Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs

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 LAT1-utilizing 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.

Tyrosine–Chlorambucil Conjugates Facilitate Cellular Uptake through L-Type Amino Acid Transporter 1 (LAT1) in Human Breast Cancer Cell Line MCF-7

“…The half-life of the bioconversion of the amide derivative 2 was up to 10 times higher compared with that of the ester 1. As with other reports, the result indicates that the amide bond is less sensitive to enzymatic hydrolysis than the corresponding ester bond [2,24]; the ester derivative is more susceptible to enzymatic bioconversion, limiting its ability to reach the target site intact in vivo.…”

L-type amino acid transporter 1 utilizing prodrugs: How to achieve effective brain delivery and low systemic exposure of drugs

“…The high concentration of prodrug 5 in both brain and liver can be achieved due to its utilization of other transporters such as organic anion-transporting polypeptides (OATPs) which are expressed in these tissues. [23,29] Previously, we revealed that OATPs were involved in the transport of LAT1-utilizing prodrugs of perforin inhibitors [17]. In summary, the data emphasizes that the small structural changes in prodrugs designed to utilize LAT1 introduce significant effects on their brain and systemic pharmacokinetics, either enhancing or limiting the brain distribution of the parent drug.…”

“…Based on these features, it has been demonstrated that several prodrugs have an ability to bind to LAT1 and exploit this transporter for BBB permeation or/and cell uptake [5][6][7][8][9]16]. However, there is limited information about systemic and brain pharmacokinetics of these prodrugs including the distribution of the prodrugs and their parent drugs in the liver and their first pass metabolism [5,8,16,17].…”

“…The ability of ketoprofen and prodrugs 1-5 to cross the BBB was studies using in situ mouse brain perfusion technique according to the method described by Gynther et al (2016) [17]. The perfusion of 10 μM solutions of ketoprofen or prodrugs 1-5 was implemented at flow rate of 2.5 mL/min at 37 °C for 60 s and was followed by the wash of brain capillaries using drug free perfusion buffer for 2 s at 4°C.…”

Insights into the Structure, Function, and Ligand Discovery of the Large Neutral Amino Acid Transporter 1, LAT1

“…The in situ rat brain perfusion technique demonstrated that prodrugs 77 – 79 significantly inhibited the cellular uptake of l -[ 14 C]-leucine (91.4 ± 1.6%, 75.7 ± 1.8% and 88.3 ± 2.3%, respectively), while prodrugs 80 and 81 showed minor reduction of l -[ 14 C]-leucine uptake (17.6 ± 6.7% and 6.2 ± 2.2%, respectively). In a study using the in situ rat brain perfusion technique, valproic acid prodrugs 82 and 83 and perforin inhibitor prodrugs 84 and 85 showed 16%, 81%, 97.4% and 59.4% inhibition of l -[ 14 C]-leucine uptake, respectively [ 121 , 122 ]. The studies described here have demonstrated that natural substrates of LAT1 can serve as an excellent template to enhance the delivery of small molecules into the brain.…”