarly. Kolliphor was added
to each solution at 10% every week and stored at 4 C.
Treatment Groups:
= Treatment Group 1, Vehicle treatment: 3-month-old C57 mice (n=10)
received daily IP
injection of LPS for 7 weeks and twice daily (BID) oral gavage (PO) treatment
of vehicle
solution for up to 4 weeks.
= Treatment Group 2, Vehicle treatment and Oxazolone treatment: 3-month-old
C57 mice
(n=10) received daily IP injection of LPS for 7 weeks and twice daily (BID)
oral gavage
(PO) treatment of vehicle solution for up to 4 weeks.
= Treatment Group 3, Compound 1 treatment and Oxazolone treatment: 3-month-
old C57
mice (n=9) received daily IP injection of LPS for 7 weeks and twice daily
(BID) oral
gavage (PO) treatment of Compound 1, 30 mg/kg, for up to 4 weeks.
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Figure 24A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 24B was
performed. Anxiety was tested in the Open Field test after 1 week of Compound
1 treatment (Figure 24B).
LPS treatment significantly increased anxiety in the Open Field, and Compound
1 strongly decreased the
increased anxiety, * p < 0.05.
Figure 25A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 25B was
performed. Cognition was tested in the Y maze after 3 weeks of Compound 1
treatment (Figure 25B). Mice
treated with LPS did not show a significant preference for the novel arm.
However, mice treated with
Compound 1 showed a significant preference for the novel arm, similar to
vehicle treated mice, * p < 0.05,
** p < 0.01.
Figure 26A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 26B was
performed. mRNA levels of the inflammatory cytokine IL-lbeta were measured by
quantitative PCR 4
weeks after Compound 1 treatment (Figure 26B). LPS treated mice showed a trend
towards increased levels
of IL-lbeta, and mice treated with Compound 1 showed a significant decrease in
IL-lbeta expression, * p
<0.05.
Figure 27A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 27B was
performed. Brain sections were immunostained for detection of CD68+ activated
microglia. Compound 1-
treated mice exhibited dramatically decreased CD68+ immunoreactivity
(decreased activated microglia) in
contrast to LPS-only-treated mice (Figure 27B).
Figure 28A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 28B was
performed. Brain sections were immunostained for detection of Iba 1 -positive
microglia. Compound 1-
treated mice exhibited dramatically decreased Iba 1+ immunoreactivity
(decreased total microglia) in
contrast to LPS-only-treated mice (Figure 28B).
Figure 29A describes the dosing paradigm for the tenth experimental group. The
section of the
paradigm highlighted by the square represents the time points at which the
assay in Figure 29B was
performed. Brain sections were immunostained for detection of GFAP-positive
astroglia. Compound 1-
treated mice exhibited dramatically decreased GFAP+ immunoreactivity
(decreased total astrocytes) in
contrast to LPS-only-treated mice (Figure 29B).
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These data indicate a potent anti-neuroinflammatory effect of Compound 1, with
therapeutic
potential for reducing neuroinflammation-induced toxicity to neurons in
diseases exhibiting
neurodegeneration or cognitive or motor decline.
Eleventh Experimental Group (See Figure 30): Forty-five (42) 2-month-old
C57BL/6 mice were
allocated to three groups: vehicle treated controls, vehicle treated LPS mice,
and Compoundl -treated LPS
mice. Vehicle or compound was given twice a day orally (PO BID) for a total of
6 doses. Histological
assessments were done on the hippocampus with microglia marker Iba-1.
Drug formulation: Compoundl was formulated in 40% HP-I3-cyclodextrin and
adjusted to pH 6.5
with NaOH (1M). Vehicle solution was formulated and adjusted for pH similarly.
Solutions were prepared
fresh weekly and stored at 4 C.
LPS formulation: LPS was formulated at 0.55mg/m1 in saline and dosed at
5mg/kg.
Treatment Groups:
= Treatment Group 1, Vehicle treatment controls: young C57BL/6 mice (n=13),
aged 2
months, received a single IP injection of saline followed by vehicle PO BID
for 3 days for
a total of 6 injections.
= Treatment Group 2, Vehicle treatment LPS: young C57BL/6 mice (n=19), aged
2 months,
received a single IP injection of LPS (5mg/kg) followed by vehicle PO BID for
3 days for
a total of 6 injections.
