Elnaz Gozalpour: Hello and welcome to this Pharmaron podcast and this episode of our DMPK Insight Podcast series. My name is Elnaz Gozalpour. I’m a member of DMPK Consultancy team which is part of our UK based Integrated Drug Discovery Platform. Today I have the honor of talking to Dr. Elena Puris and Dr. Mikko Gynther. Hello both. Thank you for joining us today for the podcast.
Elena Puris: Hi Elnaz, Nice to be here.
Mikko Gynther: Hello. Thank you for inviting us for this podcast.
Elnaz Gozalpour: Thanks. By the way of introduction Elena is an Associate professor in Neuropharmacology and Senior Researcher at the University of Eastern Finland. During her PhD at the University of Eastern Finland and postdoctoral research at Heidelberg University, Elena developed a strong interest in blood brain barrier transporters and their roles in drug discovery and disease pathogenesis. She has a multidisciplinary background in drug development and over 15 years of experience in academia and the pharmaceutical industry. Elena’s main research focus is membrane transporters and their impact on drug delivery and biochemical process in health and pathological conditions.
We have Mikko Gynther, Associate professor in Biopharmacy and a Senior Researcher at the School of Pharmacy, University of Eastern Finland. Mikko obtained his PhD in pharmacy at the University of Eastern Finland on the topic of utilizing glucose and amino acid transporters for the CNS delivery of the prodrugs. Mikko has extensive experience of 20 years in transporters mediated drug delivery and pharmacokinetics research. Mikko’s current research is focused on transporter mediated drug delivery into the brain and cancer cells and utilizing proteomics to investigate disease induced changes in drug transporters and enzymes.
Elena, Mikko, before we talk about some of your recent work, I wonder if you could share a bit with us how this journey of connecting drug transporters with drug delivery into the brain began for you. What inspired you?
Mikko Gynther: I first became interested in this topic during my Master’s thesis which was around 2004. So back then I was reading a lot of publications which criticized the approach of optimizing drug delivery to the brain by increasing the lipophilicity of the drugs. And back then it was already shown that this increase in lipophilicity actually increases the total brain concentration of the drug by increasing non-specific binding to the brain tissue and this causes that the free drug concentrations in the brain are actually not increased at all.
So how this connects to transporters is that transporters can facilitate blood brain barrier permeation of hydrophilic compounds also so utilizing them for BBB permeation would allow us not to increase the lipophilicity of the drug so we would avoid this increased non-specific binding to the brain.
Then in my PhD thesis, I investigated the possibility to utilize glucose and amino acid transporters for increased brain delivery of drugs by utilizing prodrugs. This was very interesting topic as it allowed me to investigate all the processes that are involved in this brain delivery. Not just how these transporters facilitate the blood brain barrier permeation, but how they also facilitate the intra brain distribution of this unbound drug and where this parent drug is released in the brain and so forth. So this was very interesting topic for me.
Elnaz Gozalpour: Yeah. Thank you. Elena, can you please share your story with us?
Elena Puris: I’ve always been interested in drug pharmacokinetics and how to quantitatively estimate drug delivery into the brain. And of course, transporters are the key players in drug distribution. And by the time I started my PhD project in 2016, there had been more knowledge generated about the blood brain barrier transporters, their expression in the brain, and also substrate specificity. And by that time, the myriads of prodrugs targeting transporters for drug delivery to the brain have been developed.
In addition, I was very much inspired by significant advancement in the neuropharmacokinetics field made by Professor Margareta Hammarlund-Udenaes and Professor Elizabeth de Lange and also Rena Lorian, whose work became the basis of my PhD work. Thus, in my PhD thesis, I tried to apply pharmacokinetic principles which I learned from their work, to establish a workflow for developing transporter mediated products for drug delivery to the brain.
Elnaz Gozalpour: Okay, thank you. Interesting. So recently yourself, Elena and Mikko, both of you, and your colleague Gert Fricker published an article entitled “Targeting Transporters for Drug Delivery in the Brain, Can We Do Better?” Which was part of the publication in 2022 in a special edition of Pharmaceutical Research, recognizing the work of Professor Margareta Hammarlund-Udenaes. Sorry for any wrong pronunciation. This is a comprehensive summary of literature and perhaps you could share some of your reflections on the key themes of this publication. How you brought all these information together and if you kind of categorized them in a key theme, how you would do that.
