Article pubs.acs.org/molecularpharmaceutics
Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs Mikko Gynther,*,† Darryl S. Pickering,‡ Julie A. Spicer,§ William A. Denny,§ and Kristiina M. Huttunen† †
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland Department of Drug Design and Pharmacology, Faculty of Health & Medical Sciences, University of Copenhagen, Jagtvej 160, 1165 Copenhagen, Denmark § Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand ‡
ABSTRACT: We have recently reported that by converting a perforin inhibitor into an L-type amino acid transporter 1 (LAT1)utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and their LAT1utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 μmol/kg i.p. bolus injection, the prodrugs’ unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs could not reach the brain. The unbound brain concentrations of the prodrugs after 100 μM in situ mouse brain perfusion were 521.4 ± 46.9 and 126.9 ± 19.9 pmol/g for prodrugs 1 and 2, respectively. The combination of competing transporter substrates for LAT1, L-tryptophan, and for organic anion transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 μM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug. Thus, the prodrugs enhance significantly the therapeutic potential of the parent drugs for the treatment of disorders of central nervous system in which neuroinflammation is involved. KEYWORDS: brain uptake, pharmacokinetics, prodrug, transporter, perforin inhibitor
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Therefore, perforin inhibitors have to be designed with pharmacokinetics and brain drug delivery in mind. We have recently reported that by converting a perforin inhibitor into a human L-type amino acid transporter 1 (LAT1)-utilizing prodrug, its cellular uptake into the cells that expresses LAT1 can be greatly increased.16 Sodiumindependent heterodimeric transmembrane protein LAT1 facilitates the cell entry of neutral and branched amino acids and also the disposition of several drugs in clinical use.17 The LAT1 transporter is relatively highly and selectively expressed at the blood−brain barrier18 but also in metabolically active cells, such as placental, tumor, brain, and activated immune cells.19,20 Thus, targeting perforin inhibitors via LAT1 into the cytotoxic effector cells and/or into the brain cells can result in site-selective drug delivery and improved efficacy. To gain neuroprotection via inhibition of perforin in the synaptic cleft of the target cells and cytotoxic effector cells, it is crucial to deliver a potent inhibitor across the blood−brain barrier to its site of action. However, it is yet unknown whether these inhibitors act only extracellularly or does the intracellular
mall molecule perforin inhibitors have been studied as potential immunosuppressive agents since they can inhibit the function of the cytolytic protein perforin.1−4 Perforin is a glycoprotein that forms pores to the plasma membrane and is secreted by natural killer cells and cytotoxic T lymphocytes.5−7 It is essential for protective immunosurveillance; however, perforin has also been reported to be a key component in numerous human immunopathologies; especially in diseases and disorders related to neuroinflammation and blood−brain barrier disruption such as multiple sclerosis, ischemic stroke, and cerebral malaria.8−12 Besides of the fact that the perforin mechanism of function is complex, currently, it is not fully understood at what point in the process the inhibitors interact with perforin. Exocytic delivery of perforin and granzymes from the granules of cytotoxic effector cells occurs after effector cell conjugation with the target cell.13,14 The first systemic pharmacokinetic study of small molecule perforin inhibitors revealed that the in vivo clearance of the studied inhibitors was very high as they were prone to phase I metabolic reactions, although the redoxmetabolism did not completely explain fast clearance.15 Therefore, if the perforin inhibitors are rapidly cleared from the body, they may be unable to reach their target sites at therapeutically relevant concentrations. In addition, to reach their site of action, drugs intended for the treatment of central nervous system disorders have to cross the blood−brain barrier. © XXXX American Chemical Society
Received: March 11, 2016 Revised: May 12, 2016 Accepted: June 6, 2016
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DOI: 10.1021/acs.molpharmaceut.6b00217 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
Figure 1. Schematic presentations of the prodrug bioconversion to their parent drugs.
mode and electrospray ionization; nitrogen was used as drying gas at 300 °C with a flow rate of 10 L/min, whereas the capillary voltage was 4000 V and nebulizer pressure was 40 psi. The transitions used for analyte detection with multiplereaction monitoring were 514 → 304.7, 499.5 → 191.7, 322.8 → 203.7, and 308.4 → 229.8 for compounds 1−4, respectively. The transition for the internal standard, repaglinide, was 453 → 229.9. Fragmentor voltages used for compounds 1−4 were 120, 140, 140, and 140 V, whereas the collision energies were 46, 30, 34, and 40 V, respectively. For repaglinide these parameters were 75 and 8 V. The collision cell nitrogen pressure was 2.9 × 10−5 Torr. The software used for data acquisition was Agilent MassHunter Workstation (Data Acquisition for Triple Quadrupole Mass Spectometer, version B.03.01), and the data processing and analysis was performed with Quantitative Analysis (B.04.00) software. The lower limit of quantification for the tissue samples was 0.01 nmol/g as it was the lowest drug concentration that could be determined with a relative error and precision of