= Treatment Group 3, Compoundl treatment LPS: young C57BL/6 mice (n=18),
aged 2
months, received a single IP injection of LPS (5mg/kg) followed by Compoundl
(30mg/kg) PO BID for 3 days for a total of 6 injections.
Tissue processing and histology: Mice Hemibrains were sectioned at 30 m on a
microtome at -
22 C. Brain slices were collected sequentially into 12 tubes, so that every
12th section of the hippocampus
was represented in a given tube. Brain sections were stored in cryoprotectant
media at -20 C until needed
for staining. Free floating sections were blocked in the appropriate serum at
10% serum in PBST 0.5%.
Primary antibodies Ibal (Wako, 1:1000) was incubated overnight at 4 C. The
appropriate fluorescent
secondary antibodies was applied the next day at a concentration of 1:300 for
one hour at room temperature.
Prolong Gold Mounting Media was used to coverslip the slides.
Neuroinflammation quantification: Iba-1 positive area was quantified using
thresholding on Image
Pro Premier v9.2 software as a percentage of an ROI around the entire
hippocampus. The Iba-1 positive
percent area was averaged from approximately 5 sections for each mouse.
Ordinary one-way ANOVA was
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used to test for statistical significance, with Dunnett's multiple comparisons
test post-hoc between treatment
groups.
Figure 30 shows the results of histological analysis of the mouse brains in an
acute model of LPS-
induced inflammation. It reveals a significant reduction of LPS-induced
microgliosis in the hippocampi of
mice treated with Compound 1 for three days, when both treatment with Compound
1 and LPS was
commenced on the same day. Microgliosis was measured by determining the
percentage of Iba-1 positive
area in the hippocampi. Ordinary one-way ANOVA was used to test for
statistical significance, with
Dunnett's multiple comparisons test post-hoc between treatment groups. (*
P<.05; *** P<.001).
Twelfth Experimental Group (See Figure 31): Fifteen (15) 2-month-old C57BL/6
mice were
allocated to three groups: vehicle treated controls, vehicle treated LPS mice,
and Compound 1 treated LPS
mice. Vehicle or compound was given twice a day orally (PO BID) for a total of
6 doses. Histological
assessments were done on the hippocampus with microglia marker Iba-1.
Drug formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin and
adjusted to pH 6.5
with NaOH (1M). Vehicle solution was formulated and adjusted for pH similarly.
Solutions were prepared
fresh weekly and stored at 4 C.
LPS formulation: LPS was formulated at 0.55mg/m1 in saline and dosed at
5mg/kg.
Treatment Groups:
= Treatment Group 1, Vehicle treatment controls: young C57BL/6 mice (n=13),
aged 2
months, received a single IP injection of saline then 72 hours late were dosed
vehicle PO
BID for 3 days for a total of 6 injections.
= Treatment Group 2, Vehicle treatment LPS: young C57BL/6 mice (n=19), aged
2 months,
received a single IP injection of LPS (5mg/kg) then 72 hours late were dosed
with vehicle
PO BID for 3 days for a total of 6 injections.
= Treatment Group 3, Compound 1 treatment LPS: young C57BL/6 mice (n=18),
aged 2
months, received a single IP injection of LPS (5mg/kg) then 72 hours late were
dosed with
Compound 1 (30mg/kg) PO BID for 3 days for a total of 6 injections.
Tissue processing and histology: Mice Hemibrains were sectioned at 30 m on a
microtome at -
22 C. Brain slices were collected sequentially into 12 tubes, so that every
12th section of the hippocampus
was represented in a given tube. Brain sections were stored in cryoprotectant
media at -20 C until needed
for staining. Free floating sections were blocked in the appropriate serum at
10% serum in PBST 0.5%.
Primary antibodies Ibal (Wako, 1:1000) was incubated overnight at 4 C. The
appropriate fluorescent
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secondary antibodies were applied the next day at a concentration of 1:300 for
one hour at room
temperature. Prolong Gold Mounting Media was used to coverslip the slides.
Neuroinflammation quantification: Iba-1 positive area was quantified using
thresholding on Image
Pro Premier v9.2 software as a percentage of an ROT around the entire
hippocampus. The Iba-1 positive
percent area was averaged from approximately 5 sections for each mouse.
Ordinary one-way ANOVA was
used to test for statistical significance, with Dunnett's multiple comparisons
test post-hoc between treatment
groups.
