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Metis Labs Inc.
Study number XX
Study Title
Example Final Report
Study Lead
lead scientist
Contributing Scientists
contributing staff members
Testing Facility
Metis Laboratories Inc.
20 W. Lincoln Ave
Valley Stream, NY 11580
Sponsor
sponsor
Metis Study Number
study number
Sponsor Reference Number
NA
Report Issued
issue date
CONFIDENTIAL
Page 1
Metis Labs Inc.
Study number XX
MATERIALS AND METHODS
Membranes:
membrane source
Radiolabeled Compound:
[3H] radioligand (commercial or prepared in-house)
Reference Compound:
reference compound
Binding Buffer:
50 mM Tris, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4
Wash Buffer:
50 mM Tris, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4
Compound preparation:
DMSO, 10 mM
Assay protocol
Membrane Preparation
Frozen tissue maintained at -800C was partially thawed and homogenized
in 10 volumes of cold lysis buffer (50 mM Tris, protease inhibitor cocktail)
using a IKA homogenizer. The homogenate was centrifuged at 1,000 x g
for 10 minutes at 40C to obtain supernatant. The supernatant was then
centrifuged at 40,000 x g for 20 min at 40C to pellet the membranes, the
supernatant replaced with fresh buffer and the pellet resuspended. Two
additional rounds of centrifugation and resuspension were performed to
ensure wash out of interfering endogenous ligands. The final pellet was
resuspended in buffer containing 10 % sucrose and stored at – 80 OC. A
sample of the homogenate is analyzed for protein content using the
Pierce® BCA assay
Filtration Binding:
Filtration binding assay was carried out in 96-well plates in a final volume
of 200 µL per well. To each well was added 120 µL of membranes, 40
µL of the test drug solution in binding buffer and 40 µL of radioligand
solution in binding buffer. The plate was incubated at 30 C for 90
minutes. The incubation was stopped by vacuum filtration onto 0.5 % PEI
presoaked 96-well GF/C filtermats using a 96-well Filtermate harvester
followed by four washes with ice-cold wash buffer. Filters were then dried
for 30 minutes at 50 OC. The filter was sealed in a polyethylene bag,
scintillation cocktail (Beta Scint) added, and the radioactivity counted in a
Wallac® TriLux 1450 MicroBeta counter.
Data Analysis:
IC50 values were determined using the non-linear curve fitting routines in
Prism® (Graphpad Software Inc.). Ki values were calculated from IC50
values using the formula Ki = IC50 / (1 + ([S]/Kd)) where [S] is the
radiotracer concentration used in the assay and Kd is the dissociation
constant of the radiotracer. A two-site model was used for several of the
compounds as indicated in the figure legends. A literature Kd of 15 nM
was used for Ki calculations.
CONFIDENTIAL
Page 2
Metis Labs Inc.
Study number XX
TABLES AND FIGURES
Figure. 1. Plate counts.
Figure 2. Competition binding curves
a) Compound A
1400
Compound A
Specific Binding
(CPM)
1200
1000
800
600
400
200
0
-10
-9
-8
-7
-6
-5
Log [drug] (M)
Log IC50 (M): -7.03
CONFIDENTIAL
Page 3
Metis Labs Inc.
Study number XX
b) Compound B
Specific Binding
(CPM)
1200
Compound B
1000
800
600
400
200
0
-10
-9
-8
-7
-6
-5
Log [drug] (M)
Log IC50 (M): -7.20
c) Compound C
1400
Compound C
Specific Binding
(CPM)
1200
1000
800
600
400
200
0
-10
-9
-8
-7
-6
-5
Log [drug] (M)
Log IC50 (M): Not determined
CONFIDENTIAL
Page 4
Metis Labs Inc.
Study number XX
d) Reference compound
Specific Binding
(CPM)
1200
Reference compound
1000
800
600
400
200
0
-10
-9
-8
-7
-6
-5
Log [drug] (M)
Log IC50 (M): -8.05
Figure 3. Data summary
Compound
Compound A
Compound B
Compound C
Reference
CONFIDENTIAL
Log IC50
-7.0
-7.2
ND
-8.1
IC50 (nM)
93
63
Ki (nM)
71
48
9
7
Page 5
Metis Labs Inc.
Study number XX
Signature page
Lead Scientist(s)
Title
Date
* END OF REPORT *
CONFIDENTIAL
Page 6