General discussion I

Kwan: I want to describe some of the studies we have done in Glasgow. The hypothesis is that refractory epilepsy is associated with a localized overexpression of P glycoprotein (Pgp), which restricts the access of drugs to their intended site of action. To test this hypothesis we carried out two parallel sets of experiments. The first set involved gene expression studies. We measured the expression of Mdrla and Mdrlb by quantitative RT-PCR, using an internal standard to allow absolute quantification. We looked at the distribution in different regions of normal rat brain and also a model of seizures. In the second set of studies we tried to screen whether the commonly used anti-epileptic drugs (AEDs) could be substrates for Pgp. Seven drugs were given to Mdrla knockout mice and wild-type mice. We compared the brain:plasma concentration ratios in these two different genotypes. Apart from the olfactory bulbs, which seem to express a relatively low level of the Mdrla RNA, the levels seem to be fairly uniform in the seven brain regions we looked at in normal rats. For Mdrlb, the situation is different: we only see an appreciable level of expression in the hippocampus. We tested the effects of seizures on the level of expression using the genetically epilepsy-prone (GEPR) rats. This is one of the animal models of epileptic seizure. These rats have an increased vulnerability for developing seizures, which can be induced by sound. These seizures appear to be initiated in the inferior colliculus ofthe midbrain, and then spread to the cortex to produce the phenotype of convulsion. After a single audiogenic stimulation, animals were sacrificed at four different time points and we measured the level of Mdrla mRNA in different brain regions. We found an increase in the midbrain and cortex following a single seizure. The increase can be seen at 4h and reaches a maximum at 24 h. After 7 days it has begun to decline but is still elevated.

In the knockout experiments, a single dose of each of seven drugs was given subcutaneously to Mdrla knockout mice and wild-type mice. These were sacrificed 30, 60 and 240 min later, and we measured the brain:plasma concentration ratio. There was no difference in plasma levels in the knockout and wild-type mice. However, with some drugs we did see a difference in the brain:plasma ratio. For carbamazepine the brain:plasma concentration reached a higher level 30min after injection in knockout mice than in the wild-type mice. We looked at four new drugs: vigabatrin, gabapentin, lamotrigine and topiramate. There was a significant increase in the brain:plasma concentration ratio in the knockout mice. For topiramate the brain:plasma ratio was significantly higher at 30, 60 and 240 min compared with the wild-type mice.

What does this all mean in humans? Humans only have one isoform of MDR1. Does this translate to a higher level of Pgp on a protein level? Clearly, we need more time points and pick out a range of doses to work out the temporal changes in kinetics. We need to find out whether the seizure changes we see will apply to other seizure models also.

Ling: You have used the Mdrla knockout. My suspicion is that Mdrlb will not play such an important role. There is also a triple knockout, which the Dutch group has. It will be interesting to see what role MRP1 plays. There might be a tremendous difference in terms of the ratios.

Sander: Did I understand correctly that the levels of topiramate were much higher in the knockout than the wild-type mice?

Sills: Yes. In the knockout mice there was a higher brain concentration of topiramate than in normal mice. However, the plasma topiramate levels were essentially identical. We opted to express the results as brain:plasma concentration ratios as we were concerned that any slight variation in plasma levels might be observed as variability in brain concentrations.

Kwan: We are limited by the number of animals that we can use.

Pirmohamed: How did you quantitate the concentrations in brain and plasma?

Sills: The standard drugs were analysed by an enzyme-mediated immunoassay technique that we use for plasma concentrations in patients. The newer drugs were assayed by HPLC and, in the case of topiramate, by fluorescence polarization immunoassay.

Pirmohamed: Did you have to use a different internal standard for every drug?

Sills: Where appropriate, yes.

Vezzani: Dr Löscher has shown us data about phenytoin being a substrate for Pgp. I wonder whether it would be wise to measure the drug concentration by microdialysis and not only the tissue levels. I know this is difficult in mice. Why does phenytoin change very little in these mice if it is a substrate?

Kwan: The phenytoin data are mostly in vitro. Professor Schinkel, whose laboratory generated these knockout mice, has also looked at phenytoin in the Mdrla knockout mouse, and he couldn't find a difference in vivo (Schinkel et al 1996). He suspected that this might be something to do with metabolism.

