In Vivo Receptor Occupancy
Overview: Measures the degree to which the test drug actually occupies its receptor in vivo. Receptor occupancy is
determined by measuring the ability of a systemic dose of the test drug (non-radioactive) to compete with binding of a
radiolabeled drug to the receptor. The test is useful to determine the dose or plasma concentration of the test drug
needed to reach a therapeutically effective occupancy level.
Protocol summary: Administer (unlabeled) test drug to the animal (p.o./i.v./s.c.). After a period of time for drug
absorption (depending on administration route and pharmacokinetics) inject a standard radiotracer for the targeted
receptor intravenously. 30 min later sacrifice animal. Collect blood and dissect out brain and/or other tissues. Weigh
and digest tissues. Count in scintillation counter.
Data analysis: Radioactivity levels are determined in both a receptor-rich region and a reference, receptor-deficient,
region and both converted to DPM/mg tissue. Receptor occupancy of the test (non-radioactive) drug is determined
from its ability to reduce the radioactivity in the receptor rich region relative to that in the reference (receptor deficient
region). Complete occupancy (100 %) would typically reduce this ratio to unity, i.e. no difference in counts between the
receptor-rich region and reference region.
Suggested testing: 5 doses of the test drug plus a vehicle control and one or more doses of a positive control.
n = 6 - 8 animals per dose.
Approximate prices*:
Typical setup fee: $ 2,500 + radioligand costs
Mice: $ 150/animal
Rat: $ 240/animal
*Please contact us for a final quote and turn-around time.
Example radiotracers for in vivo receptor occupancy measurements:
Overview: Measures the degree to which the test drug actually occupies its receptor in vivo. Receptor occupancy is
determined by measuring the ability of a systemic dose of the test drug (non-radioactive) to compete with binding of a
radiolabeled drug to the receptor. The test is useful to determine the dose or plasma concentration of the test drug
needed to reach a therapeutically effective occupancy level.
Protocol summary: Administer (unlabeled) test drug to the animal (p.o./i.v./s.c.). After a period of time for drug
absorption (depending on administration route and pharmacokinetics) inject a standard radiotracer for the targeted
receptor intravenously. 30 min later sacrifice animal. Collect blood and dissect out brain and/or other tissues. Weigh
and digest tissues. Count in scintillation counter.
Data analysis: Radioactivity levels are determined in both a receptor-rich region and a reference, receptor-deficient,
region and both converted to DPM/mg tissue. Receptor occupancy of the test (non-radioactive) drug is determined
from its ability to reduce the radioactivity in the receptor rich region relative to that in the reference (receptor deficient
region). Complete occupancy (100 %) would typically reduce this ratio to unity, i.e. no difference in counts between the
receptor-rich region and reference region.
Suggested testing: 5 doses of the test drug plus a vehicle control and one or more doses of a positive control.
n = 6 - 8 animals per dose.
Approximate prices*:
Typical setup fee: $ 2,500 + radioligand costs
Mice: $ 150/animal
Rat: $ 240/animal
*Please contact us for a final quote and turn-around time.
Example radiotracers for in vivo receptor occupancy measurements:
In vivo CB1 cannabinoid receptor occupancy by a cannabinoid
drug, as determined by inhibition of the corresponding
radioiodinated tracer. Values are ratios of radioactivity in
cerebellum (i.e. a CB1 receptor-rich region) to brain stem (a
reference region) and are the means ± SEM of 5 - 6 animals per
dose. Unlabeled drug reduced specific radiotracer binding by
50 % (~ 50 % receptor occupancy) at about 0.3 mg/kg. Dotted
line indicates the level of non-specific binding.
drug, as determined by inhibition of the corresponding
radioiodinated tracer. Values are ratios of radioactivity in
cerebellum (i.e. a CB1 receptor-rich region) to brain stem (a
reference region) and are the means ± SEM of 5 - 6 animals per
dose. Unlabeled drug reduced specific radiotracer binding by
50 % (~ 50 % receptor occupancy) at about 0.3 mg/kg. Dotted
line indicates the level of non-specific binding.
In vivo dopamine transporter occupancy of
cocaine and methylphenidate as determined by
inhibition of [3H]cocaine binding in the striatum.