Figure 31 shows the results of histological analysis of the mouse brains in an
acute model of LPS-
induced inflammation. It reveals a significant reduction of LPS-induced
microgliosis in the hippocampi of
mice treated with Compound 1 for three days, when Compound 1 administration
commenced after LPS had
been administered for three days prior. Microgliosis was measured by
determining the percentage of Iba-
1 positive area in the hippocampi. Ordinary one-way ANOVA was used to test for
statistical significance,
with Dunnett's multiple comparisons test post-hoc between treatment groups. (*
P<.05; *** P<.001).
3. Mouse MPTP Model of Parkinson's Disease
A mouse model using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was
used to
determine the level of improvement of MPTP-induced Parkinsonian deficits. MPTP
is a prodrug of MPP+,
which can cause permanent Parkinson's symptoms. MPP+ works by killing
dopaminergic neurons in the
brain' s substantia nigra region.
In total seventy-four (74) 8-week old male C57B1/6J mice were used in the
experiment. Animals
were divided into two separate study arms, a 12 day and 4 day study arm, and
treated similarly in both arms.
Mice were grouped into three groups within a study arm, so that one group
worked as a control, receiving
MPTP vehicle and compound vehicle, when the other two groups received MPTP
twice a day on days 1
and 2, and in addition compound vehicle or Compound 1 daily (30 mg/kg) on days
1-12 with only one dose
on Day 12 (Figure 32). After ten days of the treatment on study day 11 motor
functions of the 12 day study
arm mice were evaluated in the fine motor kinematic analysis. On study day 12
and on day 4 endpoint tissue
processing was performed. Samples were processed for hematology,
immunohistochemistry, and HPLC
measurements. Striatal levels of dopamine (DA), 3,2-dihydroxyphenylacetic acid
(DOPAC) and
homovanillic acid (HVA) were evaluated on day 12 by HPLC. Tyrosine hydroxylase
(TH)
immunoreactivity was evaluated from brain sections collected through the
substantia nigra.
Due to the large number of ninety-seven individual parameters, principal
component analysis
(PCA) was performed. The PCA combined all the parameter data together and
revealed correlations
between different parameters, providing an overall view on the fine motor and
gait characteristics. Figure
33A illustrates visualizations of the ten principal components or PCs, showing
how the original parameters
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are correlated in datasets. The more intensity (blue or red) there is for each
parameter, the more strongly
the particular parameter is implicated in the corresponding PC. The heat map
is formed from the PCA of
all the parameter data available. The PC scores are presented as an overall
gait analysis score in Figure
33B, based on differences between Group 2: MPTP + Vehicle and Group 1: Vehicle
+ Vehicle mice in all
the PC scores.
Figure 34 shows the gait property of forepaw toe clearance at study day 11.
The MPTP + vehicle-
treated group exhibited a statistically significant change in forepaw toe
clearance compared to the vehicle
+ vehicle-treated group. And the MPTP + Compound 1-treated group exhibited a
positive trend compared
to the MPTP + vehicle-treated group. Data are presented as mean + SEM (Group
1: Vehicle + Vehicle, n
= 15; Group 2: MPTP + Vehicle, n = 14; Group 3: MPTP + Compound 1 n = 13).
Statistical significances:
* p < 0.05, Group 2: MPTP + Vehicle vs. Group 1: Vehicle + Vehicle (unpaired t-
test).
Figure 35 shows the gait property of forepaw swing speed at study day 11. The
MPTP + vehicle-
treated group exhibited a statistically significant change in forepaw swing
speed compared to the vehicle +
vehicle-treated group. And the MPTP + Compound 1-treated group exhibited a
positive trend compared to
the MPTP + vehicle-treated group. Data are presented as mean + SEM (Group 1:
Vehicle + Vehicle, n =
15; Group 2: MPTP + Vehicle, n = 14; Group 3: MPTP + Compound 1 (30 mg/kg), n
= 13). Statistical
significances: * p < 0.05, Group 2: MPTP + Vehicle vs. Group 1: Vehicle +
Vehicle (unpaired t-test).
Figure 36 shows the gait property of ankle range of motion at study day 11.
The MPTP + vehicle-
treated group exhibited a statistically significant change in ankle range of
motion compared to the vehicle
+ vehicle-treated group. And the MPTP + Compound 1-treated group exhibited a
positive trend compared
to the MPTP + vehicle-treated group. Data are presented as mean + SEM (Group
1: Vehicle + Vehicle, n
= 15; Group 2: MPTP + Vehicle, n = 14; Group 3: MPTP + Compound 1 (30 mg/kg),
n = 13). Statistical
significances: * p < 0.05, Group 2: MPTP + Vehicle vs. Group 1: Vehicle +
Vehicle (unpaired t-test).
