Jukka Leppänen

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.

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.

Absorption Rate Limit Considerations for Oral Phosphate Prodrugs

AbstractPurpose. To evaluate the potential of phosphate ester prodrugs to significantly improve the absorptive flux of poorly soluble parent drugs. Methods. Absorptive transport studies of parent drugs and their prodrugs were carried out in Caco-2 cells. Prodrugs of parent drugs with variable aqueous solubilities were tested: Hydrocortisone-phosphate/Hydrocortisone, Fosphenytoin/phenytoin, TAT-59/DP-TAT-59, and Entacapone phosphate/Entacapone. Additional absorption studies were carried out in rats. Results. Absorptive fluxes of DP-TAT-59 and phenytoin increased 9.8 or 3.3-fold after dosing TAT-59 and 500 μM fosphenytoin, respectively. Hydrocortisones flux did not increase with hydrocortisone-phosphate at 100 μM. Permeability of the highly lipophilic and protein bound compound, DP-TAT-59, was significantly increased with serosal albumin. No permeability increase was observed for the other drugs with albumin. Entacapone phosphate failed to improve the flux of entacapone compared to an entacapone solution, but the prodrug solution did yield higher entacapone plasma levels in rats when compared with an entacapone suspension. Conclusion. Ideal phosphate prodrug candidates are characterized by high permeability and low solubility (BCS Class II drugs). For low dose BCS Class II drug candidates, however, no biopharmaceutical advantage may be gained. Phosphate prodrugs of parent drugs with limited permeability may fail. When screening highly lipophilic parent drugs transport studies should be done with albumin.

Discovery of Boronic Acids as Novel and Potent Inhibitors of Fatty Acid Amide Hydrolase

A series of commercial phenyl-, heteroaryl-, alkyl-, and alkenylboronic acids were evaluated for their FAAH and MGL inhibitory activities. The compounds were generally selective for FAAH, with IC50 in the nanomolar or low-micromolar range. Eight of these compounds inhibited MGL with IC50 in the micromolar range. The most potent compound, phenylboronic acid with para-nonyl substituent (13), inhibited FAAH and MGL with IC50 of 0.0091 and 7.9 microM, respectively.

Design, Synthesis and Brain Uptake of LAT1-Targeted Amino Acid Prodrugs of Dopamine

ABSTRACTPurposeDrug delivery to the brain is impeded by the blood-brain barrier (BBB). Here, we attempted to enhance the brain uptake of cationic dopamine by utilizing the large amino acid transporter 1 (LAT1) at the BBB by prodrug approach.MethodsThree amino acid prodrugs of dopamine were synthesized and their prodrug properties were examined in vitro. Their LAT1-binding and BBB-permeation were studied using the in situ rat brain perfusion technique. The brain uptake after intravenous administration and the dopamine-releasing ability in the rat striatum after intraperitoneal administration were also determined for the most promising prodrug.ResultsAll prodrugs underwent adequate cleavage in rat tissue homogenates. The prodrug with phenylalanine derivative as the promoiety had both higher affinity for LAT1 and better brain uptake properties than those with an alkyl amino acid -mimicking promoiety. The phenylalanine prodrug was taken up into the brain after intravenous injection but after intraperitoneal injection the prodrug did not elevate striatal dopamine concentrations above those achieved by corresponding L-dopa treatment.ConclusionsThese results indicate that attachment of phenylalanine to a cationic drug via an amide bond from the meta-position of its aromatic ring could be highly applicable in prodrug design for LAT1-mediated CNS-delivery of not only anionic but also cationic polar drugs.

Oxadiazole-carbonylaminothioureas as SIRT1 and SIRT2 Inhibitors

A new inhibitor for human sirtuin type proteins 1 and 2 (SIRT1 and SIRT2) was discovered through virtual database screening in search of new scaffolds. A series of compounds was synthesized based on the hit compound (3-[[3-(4-tert-butylphenyl)1,2,4-oxadiazole-5-carbonyl]amino]-1-[3-(trifluoromethyl)phenyl]thiourea). The most potent compound in the series was nearly as potent as the reference compound (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide).

N(epsilon)-thioacetyl-lysine-containing tri-, tetra-, and pentapeptides as SIRT1 and SIRT2 inhibitors.

N()-Thioacetyl-lysine-containing tri-, tetra-, and pentapeptides, based on the alpha-tubulin and p53 protein sequences, were studied as SIRT1 and SIRT2 inhibitors. The potency of the pentapeptides depended on the selection of the side chains. The removal of N- and C-terminal residues of the pentapeptides yielded tripeptides with retained SIRT1 inhibitory activity but decreased SIRT2 inhibitory activity. The most potent SIRT1 inhibitors were equipotent with the reference compound (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) with the IC(50) values of 180-330 nM.

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.

The First Bioreversible Prodrug of Metformin with Improved Lipophilicity and Enhanced Intestinal Absorption

Metformin is a potent antidiabetic agent and currently used as a first-line treatment for patients with type 2 diabetes. Unfortunately, the moderate absorption and uncomfortable gastrointestinal adverse effects associated with metformin therapy impair its use. In this study, two novel prodrugs of a biguanidine functionality containing antidiabetic agent, metformin, were designed, synthesized, and evaluated in vitro and in vivo to accomplish improved lipophilicity and, consequently, enhanced oral absorption of this highly water-soluble drug. These results represent that the more lipophilic prodrug 2a biotransformed quantitatively to metformin mainly after absorption. The enhanced oral absorption consequently promoted the bioavailability of metformin from 43% to 65% in rats. Thus, this novel prodrug may offer a solution to reduce the required daily doses of metformin, which may decrease the uncomfortable adverse effects associated with metformin therapy.