Aaro J. Jalkanen

Comparison of in vitro cell models in predicting in vivo brain entry of drugs.

Although several in vitro models have been reported to predict the ability of drug candidates to cross the blood-brain barrier, their real in vivo relevance has rarely been evaluated. The present study demonstrates the in vivo relevance of simple unidirectional permeability coefficient (P(app)) determined in three in vitro cell models (BBMEC, Caco-2 and MDCKII-MDR1) for nine model drugs (alprenolol, atenolol, metoprolol, pindolol, entacapone, tolcapone, baclofen, midazolam and ondansetron) by using dual probe microdialysis in the rat brain and blood as an in vivo measure. There was a clear correlation between the P(app) and the unbound brain/blood ratios determined by in vivo microdialysis (BBMEC r=0.99, Caco-2 r=0.91 and MDCKII-MDR1 r=0.85). Despite of the substantial differences in the absolute in vitro P(app) values and regardless of the method used (side-by-side vs. filter insert system), the capability of the in vitro models to rank order drugs was similar. By this approach, thus, the additional value offered by the true endothelial cell model (BBMEC) remains obscure. The present results also highlight the need of both in vitro as well as in vivo methods in characterization of blood-brain barrier passage of new drug candidates.

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.

Brain Pharmacokinetics of Two Prolyl Oligopeptidase Inhibitors, JTP‐4819 and KYP‐2047, in the Rat

Prolyl oligopeptidase (PREP) inhibitors are potential drug candidates for the treatment of neurological disorders, but little is known about their ability to cross the blood-brain barrier and to reach the target site. This study characterizes brain pharmacokinetics of two potent PREP inhibitors, JTP-4819 and KYP-2047. Firstly, the in vitro permeability (P(app) ) of JTP-4819 and KYP-2047 through a bovine brain microvessel endothelial cell monolayer was assessed. Then, the in vivo brain/blood ratio was determined for the total brain and plasma concentrations and also for the unbound extracellular drug concentrations after a single dose (50 μmol/kg i.p.). KYP-2047 had a significantly higher P(app) than JTP-4819. In vivo, KYP-2047 had higher total and unbound brain/blood ratios. KYP-2047 was equally distributed between the cortex, hippocampus and striatum. In the case of JTP-4819, the unbound brain extracellular concentrations could not be readily predicted from the unbound blood levels, probably because of its poor membrane penetration properties. KYP-2047 displayed a better ability to reach the intracellularly located brain PREP, and it inhibited this enzyme more effectively than JTP-4819 after an equimolar single dose. In conclusion, KYP-2047 showed better brain penetration characteristics than JTP-4819 both in vitro and in vivo. KYP-2047 is a brain-penetrating, potent and long-acting PREP inhibitor; thus, it represents a convenient pharmacological tool for assessing the potential of PREP as a drug target.

The effect of prolyl oligopeptidase inhibition on extracellular acetylcholine and dopamine levels in the rat striatum.

Prolyl oligopeptidase (PREP, EC 3.4.21.26) inhibitors have potential as cognition enhancers, but the mechanism of action behind the cognitive effects remains unclear. Since acetylcholine (ACh) and dopamine (DA) are known to be associated with the regulation of cognitive processes, we investigated the effects of two PREP inhibitors on the extracellular levels of ACh and DA in the rat striatum using in vivo microdialysis. KYP-2047 and JTP-4819 were administered either as a single systemic dose (50 μmol/kg∼17 mg/kg i.p.) or directly into the striatum by retrodialysis via the microdialysis probe (12.5, 37.5 or 125 μM at 1.5 μl/min for 60 min). PREP inhibitors had no significant effect on striatal DA levels after systemic administration. JTP-4819 significantly decreased ACh levels both after systemic (by ∼25%) and intrastriatal (by ∼30-50%) administration. KYP-2047 decreased ACh levels only after intrastriatal administration by retrodialysis (by ∼40-50%) when higher drug levels were reached, indicating that higher brain drug levels are needed to modulate ACh levels than to inhibit PREP. This result does not support the earlier hypothesis that the positive cognitive effects of PREP inhibitors in rodents would be mediated through the cholinergic system. In vitro specificity studies did not reveal any obvious off-targets that could explain the observed effect of KYP-2047 and JTP-4819 on ACh levels, instead confirming the concept that these compounds have a high selectivity towards PREP.

Inhibition of prolyl oligopeptidase by KYP-2047 fails to increase the extracellular neurotensin and substance P levels in rat striatum

Prolyl oligopeptidase (PREP, EC 3.4.21.26) hydrolyzes neuropeptides, such as neurotensin and substance P in vitro, but its importance in the in vivo metabolism of these peptides has not been proved. This is the first report where intracerebral microdialysis combined with highly sensitive radioimmunoassay has been used to investigate the effect of PREP inhibition on the brain extracellular peptide levels in conscious rats. We show that PREP inhibition by KYP-2047 (50μmol/kg=17mg/kg, intraperitoneally, that effectively inhibits PREP in the brain), has no effect on the neurotensin and substance P levels in the striatum extracellular space. This provides a further piece of evidence in support of the proposition that PREP is not significantly responsible for the in vivo cleavage of substance P or neurotensin, and that occasional positive cognitive effects associated with some PREP inhibitors are not mediated through elevated extracellular levels of these peptides. Direct regulation of peptide processing by PREP is not likely because the enzyme is located intracellularly and the peptide substrates are mostly extracellular.