Sills: The question is really about whether we might see different effects with microdialysis as opposed to measuring tissue levels. I don't think there's much doubt about this. Ours is a simple study, employing crude techniques, designed to provide rapid answers to some basic questions. The results are, accordingly, preliminary but offer a platform for future experiments. I don't deny that the use of microdialysis might have given us clearer data. I also suspect that if we had extended the time profile of our experiments, we might have seen a greater difference in the phenytoin results. By using gross tissue measurements we are required to use sufficient doses to be able to detect the drug. With microdialysis it is possible to measure much lower concentrations. In our study, there is a possibility that, even if the drugs are substrates, we may be swamping the transporters with the amount of drug that we are using.

Bates: I am not terribly worried about the discrepancy. You do have a small difference, and you were working with a small number of animals. There are also species differences to take into account. We have already agreed that phenytoin is probably a poor Pgp substrate. The fact that it may be a Pgp substrate is verified by Wolfgang Loscher's data. If you expanded your study to 10 animals you might find that the small difference you see becomes statistically significant. The fact that you didn't get a difference in plasma also doesn't bother me, if you think about data with the knockout mice, where the orthologue for MDR1 has been deleted. For vinblastine, there is at most a twofold difference in the plasma and a 22-fold difference in the CNS, relative to the wild-type. Vinblastine is an excellent Pgp substrate.

Vezzani: I would like to describe our data showing that seizures induce Pgp protein when they are induced in otherwise normal animals. We don't use genetically modified mice but rather naive mice that receive a convulsant drug. We injected kainic acid into mice that have been prepared with chronically implanted electrodes. Seizures were recorded by EEG analysis and were often associated with behavioural convulsions. These seizures were recorded for about 90min. We killed the animals and measured Mdrla by quantitative RT-PCR. There was a significant increase in Mdrla transcript as soon as 3 h after the induction of seizure activity. This was a maximal effect: the transcript was up-regulated at about the same level for 24 h and then dropped to control levels after 72 h. Thus seizure activity per se can increase the expression of the protein in critical areas. This work was done of the hippocampus, which is an area involved in limbic seizures. This is a model of transient seizure activity; we are now studying whether the protein level is elevated in models of spontaneously recurring seizures. We have also looked at the expression of MRP1 in the same tissues. We haven't seen any induction in the transcript of this protein in these mice. We also addressed the possibility that prolonged anticonvulsant drug treatment could change the protein expression in brain tissue. This work was done in naive mice. We injected phenytoin every 12 h for four consecutive days at a dose of 30mg/kg. Then we sacrificed the animals at different time points after the last administration. At 6 h after the last phenytoin administration, the MDR1 transcript was not significantly changed by this repetitive treatment. From these preliminary results it seems that there is no evidence of up-regulation of the pump by the drug. In the animals that overexpress the Pgp protein after acute seizures, we are studying whether there is a difference compared with controls in brain concentrations of anticonvulsant drugs after their systemic administration.

Abbott: Which AEDs did you try?

Vezzani: Only phenytoin. We have done some experiments with carbamazepine, but I don't have the results yet.

Wood: What is the appropriate control for seizures? If you are looking at expression of something in brain tissue after seizure, the assumption is that there is a specific effect of the seizure. But it seems that any kind of trauma might do something: you need some control for the seizure experiment.

Meldrum: This is where our results with the GEPR rats are particularly valuable, because we get the same results as Dr Vezzani does following kainate, and we see the same time-course of increase. With kainate, there is histological damage, but for the GEPR rats we use just a modest sound stimulus that is repeatable and does not cause pathology. It seems reasonable to say that it is a consequence of the local seizure activity and essentially nothing else. I don't think you need some other control.

Kwan: For the GEPR model there was no change in other brain areas that are not involved in the seizure pathway.

Meldrum: We are convinced that it is the local seizure activity, not any systemic effects.

Schmutz: You mentioned that you measured at 3, 6, 24 and 72 h after seizure induction in your kainate model. In between these time points was there ongoing seizure activity?

Vezzani: No. In this kind of model we use transient seizure activity that lasts for 90 min. Then the activity disappears and there are no more seizures.