Figure 37 reports the short-term effects of MPTP and Compound 1 on T-cell
infiltration into the
brain. The total number of CD3 positive T-cells counted in the substantia
nigra from three sections of 30
m thickness for each mouse on Day 4 of the study are presented. Data shown are
mean s.e.m; ***P<
0.001; one-way ANOVA, Sidak's multiple comparison test post-hoc. Mice treated
with MPTP +
Compound 1 showed a trend towards less CD3-positive cells, indicating a trend
towards less T-cell
infiltration into the brain.
Figure 38 reports the short-term effects of MPTP and Compound 1 on
microgliosis on study Day
4. Figure 38A depicts the degree of CD68-positive area measured in the
striatum from three sections of
30 m thickness for each mouse on Day 4 of the study. Figure 38B depicts the
degree of CD68-positive
area measured in the substantia nigra pars compacta from three sections of 30
m thickness for each mouse
from Day 4 of the study. These data demonstrate that there is marked
microgliosis in the substantia nigra,
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the primary site of neuronal loss observed in Parkinson's disease, but not in
the striatum, the main brain
region where neurons from the substantia nigra project¨suggesting that
treatment affects the central site
of disease pathology but not secondary projection areas.
4. Synuclein Transgenic Mouse Model of Parkinson's Disease
A synuclein transgenic mouse model was used to determine the effects of
chronic CCR3 inhibition
by Compound 1 and its ability to slow, halt, or reverse the progression of
Parkinson' s-like symptoms. This
model over-expresses human a-synuclein can test whether an interventional
treatment can prevent a-
synuclein-induced behavioral and pathological effects. To select an optimal
synuclein model for testing,
plasma eotaxin levels were measured as a biomarker in multiple synuclein mouse
models at different ages.
The models included A53T, DxJ9M, and Line 61. Plasma from 6-month-old Line 61
synuclein transgenic
mice were measured by ELISA assay to detect plasma eotaxin levels as a
biomarker. Line 61 mice (QPS
Neuro, Grambach, Austria) at 6 months of age exhibited a transgene-induced
increase in eotaxin levels
(Figure 39), showing that plasma eotaxin levels may serve as an appropriate
clinical biomarker to select
treatment populations.
Thirty-three (33) male 4.5-month-old alpha-synuclein transgenic mice (Line 61)
were allocated into
two groups (Group A, n=17, Compound 11 mg/mL; Group B, n=16, Vehicle) and a
group of 15 non-
transgenic age-matched littermates (Group C, Vehicle) were treated via their
drinking water for 6 weeks.
Animals received either vehicle or Compound 1. The animals' behavior was
evaluated at the end of the
treatment period. Subsequently, tissue processing was performed.
Figure 40 reports the results from the wire suspension test. The mean wire
suspension times per
group are shown, with animals from Group C (non-transgenic, vehicle-treated)
exhibiting a significantly
higher wire suspension time. Animals from Group A (transgenic, Compound-1
treated) exhibited a
significantly higher wire suspension time compared to Group B (transgenic,
vehicle-treated). Data are
displayed as mean + SEM of all animals per group; ***P<0.001; Dunn's post-
test; *P<0.05 Mann Whitney
test for group A vs. group B). These data demonstrate a dramatic loss of
function in ability to hang on to
the wire in synuclein mice, which was at least partially rescued by Compound 1
treatment.
Figure 41 reports the results from the grip strength test. The mean maximum
grip force II] per
group is shown. Animals from Group A and Group C (transgenic, Compound 1-
treated and non-transgenic,
vehicle-treated, respectively) showed a significantly higher grip force
compared to Group B (transgenic,
vehicle-treated). Data are displayed as mean + SEM of all animals per group.
Groups were compared to
vehicle-treated transgenic animals (group B); one-way ANOVA followed by
Bonferroni's post-test. These
data demonstrate a statistically significant deficit in synuclein mice in
forelimb strength, that was
completely rescued by Compound 1 treatment.
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Figure 42 reports results from the beam walk test. The number of animals per
group for each trial
which were able to traverse the beam completely are shown. No animal from
Group B (transgenic, vehicle-
treated) was able to traverse the fifth beam (most difficult), indicating that
Group A transgenic mice treated
with Compound 1 performed better than Group B transgenic mice treated with
vehicle, and improved
performance on the beam walk test closer to non-transgenic controls.
