Objectives

The  main objective is to evaluate the feasibility of the new radiotracers developed in Workpackage 01 as tools to image P-gp with PET and SPET. In addition, it has to be shown that they are superior to the present gold standard, verapamil. The new radiotracers should be able to reflect the in vivo distribution and activity of P-gp and this will have to be related to actual brain uptake and distribution of CNS active drugs. The ultimate test is to see whether the radiotracers are able to discriminate between drug-resistant and drug-responsive subjects. All these aspects will be investigated in naive rats, in rat models of chronic epilepsy and in subgroups of drug-resistant and drug-responsive rats.

 

Workpackage description

Background
The development of radiotracers of high-affinity inhibitors of P-gp aims to provide biomarkers for the expression and distribution of the transporter. The intensity of the PET or SPET signal should increase under conditions where the expression of P-gp is increased. At the same time, the uptake of substrates of the transporter should be decreased. As the radiotracer cannot reflect the transport process itself, this has to be confirmed independently by using a suitable P-gp substrate. Furthermore, evaluation of the new radiotracers requires comparison to existing biomarkers, such as (R)-[11C]-verapamil.
Mesial temporal lobe epilepsy in humans is often associated with pharmacoresistance and P-gp upregulation. This condition, including increased P-gp expression can be mimicked in rats by induction of status epilepticus. This animal model is therefore useful to evaluate the new radiotracers.

Task 1:
P-gp expression and activity in naive rats evaluated with quinidine (P02-VUmc, P10-LACDR) and with (R)-[11C]-verapamil
Passage of quinidine across the blood-brain barrier is very sensitive to P-gp functionality and quinidine is therefore a useful model compound. The pharmacokinetics (PK) will be characterized after i.v. administration. Arterial sampling and  intracerebral microdialysis will be applied to determine the concentration time profile in plasma and extracellular brain fluid. At termination of the experiment whole brain concentrations will be determined as well. From these data the optimal dosing schedule for later experiments will be designed.  (R)-[11C]-verapamil binding will be determined with PET under the same conditions to assess P-gp expression. This will allow establishment of the relation between quinidine PK  and P-gp expression in healthy animals.

Task 2:
Modulation of (R)-[11C]-verapamil uptake and quinidine uptake  in naive rats by P-gp inhibitors
In naive rats P-gp will be inhibited by tariquidar, an effective blocker of the transporter. This should increase the intensity of the (R)-[11C]-verapamil signal and simultaneously increase the brain concentration of quinidine. Decreased efflux from the brain will be reflected by the dialysate concentrations and by alterations in PK parameters. This is an important step to validate the use of quinidine PK as an independent method to quantify the expression and activity of P-gp.

Task 3:
Characterization of newly developed radiotracers in naive rats
Binding of [11C]laniquidar and other radiotracers will be determined with PET or SPET in the presence or absence of tariquidar  and this will be compared to (R)-[11C]-verapamil binding and the brain PK of quinidine.

Task 4:
Biological evaluation of radiotracers in animal models for chronic epilepsy
Induction of status epilepticus (SE) initially induces prolonged seizure activity of several hours after which seizures subside. Following a silent period of 1-2 weeks spontaneous seizures start to develop which persist for months. Expression of P-gp is permanently increased after SE, with a peak approximately after one week. At this time point the experiments described in task 3 will be repeated The effects of P-gp inhibition should then be most conspicuous and reflect the maximal capacity of the transporter.

Task 5:
Biological evaluation of radiotracers in animal models of drug resistance
Groups of epileptic rats exhibiting pharmacoresistance or pharmacosensitivity for antiepileptic drugs will be identified in Workpackage  04. These rats are likely to show differences in transporter expression. The new radiotracers tested in tasks 3 and 4 will be investigated in these animals. For experiments taking place at VUmc animals will be made available by LMU.