Ling: The interesting observation here is the fact that MDR1 can be induced under some kind of 'trauma' situation. This is significant. However, what happens in the mouse or rat may not be happening in humans. What we have found, at least in tissue culture, is that in some cell lines using drugs as an insult could induce MRP, other cell lines may not. It could be tissue, cell line or even species specific.

Vezzani: The best thing would be to try different experimental models of seizures. This is just a starting point — a proof of principle.

Sills: You showed that phenytoin doesn't increase MDR1 expression. It would appear from our discussions that phenytoin is a weak Pgp substrate. Do any of the oncologists have evidence which suggests that stronger substrates can induce expression?

Newman: It has been seen in the clinic (Abolhoda et al 1999).

Bates: There is a lot of circumstantial evidence. You see in the clinic that people who have been treated have higher levels. There are the promoter models where we give drugs and we see increased promoter activity for Pgp. Then there is the differentiation model that shows there is inducible Pgp. But it is really hard in a cancer population to sort out induction versus selection. The data are not that strong, but people who do promoter work would say that the drugs can induce Pgp. There is a nice clinical paper showing induction in sarcoma (Abolhoda et al 1999). Patients with lung metastases from sarcoma went to surgery, had doxorubicin administered by isolated perfusion, and had biopsies performed before and after the perfusion. MDR1, as measured by RT-PCR, was induced over the course of the 50 min perfusion. This is a good induction model.

Sills: Is the level of induction related to the affinity for the transporter?

Brinkmann: I think the level of induction is more defined by the affinity for the transcription factors.

Ling: The way I read it is that it is possible that these drug transporters can be induced. The challenge now is to determine whether or not this happens in a clinical situation.

Pirmohamed: Is all induction related to transcriptional activation? Is there any post-translational modification going on, such as phosphorylation, which makes the protein more stable and perhaps more active?

Vezzani: We only measured the transcript in our experiments.

Pirmohamed: In oncology studies, have people looked not only at the increase in mRNA but also protein stabilization?

Newman: Serum concentration has been shown to influence Pgp stability in cell culture models.

Varadi: We made mutants knocking out all the phosphorylation sites. We found that there was no difference in ATPase activity stimulated by drugs.

Wood: The fact that rifampin does it would suggest that it is. Rifampin clearly induces Pgp in humans.

Bates: What are the rifampin data?

Wood: There are data with fexofenadine showing an alteration in the kinetics of fexofenadine that is not metabolized. It is a Pgp substrate. It would appear that this is due to the rifampin inducing Pgp.

Pirmohamed: St John's wort also induces Pgp.

Ling: Would it be useful to explore where the experimental models are going in this whole area? In particular, I would like to ask Wolfgang Löscher about whether it will be easy to home in on the relevant molecules or genes using a screening approach like the chip arrays that Peter Atadja described earlier. Or will it be better to characterize responders and non-responders at a physiologoical level, such as by using PET (positron emission tomography) imaging?

Löscher: We have to posit hypotheses which we can test directly. Related to the main topic of this meeting, we have looked for different multidrug transporters at the gene level in different regions of the hippocampus, for example. I am a little frustrated. If it really is the case that overexpression of these transporters is transiently induced by a seizure and then returns to normal, it would have little relevance for pharmacoresistance. In a pharmacoresistant brain the drug is there. If there is no seizure, there cannot be any induction of transporter. If there is a seizure, there must be another reason for it because the drug is already there. Perhaps overexpression of drug transporters is only an epiphenomenon. We are more interested in more permanent alterations. We kindle animals and wait for a week or two before we look at the mRNA levels for different drug transporters. Only then, if there is a stable increase, can we explain something. These transient effects cannot explain pharmacoresistance.

Vezzani: The change we showed is only transient because the model itself is transient. But in spontaneously epileptic rats, perhaps the level of the protein would remain elevated, even if the seizures are subclinical. As long as there is some epileptic activity in the crucial areas, this may be sufficient to increase the pump expression to a level that has functional relevance.

Ruetz: There is an interesting observation in tissue culture where cells are selected under drug pressure. This can result in cells with differing levels of resistance. If the drug pressure is removed, the Pgp levels decrease, but they settle at a slightly higher level than before selection began. If you keep this culture, the levels are permanently elevated. This translates into a slight increase in resistance to anticancer drugs in classical assays. There is the possibility that a cell reaches a higher level of Pgp expression.

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