Figure 43 reports the results of the five trials for beam walk in slips or
footfalls made in each trial
for three groups of mice (group A, transgenic Compound 1-treated; group B,
transgenic vehicle-treated;
and group C, non-transgenic vehicle-treated). Graphs represent the mean number
of slips [n] per group,
and each graph represents one trial (1-5). Data are mean + SEM of all animals
per group. Groups were
compared to group B; one-way ANOVA followed by Bonferroni's post-test. These
data clearly show that
synuclein overexpressing mice are highly impaired in their ability to traverse
the beams, and treatment with
Compound 1 at least partially rescues this effect, showing a dramatic
improvement in motor function.
Figure 44 reports eosinophil count from peripheral blood. Figure 44A reports
the percentage of
eosinophils in peripheral blood from three groups of mice (Tg Cmpd 1 =
transgenic Compound 1-treated;
Tg Veh = transgenic vehicle-treated; nTG Veh = non-transgenic vehicle-
treated). Data were compared by
t-test. Figure 44B reports the absolute eosinophil count in peripheral blood
from three groups of mice (Tg
Cmpd 1 = transgenic Compound 1-treated; Tg Veh = transgenic vehicle-treated;
nTG Veh = non-transgenic
vehicle-treated). Data were compared by t-test. These data demonstrate that
the Parkinson's Disease model
of synuclein overexpression results in decreased eosinophils, which are
restored to levels in non-transgenic
mice with Compound 1 treatment, suggesting beneficial immune modulation in
this model of Parkinson's
disease. This also suggests that determining eosinophil levels in Parkinson's
Disease patients treated with
Compound 1 can be a biomarker for the disease, including determining the level
of progression, stasis, or
regression of the disease, as well as treatment efficacy.
Figure 45 reports the effect of Compound 1 on neuroinflammation. Figure 45A
reports the CD68
positive area quantified in the hippocampus of: non-transgenic, vehicle-
treated mice; transgenic, vehicle
treated mice; and transgenic, Compound 1-treated mice (n=14, 12, and 15,
respectively). Figure 45B
reports the CD68 positive area quantified in the striatum of: non-transgenic,
vehicle-treated mice;
transgenic, vehicle treated mice; and transgenic, Compound 1-treated mice
(n=15, 11, and 16, respectively).
Figure 45C reports the Ibal positive area quantified in the hippocampus of:
non-transgenic, vehicle-treated
mice; transgenic, vehicle treated mice; and transgenic, Compound 1-treated
mice (n=14, 13, and 16,
respectively). Figure 45D reports the Ibal positive area quantified in the
striatum of: non-transgenic,
vehicle-treated mice; transgenic, vehicle treated mice; and transgenic,
Compound 1-treated mice (n=15, 11,
and 16, respectively). Data are mean +/- s.e.m.; *P<0.05. Figure 45E reports
the GFAP positive astrocytes
quantified in the hippocampus of: non-transgenic, vehicle-treated mice;
transgenic, vehicle treated mice;
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and transgenic, Compound 1-treated mice (n=14, 13, and 15, respectively).
Figure 45F reports the GFAP
positive area quantified in the striatum of: non-transgenic, vehicle-treated
mice; transgenic, vehicle treated
mice; and transgenic, Compound 1-treated mice (n=15, 13, and 15,
respectively). Data are mean +/- s.e.m.;
*P<0.05. Figure 45G reports the Iba-1 positive area quantified in the pars
compacta of the substantia nigra
of non-transgenic, vehicle-treated mice; transgenic, vehicle-treated mice; and
transgenic, Compound 1-
treated mice.
These data demonstrate that, while synuclein overexpression does not result in
dramatic microgliosis
evidenced by CD68 and Iba 1 , or astrocytosis evidenced by GFAP
immunoreactivity, treatment with
Compound 1 decreases microgliosis by both markers, and astrogliosis by GFAP,
potentially having a global
anti-inflammatory effect, i.e. not simply on one inflammatory cell type.
Furthermore, the striatum was
impacted more than the hippocampus, demonstrating that regions relevant for
Parkinson's disease are
specifically reduced in microgliosis and astrogliosis markers. This is further
evidenced by the effect of
Compound 1 on reversing microgliosis on the pars compacta of the substantia
nigra.
