Mikko Gynther

Large Neutral Amino Acid Transporter Enables Brain Drug Delivery via Prodrugs

The blood-brain barrier efficiently controls the entry of drug molecules into the brain. We describe a feasible means to achieve carrier-mediated drug transport into the rat brain via the specific, large neutral amino acid transporter (LAT1) by conjugating a model compound to L-tyrosine. A hydrophilic drug, ketoprofen, that is not a substrate for LAT1 was chosen as a model compound. The mechanism and the kinetics of the brain uptake of the prodrug were determined with an in situ rat brain perfusion technique. The brain uptake of the prodrug was found to be concentration-dependent. In addition, a specific LAT1 inhibitor significantly decreased the brain uptake of the prodrug. Therefore, our results reveal for the first time that a drug-substrate conjugate is able to transport drugs into the brain via LAT1.

Prodrug Approaches for CNS Delivery

Central nervous system (CNS) drug delivery remains a major challenge, despite extensive efforts that have been made to develop novel strategies to overcome obstacles. Prodrugs are bioreversible derivatives of drug molecules that must undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which subsequently exerts the desired pharmacological effect. In both drug discovery and drug development, prodrugs have become an established tool for improving physicochemical, biopharmaceutical or pharmacokinetic properties of pharmacologically active agents that overcome barriers to a drug’s usefulness. This review provides insight into various prodrug strategies explored to date for CNS drug delivery, including lipophilic prodrugs, carrier- and receptor-mediated prodrug delivery systems, and gene-directed enzyme prodrug therapy.

Glucose promoiety enables glucose transporter mediated brain uptake of ketoprofen and indomethacin prodrugs in rats.

The brain uptake of solutes is efficiently governed by the blood-brain barrier (BBB). The BBB expresses a number of carrier-mediated transport mechanisms, and new knowledge of these BBB transporters can be used in the rational targeted delivery of drug molecules for active transport. One attractive approach is to conjugate an endogenous transporter substrate to the active drug molecule to utilize the prodrug approach. In the present study, ketoprofen and indomethacin were conjugated with glucose and the brain uptake mechanism of the prodrugs was determined with the in situ rat brain perfusion technique. Two of the prodrugs were able to significantly inhibit the uptake of glucose transporter (GluT1)-mediated uptake of glucose, thereby demonstrating affinity to the transporter. Furthermore, the prodrugs were able to cross the BBB in a temperature-dependent manner, suggesting that the brain uptake of the prodrugs is carrier-mediated.

Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery.

Central nervous system (CNS) drug delivery is a major challenge in drug development because the blood-brain barrier (BBB) efficiently restricts the entry of drug molecules into the CNS at sufficient amounts. The brain uptake of poorly penetrating drugs could be improved by utilizing the transporters at the BBB with a prodrug approach. In this study, we designed four phenylalanine derivatives of valproic acid and studied their ability to utilize a large amino acid transporter 1 (LAT1) in CNS delivery with an aim to show that the meta-substituted phenylalanine prodrugs bind to LAT1 with a higher affinity compared with the affinity of the para-substituted derivatives. All of the prodrugs crossed the BBB carrier mediatedly via LAT1 in in situ rat brain perfusion. For the first time, we introduced a novel meta-substituted phenylalanine analogue promoiety which improved the LAT1 affinity 10-fold and more importantly the rat brain uptake of the prodrug 2-fold compared with those of the para-substituted derivatives. Therefore, we have characterized a new prodrug design idea for CNS drug delivery utilizing a transporter-mediated prodrug approach.

Brain uptake of ketoprofen-lysine prodrug in rats

The blood-brain barrier (BBB) controls the entry of xenobiotics into the brain. Often the development of central nervous system drugs needs to be terminated because of their poor brain uptake. We describe a way to achieve large neutral amino acid transporter (LAT1)-mediated drug transport into the rat brain. We conjugated ketoprofen to an amino acid l-lysine so that the prodrug could access LAT1. The LAT1-mediated brain uptake of the prodrug was demonstrated with in situ rat brain perfusion technique. The ability of the prodrug to deliver ketoprofen into the site of action, the brain intracellular fluid, was determined combining in vivo and in vitro experiments. A rapid brain uptake from blood and cell uptake was seen both in in situ and in vivo experiments. Therefore, our results show that a prodrug approach can achieve uptake of drugs via LAT1 into the brain intracellular fluid. The distribution of the prodrug in the brain parenchyma and the site of parent drug release in the brain were shown with in vivo and in vitro studies. In addition, our results show that although lysine or ketoprofen are not LAT1-substrates themselves, by combining these molecules, the formed prodrug has affinity for LAT1.

Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1).

L‐Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood–brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier‐mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q2=0.53, R2=0.75, Q2 SE=0.77, R2 SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L‐tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1‐targeted high‐affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1‐binding prodrugs and other compounds that bind to LAT1.

Does increasing the ratio of AMPA-to-NMDA receptor mediated neurotransmission engender antidepressant action? Studies in the mouse forced swim and tail suspension tests.

Monoamine-based antidepressant drugs increase α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function and decrease N-methyl-d-aspartate receptor (NMDAR) function. The NMDAR antagonist ketamine shows potent antidepressant action in humans and the antidepressant-like effects of ketamine and monoamine-based antidepressants in rodents depend on increased AMPAR throughput. Further, the antidepressant-like effects of monoamine-based antidepressants are enhanced by AMPAR potentiation and by NMDAR antagonism. This has led to a hypothesis that antidepressant efficacy involves an increases ratio of AMPAR-to-NMDAR-mediated neurotransmission. To further elucidate the interaction of AMPAR, NMDAR and monoamine transmission we tested combinations of the AMPAR positive allosteric modulator (AMPA potentiator), (R,R)-N,N-(2,20-[biphenyl-4-40-diyl]bis[propane-2,1-diyl])dimethanesulfonamide (PIMSD), with: the uncompetitive NMDAR antagonist MK-801; nicotine, which has potent glutamate-releasing properties; and the selective serotonin reuptake inhibitor escitalopram using the mouse forced swim (mFST) and tail suspension tests (mTST). MK-801, nicotine or escitalopram did not induce antidepressant-like effects in either of the two tests. PIMSD enhanced the effect of MK-801 in the mFST, supporting the hypothesis that increasing AMPAR-to-NMDAR-mediated neurotransmission conveys antidepressant action. Nicotine-induced glutamate release simultaneously activates NMDARs and AMPARs and showed no net effect in the mFST when given alone. However, increasing the ratio of AMPAR-to-NMDA-R transmission by favouring AMPAR throughput with PIMSD revealed an antidepressant-like action of nicotine in the mFST. PIMSD also enhanced the effect of escitalopram treatment in the mFST and mTST, supporting existing evidence and suggesting a synergistic effect of simultaneously facilitating monoamine transmission and increasing the ratio of AMPAR-to-NMDAR throughput. No synergistic effects of the PIMSD+MK-801 or PIMSD+nicotine were found in the mTST, indicating a differential sensitivity of mFST and mTST when investigating glutamate-based antidepressant mechanisms. This study corroborates existing evidence that there may be an unexploited therapeutic potential in treating depression by directly increasing the ratio of AMPAR-to-NMDAR neurotransmission, possibly in combination with monoamine-based mechanisms.

A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentiates Antiproliferative Drug Efficacy in Cancer Cells

The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 μM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.

L-Type amino acid transporter 1 (lat1)-mediated targeted delivery of perforin inhibitors.

Perforin is a cytolytic pore-forming glycoprotein secreted by cytotoxic effector cells. It is a key component of the immune response against virus-infected and transformed cells and has been implicated in a number of human diseases. Perforin activity can be inhibited by small-molecular-weight compounds, although less is known about their delivery to the site of action. Therefore, in the present study, it was explored if perforin inhibitors could be efficiently and site-selectively delivered firstly into the cytotoxic effector cells and secondly into lytic granules, in which perforin is stored. This was accomplished by designing and synthesizing four prodrugs of perforin inhibitors that could utilize l-type amino acid transporter (LAT1), since activated immune cells are known to over-express LAT1. The results demonstrate that cellular uptake of perforin inhibitors can be increased by LAT1-utilizing prodrugs (into human breast adenocarcinoma cells (MCF-7)). Furthermore, these prodrugs were also able to deliver perforin inhibitors into the cell organelles having lower pH (rat liver lysosomes). Therefore, by using these prodrugs, intracellular mechanisms of perforin inhibitory activity can be studied more thoroughly in future. Moreover, this prodrug approach can be applied for other drugs that would benefit from targeted delivery into cells expressing LAT1, such as cancer.

Optimization of 1,2,5-Thiadiazole Carbamates as Potent and Selective ABHD6 Inhibitors

At present, inhibitors of α/β‐hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5‐thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4‐morpholino‐1,2,5‐thiadiazol‐3‐yl cyclooctyl(methyl)carbamate (JZP‐430) potently and irreversibly inhibited hABHD6 (IC50=44 nM) and showed ∼230‐fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off‐targets of related compounds. Additionally, activity‐based protein profiling indicated that JZP‐430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP‐430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.