W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1045
Test compounds were assayed in six-point dose response exper-
iments. The highest compound concentration tested was 100 M,
4.14. Plasma concentration and blood brain barrier penetration
of 26
l
which was then diluted by approximately one-third with each sub-
sequent dose. After 24 h incubation with the compounds, MG132
was added at a final concentration of 100 nM, a dose that produces
an approximately 70% loss of viability. Cell viability was measured
48 h later using the fluorescent viability probe, Calcein-AM (Molec-
ular Probes, Invitrogen, Carlesbad, CA). Briefly, cells were washed
twice with PBS, Calcein-AM was added at a final concentration of
Compound 26 was administered in mice as a single bolus intra-
peritoneal injection of 50 mg/kg. A group of 18 mice were used and
blood and brain tissue samples were immediately quenched in li-
quid nitrogen and stored at ꢀ80 °C for analysis. Samples were col-
lected at 0 h (no drug), 1 h, 3 h, 6 h, 12 h, and 24 h.
1 lM for 20 min at room temperature, and fluorescence intensity
4.15. Effect of 26 in ALS mouse model
was then read in a POLARstar fluorescence plate reader (BMG).
Compounds that restored viability at any dose to a level equal or
higher than 5 standard deviations from MG132 controls were con-
sidered active.
A dose–response curve was implemented with 5 cohorts of
mice (littermate wild type untreated mice, untreated mutant
SOD 1 G93A mice, and 3 treated cohorts of mutant SOD 1 G93A
mice at 1 mg/kg, 10 mg/kg, and 20 mg/kg). Mice were weighed
and administered drug daily via intraperitoneal injection at the
same time of day. The mice were monitored twice daily for any ad-
verse events and were euthanized at end stage disease using the
righting mechanism as a surrogate measure of death.
4.11. Radioligand binding assays
These studies were carried out by MDS Pharma Services.
4.12. Effect of compound 26 on hERG potassium channels
Acknowledgments
FASTPatch hERG screen assay was carried out by ChanTest Cor-
poration (Cleveland, OH). Cloned hERG potassium channels were
expressed in human embryonic kidney cells (HEK293).
We thank the National Institutes of Health [1R43NS057849],
the ALS Association (TREAT program), the Department of Defense
[AL093052], and the Veterans Administration Pittsburgh Health-
care System, 7180 Highland Drive, Pittsburgh, PA for their gener-
ous support of the research project. The authors are grateful to
Biogen Idec, Inc. for carrying out the in vivo rat PK profiling tests,
in vitro P450 reversible inhibition study, the plasma binding
affinity assay, the hERG inhibition assay, and for funding to MDS
Pharma Service (now Ricerca Biosciences, LLC), who carried out
the LeadProfilingScreen of 26.
4.13. General procedures for in vivo experiments
Male transgenic ALS mice of the G93A H1 high-expressor strain
(Jackson Laboratories, Bar Harbor, ME, USA) were bred with fe-
males having a similar background (B6/SJLF1). Offspring were gen-
otyped using a PCR assay on tail DNA. To ensure homogeneity of
the cohorts tested, we developed a standardized method by which
to select mice. Mice were randomized from 24 litters all within
4 days of age from the same ‘f’ generation removed from the found-
ing mice in our colony. Bodyweights were taken at 20 days, and
mice were equally distributed according to weight within each
experimental cohort. Mice under 8 g at 20 days were excluded
from the experiments. Only male mice were used in the treatment
studies because we have observed gender differences in survival in
the G93A transgenic ALS mouse model.9 These experiments were
carried out in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals, and were
approved by both the Veterans Administration and the University
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Animal Care Committees. Based upon previous studies,10,11
a
dose–response study was performed. Groups (n = 10) of wild-type
(WT), untreated G93A, and G93A mice received 1, 10, and 20 mg/kg
of 26 injected intraperitoneally once a day in the morning. Mice
were observed twice daily, morning and afternoon, for any adverse
events. Compound 26 was dissolved in phosphate-buffered saline
(PBS) with 0.1% DMSO, and fresh solutions were prepared daily.
Treatment was started at 42 days, as our studies demonstrate that
clinical disease onset begins on or about this age. The ALS mice
were euthanized when they were unable to right themselves after
being placed on their back for 30 s. Some deaths occurred over-
night and were recorded the following morning. Two independent
observers who were blinded to the treatment assignment agreed
when animals should be euthanized. Survival data were analyzed
by means of Kaplan–Meier survival curves.
11. Ryu, H.; Ferrante, R. J. MRMC 2007, 7, 141.
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