Values are ratios of radioactivity in striatum
(receptor-rich region) to cerebellum (reference
region) and are the means ± SEM of 5 - 6
animals per dose. Both cocaine and
methylphenidate reduced specific [3H]cocaine
binding with 50 % inhibition (~ 50 % receptor
occupancy) at about 0.25 mg/kg for both drugs.
Dotted line indicates the level of non-specific
binding, as determined by injection of a
saturating dose of a high-affinity cocaine
analogue. This is slightly above 1.0 for
[3H]cocaine due to differences in lipid solubility
between striatum and cerebellum.
cocaine and methylphenidate as determined by
inhibition of [3H]cocaine binding in the striatum.
Values are ratios of radioactivity in striatum
(receptor-rich region) to cerebellum (reference
region) and are the means ± SEM of 5 - 6
animals per dose. Both cocaine and
methylphenidate reduced specific [3H]cocaine
binding with 50 % inhibition (~ 50 % receptor
occupancy) at about 0.25 mg/kg for both drugs.
Dotted line indicates the level of non-specific
binding, as determined by injection of a
saturating dose of a high-affinity cocaine
analogue. This is slightly above 1.0 for
[3H]cocaine due to differences in lipid solubility
between striatum and cerebellum.
| Radiotracer |
Receptor |
Receptor region |
Reference region |
Suggested positive control |
| [3H]Raclopride [3H]SCH 23390 [3H]N-methylspiperone [3H]WAY100635 [3H]WIN 35,428 [3H]Paroxetine [125I]AM251 [3H]Anandamide |
Dopamine D2 Dopamine D1 5-HT2A 5-HT1A Dopamine transporter Serotonin transporter Cannabinoid CB1 Fatty acid amide hydrolase |
Striatum Striatum Cortex Cortex Striatum Cortex Cerebellum Whole brain* |
Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Brain stem NA |
1 mg/kg i.v. haloperidol 0.3 mg/kg i.v. SCH23390 1 mg/kg i.v. ritanserin 1 mg/kg i.v. WAY100635 2 mg/kg i.v. beta-CIT 3 mg/kg i.v. paroxetine 1 mg/kg i.v. SR141716A 12 mg/kg i.v. CAY10435 |
*assay measures in vivo brain anandamide metabolism. For assay details see Glaser, Gatley and Gifford J. Pharmacol. Exp. Ther. 316
(2006) 1088-97
Custom Services: Receptor-radiotracer combinations listed above were selected on the basis of having been
determined in the laboratories of the co-founders as giving receptor-specific binding signal sufficient to enable in vivo
occupancy measurements by competing drugs in mice. We will be happy to evaluate other radiotracer-receptor
combinations upon request. In the event no specific binding signal is obtained, clients will be charged for the setup
and material costs only and unused drugs returned.
Ex vivo receptor occupancy determinations: For drugs with high affinity to their targeted receptor, an estimate of
receptor occupancy can instead be made in some cases using an ex vivo approach. In this technique the unlabeled
test drug is administered to the animal. However, receptor occupancy is subsequently measured in vitro immediately
following sacrifice of the animal using either a homogenate radioligand binding assay or with in vitro autoradiography.
A short non-equilibrium incubation time in the radiotracer is used to avoid appreciable dissociation of the unlabeled
test drug before the termination of the assay.
Example data:
(2006) 1088-97
Custom Services: Receptor-radiotracer combinations listed above were selected on the basis of having been
determined in the laboratories of the co-founders as giving receptor-specific binding signal sufficient to enable in vivo
occupancy measurements by competing drugs in mice. We will be happy to evaluate other radiotracer-receptor
combinations upon request. In the event no specific binding signal is obtained, clients will be charged for the setup
and material costs only and unused drugs returned.
Ex vivo receptor occupancy determinations: For drugs with high affinity to their targeted receptor, an estimate of
receptor occupancy can instead be made in some cases using an ex vivo approach. In this technique the unlabeled
test drug is administered to the animal. However, receptor occupancy is subsequently measured in vitro immediately
following sacrifice of the animal using either a homogenate radioligand binding assay or with in vitro autoradiography.
A short non-equilibrium incubation time in the radiotracer is used to avoid appreciable dissociation of the unlabeled
test drug before the termination of the assay.
Example data:
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Preclinical contract research services
InvivoPharm: Receptor occupancy CRO service
© 2007, 2008 Invivopharm Inc.