Figure 46 reports the effect of Compound 1 on circulating levels of IL-4 and
IL-6 cytokines in: non-
transgenic, vehicle-treated mice; transgenic, vehicle treated mice; and
transgenic, Compound 1-treated mice
(n=14, 15, and 17, respectively). Figure 46A reports the levels of IL-4
measured in terminal cardiac plasma
of all three groups. Figure 46B reports the levels of IL-6 measured in
terminal cardiac plasma of all three
groups. Data shown are mean +/- s.e.m; *P<0.05, **13<0.01; one-way ANOVA,
Dunnett's multiple
comparison test post-hoc. These alterations in key cytokines indicates a
general effect of Compound 1
treatment on proteins involved in immune function and inflammation.
5. T-Cell Infiltration into The Brain
(a) Tissue processing and histology
Mice were sacrificed two hours after the last PO administration on the day
following 9 days of
dosing. Anesthesia was induced by 2,2,2-tribromoethanol. Mice were
subsequently transcardially perfused
with 1% EDTA in PBS followed by 4% PFA in PBS. The brains were dissected and
cut sagitally in two
even halves and drop fixed in 4% PFA in PBS. After two days of fixation, the
hemibrains were transferred
to a 30% sucrose in PBS solution and changed after two days. Plasma was
collected and stored on dry ice.
Hemibrains were sectioned sagitally at 35tim on a microtome at -22 C. Brain
slices were collected
sequentially into 12 tubes, so that every 12th section of the brain was
represented in a given tube. Brain
sections were stored in cryoprotectant media at -20 C until needed for
staining. Free floating sections were
blocked in the appropriate serum at 10% serum in PB ST 0.5%. Primary
antibodies were incubated overnight
at 4 C or room temperature as described below.
Rat anti-CD8a (63-0081-80, Thermo Fisher Scientific) was used at a
concentration of 1:100 at room
temperature, rat anti-CD3 (555273, BD Biosciences) was used at a concentration
of 1:100 at room
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temperature, rabbit anti-Iba-1 (016-26721, Wako) was used at a concentration
of 1:1000 at 4 C, rat anti-
CD68 (MCA1957, Bio-Rad) was used at 1:1000 at 4 C , and Dylight 448/594
labeled Lycopersicon
Esculentum (Tomato) Lectin (DL-1177, Fisher Scientific) was used at a
concentration of 1:200 at room
temperature. The appropriate fluorescent secondary antibodies (Alexa-
488/555/647, Invitrogen) were
applied the next day at a concentration of 1:300 for one hour at room
temperature. Prolong Gold Mounting
Media was used to coverslip the slides. Images were acquired on the Hamamatsu
Nanozoomer 2.0HT slide-
scanner at 20x.
(b) T-cell infiltration quantification
CD3 and CD8 positive cells were counted from images acquired on the Hamamatsu
slide scanner
from the cerebellum. The total number of CD3 and CD8 positive cells in the
cerebellum was summed from
approximately 5 sections of 350m each. Ordinary one-way ANOVA was used to test
for statistical
significance, with Dunnett's multiple comparisons test post-hoc between
treatment groups.
(c) Neuroinflammation quantification
Iba-1 and CD68 positive area was quantified using thresholding on Image Pro
Premier v9.2
software as a percentage of an ROT around the entire cerebellum. The Iba-1 and
CD68 positive percent area
was averaged from approximately 5 sections for each mouse. Ordinary one-way
ANOVA was used to test
for statistical significance, with Dunnett's multiple comparisons test post-
hoc between treatment groups.
Treatment Groups
Two-month-old C57B1/6 mice were divided into three treatment groups: vehicle
controls, vehicle
treated EAE (Experimental Autoimmune Encephalomyelitis, see Methods Mol Biol.
2012; 900: 381-401,
herein incorporated by reference in its entirety), and Compound 1 treated EAE
. EAE was induced on day
0 with subcutaneous (SQ) injection of MOG+CFA emulsification and intravenous
injection (IV) of
Pertussis toxin (PT). An additional injection of PT occurred on day 2. Vehicle
or Compound 1 dosing
occurred via oral gavage (PO) on day 0 and continued twice daily (BID) till
day 9. Mice were taken down
2 hours following the last PO dose.
Drug formulation: Compound 1 was formulated in 40% HP-13-cyclodextrin and
adjusted to pH 6.5
with NaOH (1M). Vehicle solution was formulated and adjusted for pH similarly.