Elena Puris: Yes, we were honored to be invited to contribute an article to this special issue dedicated to Margareta, who of course played a significant role in development of me as a scientist, but also, I’m sure, other scientists as well. And for a long time, me and Mikko had the idea of writing a review article about transport immediate drug delivery to the brain and this review article, what you mentioned was aimed to not only summarize the reports of drugs, prodrugs and nanocarriers developed to utilize transporters for drug delivery to the brain, but to actually critically evaluate the studies and maybe shed light on current knowledge about the approach and also to find the research gaps for the future studies. And as it said, for this much wisdom, there is much sorrow.
While collecting the information for this article, we realized that although many studies were performed, there are little useful information for understanding this approach. And therefore, in the end, the idea was to provide a systematic workflow for developing transportation utilizing drug delivery systems to the brain to help scientists to perform their experiments while developing these drug delivery systems targeting transporters at the blood brain barrier.
Elnaz Gozalpour: Good. Thank you. Mikko, what your thoughts are about this?
Mikko Gynther: Yes. So our aim was to understand why this, although this approach seems very promising, this targeting transporters for brain delivery, why there aren’t any successful applications in clinical use yet. We know that there are more than 100 transporter targeted drug delivery systems published to date. But still there is a lack of success really here to understand why is it so, we first laid out some basic parameters that we think that are crucial to be investigated in order to tell that can we reach enhanced brain delivery by this transporter targeting drug delivery systems. And then we looked into literature to see how many of these articles or reports about these drug delivery systems actually have studied these basic parameters, which in our opinion should be studied. And we found that the available information was very scattered.
For some drug delivery systems, some parameters were investigated, and for another drug delivery system, other parameters, but very little of comprehensive studies were made. And for example, I can tell that very important feature that should be investigated is the capability of these drug delivery systems to bind and interact with the targeted transporter. But this was very poorly investigated in many of the studies. Another key thing missing was the thorough investigation of brain pharmacokinetics. So there were these key parameters which lacked evidence in many of these studies that we found.
Elnaz Gozalpour: Yeah, thank you. I think you touched the basis of my next question as well. As you mentioned, in this comprehensive review that you’ve gathered together, there are stories of kind of unsuccessful application of transporters in drug discovery as well. Would you be able to mention or list the reason behind that? Why? Is it because of lack of knowledge? Are there any other reasons? I know, Mikko, you touched some of the reasons for that, why it hasn’t been successful yet.
Elena Puris: The main idea of transporter mediated drug delivery is to design a drug, prodrug or nanocarrier that the targeted transporter at the blood brain barrier will recognize to deliver the drug across the blood brain barrier by using this transporter. And thus the information about the utilization of this transporter for cellular uptake to the brain endothelial cells, but also importantly, efflux from the brain endothelial cells is very important. And when studying this information about the reported prodrugs which were claimed to utilize the transporters, the transport mechanism hasn’t been investigated for more than a third part of compounds. And among those for half of the compounds, the binding to the transporter, either using competitive cellular uptake assays or in situ brain perfusion in rodents with the corresponding transporter substrates were reported.
And as we know, the binding to the transporter doesn’t guarantee the utilization of the transporter for cellular passage. Therefore, ideally, to show that the compound process the cell membrane by transporter, the uptake of the compound in the presence and absence of the selective transporter inhibitor should be evaluated. And among reviewed cases, this information was provided for limited number of compounds. So here it comes that it’s not only important to attach your compound to some molecule which is known to be a substrate. It’s not enough. You need to really demonstrate that your final molecule, your prodrug or nanocarrier, will utilize this transporter for cellular uptake and preferably at the blood brain barrier.
Elnaz Gozalpour: Yeah, thank you very much. Mikko, do you have anything to add?
Mikko Gynther: Yeah, I can maybe add a bit on the perspective of nanoparticles and liposomes. So there the situation was even worse when you look at this, how it was investigated, whether they actually utilized this targeted transporter or not. So majority of the studies lacked statistical significance when they compared, let’s say a nanocarrier endocytosis into cells in the presence and without transporter inhibitor, or that this difference between these different treatments was very low. This can be either due to that this competing substrate or inhibitor doesn’t have high enough affinity to compete with this binding of the nanocarrier to the transporter, or simply that the nanocarriers actually enter the cells by a different mechanism than binding to this targeted transporter and thus triggering this endocytosis process.
Elnaz Gozalpour: Yeah, good, thank you very much.
Elena Puris: I would like to add to what Mikko said, that another important aspect of the transporter mediated prodrug and nanocarrier drug delivery strategy is knowledge about the mechanism of the release of parent drug and also site of the release of parent drug within the brain. And this information was practically missing for the majority of studies what we reviewed in our article. And I think that the main reason for that is the main focus of the researchers still has to like to investigate the mechanism of transport. Although another important step, release of parent drug, is still missing and this mechanism is really important. So in fact transport immediate drug delivery is not only delivering of prodrug on nanocarrier, but also release in correct place. Therefore there are many techniques available such as pharmacokinetic studies combined with cerebral microdialysis. But these are very laborious techniques.
And maybe this fact that these are laborious and time consuming techniques is the reason why this information wasn’t reported for the compounds which were reviewing.
Elnaz Gozalpour: Yeah, I think you’ve provided very comprehensive list of subjects for next generation research. Thank you very much for that. I think most of our audience are familiar with Kp,uu values. I don’t need to explain that when we are talking about blood brain barrier. So we know that blood brain barrier transporters, we think based on your publication that blood brain barrier transporters can enhance brain Kp,uu. When you’re talking about the uptake transporters, which we are talking about this topic of transporters as a drug delivery method. So we expect drug transporters to enhance this Kp,uu value. This is an interesting prospect for drug design incorporating both target potency and uptake transporter across blood brain barrier.
With having this in mind, what would you say are some of the important research areas that would help drug designers exploit uptake transporters more deliberately and more thoroughly, rather than say today where the uptake is arguably more by random findings sometimes. So how you would describe that?
Elena Puris: In my opinion, the most important part for developing drug delivery systems into the brain is to systematically investigate pro drug or nanocarrier in terms of drug delivery efficacy. And therefore, before developing any drug delivery system, it’s very crucial to ask the following question. So which drug do I want to deliver to the brain? Is there any issue with drug delivery or some other aspect affecting the pharmacological efficacy of the drug? Where do I want to deliver the drug? Is it extracellular fluid or is it intracellular compartment of the brain? And after crossing the brain, of course we need to know the information where we want to measure our drug in extracellular compartment or intracellular compartment, because it will affect the methods which we will apply investigation of that. And is the target actually in extracellular or intracellular compartment?
So this information will affect everything. And these are very important questions which researchers should ask early enough before they start developing anything. Because after that, after they know where is target and where they are targeting their component, they will understand which transporters it would besides of the blood brain barrier and also parenchyma cells and some transportors expressed only at the blood brain barrier. And so in different situations you need to consider different transporters.
Elnaz Gozalpour: Thank you, Elena. Mikko, what are you thinking about research areas here?
Mikko Gynther: Yeah, I agree with Elena that the most crucial thing is to really understand where in the brain you are targeting the drug. And then this affects of course, the selection of transporters that you want to utilize possibly, but also how you will study the brain pharmacokinetics, because different techniques can be applied depending, are you interested in targeting the intracellular space or the extracellular space, for example, in the brain? We have all the techniques available for this, but of course they are quite technically demanding sometimes and time consuming. So it’s just that we need to know how to use them and when to use them and. Yeah, and unfortunately there are really no shortcuts here, so we need to use this quite difficult techniques sometimes here.
But this is extremely important because like you mentioned that these transporters can facilitate higher Kp,uu values in the brain. But because the same transporters can be expressed also in the brain parenchyma cells, we can also increase the Kp,uu,cell, meaning that the unbound drug concentration in the intracellular compartment in the brain. And of course, if we don’t utilize correct techniques to measure these concentrations from the brain, we might miss important phenomenon such as this increased Kp,uu,cell values.
Elnaz Gozalpour: Yeah, both of you are talking about intracellular Kp,uu value, which is a concept that most of the time at drug discovery or drug development procedures, we don’t talk about it, we talk about brain Kp,uu value, CSF Kp,uu value. Would you be able to elaborate a little bit on intracellular Kp,uu value? Maybe with an example for us it would be quite good.
Elena Puris: Yes, certainly. So let’s say we target some transporter highly expressed at the blood brain barrier. We need to consider also that is it expressed in the brain bearing chemo cells, because once the compound or drug delivery system crosses the blood brain barrier, then transporters also decide how this compound is distributed within the brain. Often these same transporters that we target the blood brain barrier, they are present in brain bearing chemo cells and they can facilitate then the drug accumulation in the brain intracellular space. In this case, Kp,uu value might actually not increase much. It might be quite modest. But Kp,uu,brain or Kp,uu,cell would be then increased because the compound would be accumulated in the brain intracellular space.