Solutions were prepared
fresh weekly and stored at 4 C.
EAE emulsion: MOG 35-55 (Anaspec AS-60130-5) was reconstituted in PBS at
4mg/ml. M.
Tuberculosis H37 Ra (Fisher Scientific DF3114-33-8) was dissolved in
Incomplete Freud's adjuvant at
4mg/ml. These two solutions were then emulsified using glass syringes (Thermo
Scientific Male Luer-LOK
Priming Syringes 03-170-301) and 3-way stop cock (Cadence 6001). Solutions
were prepared just before
dosing.
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Pertussis toxin: Pertussis toxin (Sigma P7208-5OUG) was dissolved at
0.002mg/m1 in saline and
injected 100u1 IV on the day of induction and again two days later.
Treatment Groups:
= Treatment Group 1, Vehicle treatment: Young C57BL/6 mice (n=5), aged 2.5
months,
received 100u1 of Vehicle PO BID for 9 days, starting 2 hours after SQ saline
injection and only 1 injection
on the first and last days for a total of 19 treatment injections.
= Treatment Group 2, Vehicle treatment with EAE: Young C57BL/6 mice (n=10),
aged 2.5
months, received 100u1 of Vehicle PO BID for 9 days, starting 2 hours after
EAE induction and only 1
injection on the first and last days for a total of 19 treatment injections.
= Treatment Group 3, Compound 1 treatment with EAE: Young C57BL/6 mice
(n=10), aged
2.5 months, received 100u1 of Compound 1 (30mg/kg) PO BID for 9 days, starting
2 hours after EAE
induction and only 1 injection on the first and last days for a total of 19
treatment injections.
EAE induction resulted in an increase in CD3 and CD8-positive infiltrating T-
cells in the
cerebellum. These T-cells numbers were significantly reduced with treatment
with Compound 1. EAE
induction also resulted in a significant increase in microgliosis in the
cerebellum, which was also
significantly rescued with Compound 1 treatment after 9 days. Figure 47A shows
an increase in CD3
positive infiltrating T-cell in the cerebellum which is significantly reduced
following treatment with
Compound 1 for 9 days. Figure 47B shows an increase in CD8 positive
infiltrating T-cell in the cerebellum
which is significantly reduced following treatment with Compound 1 for 9 days.
Figure 47C shows a
significant increase Iba-1 positive area in the cerebellum after EAE, which
was significantly reduced in the
cerebellum after 9 days of treatment. Figure 47D shows a significant increase
CD68 positive area in the
cerebellum after EAE, which was significantly reduced in the cerebellum after
9 days of treatment.
d. Examples in Humans
1. Eotaxin Levels and Aging
Levels of human eotaxin-1 were determined using a commercially-available
affinity-based assay
(SOMAscan, SomaLogic, Inc., Boulder, Colorado). Blood plasma samples were
collected from 18, 30,
45, 55, and 66-year-old donors and for testing by SomaLogic using a SOMAscan
aptamer-based affinity
assay that tested, among other things, for relative levels of human eotaxin-1.
Eotaxin-1 levels were
determined and plotted by age group (Figure 45). Eotaxin-1 relative
concentrations increased with age,
indicating that the eotaxin-1 pathway, including its primary receptor, CCR3,
is a target to treat aging-
associated disease such as neurodegenerative disease and cognitive decline.
2. Human Biomarker Assays
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Whole blood from humans treated with Compound 1 was incubated with human
recombinant
eotaxin-1 to trigger eosinophil shape change (Figure 48A) or CCR3 receptor
internalization (Figure 49B).
Both assays showed a robust concentration-dependent effect of Compound 1 on
the respective
functional biomarker readouts.
Together, the results obtained from the ESC and CCR3 receptor internalization
assays confirm
Compound 1 acts as an inhibitor of the human eotaxin-1 pathway. In particular,
Compound 1 can act as a
potent inhibitor of CCR3 by binding to that receptor.
Further, the data obtained in Figure 44 shows that in a mammalian transgenic
Parkinson's Disease
model, eosinophils were expressed at lower levels than non-transgenic animals,
which was reversed with
Compound 1 administration. This data, applied to a Parkinson's Disease patient
could therefore help in
diagnosing, monitoring, and determining the prognosis of the disease.