Mikko Gynther: So we have an example of lysine product of ketoprofen which utilizes amino acid transporter at the blood brain barrier but also in the brain parenchymal cells. And we compared the delivery of ketoprofen to brain compared to ketoprofen dosing and we didn’t find much of difference in Kp,uu values between the released ketoprofen and ketoprofen dosing. But there was more than hundred fold higher Kp,uu,cell for ketoprofen released from the prodrug compared to ketoprofen dosing. And if we would have been only investigating the Kp,uu value, we would have completely missed this increased accumulation to the intracellular site which is actually the target site of ketoprofen in the brain.
Elena Puris: I could support Mikko with another example which we also observed similar phenomena with phenylalanine prodrug of ketoprofen which was also targeting amino acid transporter at the blood brain barrier. And in our study in mice in pharmacokinetic analysis demonstrated that lower brain to plasma distribution of ketoprofen prodrug was observed compared to parent drug itself after IP injection. And someone would think that probably this prodrug is not effective and maybe not to go with this prodrug for another studies. However, when we used brain slice method and investigated intra brain distribution for this product, we found that Kp,uu cell value was higher than unity for this prodrug, meaning that there is active uptake intracellular compartment of the brain. Well, for ketoprofen itself it was around 0.1, meaning that it’s very low.
And of course we observed much higher release of the ketoprofen in the brain which was like distribution of release ketoprofen in mice between brain to plasma was much higher compared to parent drug dosing. And as Mikko already mentioned, for development of transporter mediated drug delivery systems, it’s very important not only to measure blood brain barrier permeation but also intra brain distribution. Unfortunately, among reviewed reports, only these two cases which we mentioned now with Mikko investigated intra brain distribution.
Elnaz Gozalpour: Thank you. That was quite interesting examples to know. When you’re talking about brain compartments and also intracellular Kp,uu value, what do you think the key and emerging technologies are in this area? Because every day we are observing different technologies coming on board. So for this purpose, what would be usable?
Mikko Gynther: I think at least in my opinion, this recent advances in sensitive LC-MS based proteomics approach is very important. So we are able to detect and absolutely quantitate more and more transporters at the blood brain barrier but also in the brain parenchymal cells. And once we know the absolute abundance of these transporters in these cells, it will be easier for us to predict which or decide rather which transporters we want to target for brain delivery. And also then to predict how our compounds or drug delivery systems are distributed within the brain. So we can, let’s say, make more rational decisions on the selection of the targeted transporters.
Elena Puris: In my opinion, important part here is to educate scientists in terms of how to apply various techniques for evaluation of drug delivery efficacy. Because in my opinion this is very limiting part. And currently, as Mikko already mentioned previously, that all the methods are available and among those cerebral microdialysis technique combined with pharmacokinetic study is still a gold standard method for evaluation of drug delivery efficacy to the brain. In addition, there are currently other techniques such as brain slice method and combinatory mapping approach introduced by Rena Lorian. And these are really great tools for evaluating Kp,uu cell and intra brain distribution.
And I think the researchers focusing on developing drug delivery systems into the brain really have to learn how to use these techniques and apply them and publish the data using these methods, correct methods and correct interpretation of the data based on these techniques.
Elnaz Gozalpour: Thank you, Elena. We talked about topics of this area, we talked about the techniques at this area. Let’s look at it from drug design perspective. The aim of drug design is to treat diseases. What do we know about the impact of diseases on the expression of blood brain barrier transporters? What is the latest knowledge about that?
Elena Puris: Yeah, the changes in functional expression of transporters at the blood brain barrier in pathological condition can indeed affect drug delivery to the brain in the disease state. And therefore, I think it’s very important that we understand what are the changes in various conditions at the blood brain barrier in targeted transporter expression and then we can translate this knowledge from health to the disease state. And the majority of compounds targeting blood brain barrier transporters have been tested in healthy animals. It’s common to do pharmacokinetic studies in healthy animals. However, our recent studies in AD rat and mouse models revealed significant alterations in several nutrient transporters including amino acid transporter and glucose transporter, which are currently a hot topic for targeting at the blood brain barrier and which were mainly used for targeting at the blood brain barrier.
And importantly, we have seen that the changes at the blood brain barrier transporter expression were model specific and dependent on the stage of the disease. So it makes the picture even more complicated. So you need to understand which disease you want to treat, but also at which stage of the disease you want to apply your approach. And unfortunately, the quantitative information about the changes in expression of the blood brain barrier transporters in various diseases in human still limited. Therefore I think more knowledge should be collected on this. And importantly, what we also observed that there are sex differences in changes in expression of blood brain barrier transporters, which make the situation even more complicated.
So it’s very important to deep into this area and with new available methods such as lipid chromatography, mass spectrometry, proteomics, we can really understand better quantitative changes in transporter expression and how to correlate them between various models and disease states and to help translatability of the data.
Elnaz Gozalpour: Thanks. It’s quite interesting subject. I need to move on to the next question of me. Let’s assume we are targeting a disease, we are designing assay cascade to be able to apply one of the transporters for drug delivery. So we should consider, to my eyes, we should consider two target uptake transporter and then the main target for drug, let’s say drug target. How would you design your assay cascade? How your cascade look like? Would you elaborate a little bit on that?
Mikko Gynther: So I think the most important thing, like Elena previously mentioned that we need to know where the target site is, where we want to target the drug, in which compartment, and then we need to know about the transporters that are available, preferably in the disease state. After this has been established, then we can move on to the assays that we would use here. So first thing of course would be to optimize the utilization of the transporter. So we would need to investigate these interactions with the targeted transporters to determine the transport kinetics, meaning affinity and transport velocity. So these are both important factors, as affinity has to be good enough that the compounds can compete with the endogenous substrates. And also the transport velocity should be high enough so we have efficient delivery.
So we can sometimes have good interaction as such with the transporter. But the transport velocity is very low. So this should be first investigated. And here preferably we would use transfected cell lines. Then the role of the targeted transporters for blood brain barrier permeation should be investigated by in situ brain perfusion technique in mice or rats, because with this technique you can both get the permeability rate, but also you can very precisely determine the permeability mechanism, whether your transporter that you are targeting is actually involved in the permeability or not. After that we would go for systemic and brain pharmacokinetics. Here we would sample both the whole tissue concentrations and also use microdialysis. With this techniques we can then determine Kp,uu values and also Kp,uu,cell.
Of course this depends whether you are targeting the intracellular compartment or do you want to target the extracellular compartment in the brain. And importantly we would also sample the off target tissues because when we target transporters we don’t only change the blood brain barrier permeation, but we also affect the systemic pharmacokinetics. So we can and also for some reason also possible that we increase the delivery to some off target tissue. So this should be checked definitely. And like Elena mentioned earlier that these pharmacokinetic experiments should be done in a relevant disease model.
Elnaz Gozalpour: Thank you, I would like to continue. It’s an interesting topic but before wrapping up the podcast because we are also time limited. Both of you, do you think there are any important translational aspects that you need to address here and if there are, how you would achieve those translational aspects in this area?
Elena Puris: Yes, indeed there are many translational aspects which needs to be considered by developing transporter mediated drug delivery systems to the brain. And first of all as it has been mentioned, functional expression of transporters at the blood brain barrier and parenchymal cells should be investigated in disease state. And moreover recently we observed sex differences as I mentioned. So in these terms it preferable that sex differences are also investigated and then correct model is used for evaluation of drug delivery efficacy. And importantly not to forget that as I mentioned before that the first step is to cross the blood brain barrier, but then the second step is to release the parent drug at site of action. And therefore there is another aspect, translational aspect such as release of the prodrug for example. So we need to know what is the mechanism.
So if it’s enzymatic by transformation, then we need also to understand translational aspects and species differences in release of the parent drug.
Mikko Gynther: I can give maybe an example to this last point that Elena made. So we have seen species differences in this brain pharmacokinetics of transporter utilizing broad drugs, although there is no species difference really in the expression of the targeted transporter between these species. So I earlier mentioned this lysine product of ketoprofen. So we did first study in rats and we saw this very nice results of delivery of ketoprofen and release of it in the intracellular compartment in the brain. And when we repeated this study in mice, we actually didn’t see any released ketoprofen in the brain intracellular compartment and when we looked at the differences in transporter expression we didn’t really see anything. So it has to be something related to the release of the parent drug within the brain and species differences there.
And if we can see this kind of high and significant differences between mice and rats we can assume that there can be really differences also between these rodents and rodent models and then human situation.
Elnaz Gozalpour: Thank you very much. Unfortunately we’ve come to the end of the podcast. Elena, Mikko, it was pleasure to talk to you. Thank you very much. It was very insightful and informative. Thank you very much for your time and attendance and thank you to the audience who were listening to this. Please look out for more topics coming to DMPK Insights podcast. Have a good day and bye for now.
Elena Puris: Thank you so much for inviting. It was pleasant to be here.
Elnaz Gozalpour: You’re very welcome.
Mikko Gynther: Thank you for the opportunity to share our knowledge on this topic.