3. Treatment of Subjects with Parkinson's Disease Using Compound 1
Subjects diagnosed with Parkinson's Disease are given either 400 mg of
Compound or placebo
twice per day (BID) orally. Subjects are treated for 12 weeks and follow-up is
performed for a subsequent
2 weeks. Subjects are assessed of the effects of Compound 1 on motor function
in practically-defined off-
medication state which is greater than or equal to 12 hours off of levodopa.
Subjects are also assessed by
flow cytometric, pharmacogenomic, and biomarker analyses that are conducted on
blood and blood plasma
samples, including eosinophil levels. Gait analysis is assessed using the Zeno
Walkway. Bradykinesia,
tremor, general activity, and sleep are assessed using a wearable device.
Changes in baseline (day 1) motor function during the practically defined off-
medication state at
week 12 is determined using Movement Disorder Society's Unified Parkinson's
Disease Rating Scale
(MDS-UPDRS), Part 3. Change in baseline (day 1) in clinical function, motor
function, and activities of
daily living at week 12 during the on-medication state is assessed by: MDS-
UPDRS Parts 1-4; Montreal
Cognitive Assessment (MoCA); Schwab and England Activities of Daily Living (SE-
ADL) Scale; Clinical
Impression of Severity Index ¨ PD (CISI-PD); PD Quality of Life Questionnaire-
39 (PDQ-39); Sheehan-
Suicidality Tracking Scale (S-STS); and 10-meter timed walk (also assessed in
off-medication state).
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It is to be understood that this invention is not limited to particular
aspects described, as such may
vary. It is also to be understood that the terminology used herein is for the
purpose of describing
particular aspects only, and is not intended to be limiting, since the scope
of the present invention will be
limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening
value, to the tenth of
the unit of the lower limit unless the context clearly dictates otherwise,
between the upper and lower limit
of that range and any other stated or intervening value in that stated range,
is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included in the
smaller ranges and are also encompassed within the invention, subject to any
specifically excluded limit
in the stated range. Where the stated range includes one or both of the
limits, ranges excluding either or
both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as
commonly understood by one of ordinary skill in the art to which this
invention belongs. Although any
methods and materials similar or equivalent to those described herein can also
be used in the practice or
testing of the present invention, representative illustrative methods and
materials are now described.
All publications and patents cited in this specification are herein
incorporated by reference as if
each individual publication or patent were specifically and individually
indicated to be incorporated by
reference and are incorporated herein by reference to disclose and describe
the methods and/or materials
in connection with which the publications are cited. The citation of any
publication is for its disclosure
prior to the filing date and should not be construed as an admission that the
present invention is not
entitled to antedate such publication by virtue of prior invention. Further,
the dates of publication
provided may be different from the actual publication dates which may need to
be independently
confirmed.
It is noted that, as used herein and in the appended claims, the singular
forms "a", "an", and "the"
include plural referents unless the context clearly dictates otherwise. It is
further noted that the claims
may be drafted to exclude any optional element. As such, this statement is
intended to serve as antecedent
basis for use of such exclusive terminology as "solely," "only" and the like
in connection with the
recitation of claim elements, or use of a "negative" limitation.
As will be apparent to those of skill in the art upon reading this disclosure,
each of the individual
aspects described and illustrated herein has discrete components and features
which may be readily
separated from or combined with the features of any of the other several
aspects without departing from
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the scope or spirit of the present invention. Any recited method can be
carried out in the order of events
recited or in any other order which is logically possible.
Although the foregoing invention has been described in some detail by way of
illustration and
example for purposes of clarity of understanding, it is readily apparent to
those of ordinary skill in the art
in light of the teachings of this invention that certain changes and
modifications may be made thereto
without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention.
It will be appreciated
that those skilled in the art will be able to devise various arrangements
which, although not explicitly
described or shown herein, embody the principles of the invention and are
included within its spirit and
scope. Furthermore, all examples and conditional language recited herein are
principally intended to aid the
reader in understanding the principles of the invention and the concepts
contributed by the inventors to
furthering the art and are to be construed as being without limitation to such
specifically recited examples
and conditions. Moreover, all statements herein reciting principles, aspects,
and aspects of the invention as
well as specific examples thereof, are intended to encompass both structural
and functional equivalents
thereof.
Additionally, it is intended that such equivalents include both currently
known equivalents and
equivalents developed in the future, i.e., any elements developed that perform
the same function, regardless
of structure. The scope of the present invention, therefore, is not intended
to be limited to the exemplary
aspects shown and described herein. Rather, the scope and spirit of present
invention is embodied by the
appended claims.
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