Different Effects of Scopolamine and Inhibition of Prolyl Oligopeptidase on Mnemonic and Motility Functions of Young and 8- to 9-Month-Old Rats in the Radial-Arm Maze

Prolyl oligopeptidase (POP) has been connected to memory and mood through regulation of the brain levels of its biologically active peptide substrates and phosphatidylinositol system. This is the first study in a radial-arm maze of the effects of a single dose of a novel potent prolyl oligopeptidase inhibitor, KYP-2047 (5 mg/kg, dissolved in 5% Tween 80), on memory and learning of scopolamine-treated (0.4 mg/kg, dissolved in saline) rats. Habituated (days 1 and 2) and trained (days 3-11) young (3 months) and old (8-9 months) male Wistar rats were given (i) saline + Tween, (ii) saline + KYP-2047, (iii) scopolamine + Tween or (iv) scopolamine + KYP-2047 30 min. prior to testing their memory. Food rewards located in four randomly chosen arms of the maze. The rat had 10 min. to find and eat the rewards. Time spent in the maze, visits to each arm and number of eaten rewards were measured. Old rats made generally more errors, spent more time and visited fewer arms per minute in the maze than young rats. The memory- and function-impairing effects of scopolamine were also seen more clearly in old than young rats. KYP-2047 had no or only a marginal effect on memory of either age group, but when given without scopolamine, it slightly increased the maze motility of young rats and decreased the motility of old rats. In a separate locomotor activity test, KYP-2047 enhanced the motility of young rats supporting a suggested role of POP in motor functions.

Localization of prolyl oligopeptidase in the thalamic and cortical projection neurons: A retrograde neurotracing study in the rat brain

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyses proline-containing peptides shorter than 30-mer. POP is believed to be associated with cognitive functions via neuropeptide cleavage. POP has been also connected to the inositol 1,4,5-triphosphate (IP(3)) signalling but the effects of POP-inhibition to the IP(3) accumulation in vivo are still unclear. However, little is known about the physiological role of POP in the brain. We have previously found that in the rat brain POP was specifically expressed in the pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and corresponding projection areas in thalamus. Using a retrograde neurotracer we have now visualized the localization of POP in thalamocortical and corticothalamic projection neurons in ventrobasal complex and medial geniculate nucleus of thalamus and somatosensory/motor and auditory cortices. We observed that both in thalamus and cortex over 50% of projection neurons contained POP. These results support the hypothesis that POP is involved in thalamocortical and corticothalamic signal processing. We also propose, based on our neuroanatomical findings and literature, that POP may take part in the thalamocortical oscillations by interacting with IP(3) signalling in cells.

KYP-2047 Penetrates Mouse Brain and Effectively Inhibits Mouse Prolyl Oligopeptidase

Recent studies have indicated that specific prolyl oligopeptidase (PREP) inhibitors can modulate inflammation, angiogenesis and neurodegeneration. As most diseases that may be potential targets for PREP inhibitors are being modelled in mice, it is essential to evaluate the pharmacological properties of investigative PREP inhibitors in mice. This study characterizes the single‐dose brain pharmacokinetics and PREP inhibitory action of a potent PREP inhibitor, KYP‐2047, in wild‐type C57 mice. KYP‐2047 penetrated into the mouse brain rapidly (tmax≤10 min.) and achieved pharmacologically active concentrations after a single dose of 15 or 50 μmol/kg i.p. The brain/blood AUC ratio was 0.050 and 0.039 after 15 and 50 μmol/kg i.p., respectively. KYP‐2047 produced efficient brain PREP inhibition at both doses; 15 μmol/kg blocked PREP activity fully for 30 min., and it took 12 hr for the activity to recover, whereas 50 μmol/kg inhibited brain PREP activity fully for 1 hr, and most, 84%, of the activity had been restored by 12 hr. Both doses completely blocked PREP activity in liver for at least 1 hr, and only about 25% the activity was recovered within 12 hr. The pharmacokinetics and inhibition kinetics of KYP‐2047 in mice were found to be similar as those previously reported in rats and indicate that KYP‐2047 would need to be administered twice per day to achieve continuous brain PREP inhibition in mice. In conclusion, KYP‐2047 is a suitable pharmacological tool with which to assess the effects of PREP inhibition in mice.

Brain Pharmacokinetics of Ganciclovir in Rats with Orthotopic BT4C Glioma

Ganciclovir (GCV) is an essential part of the Herpes simplex virus thymidine kinase (HSV-tk) gene therapy of malignant gliomas. The purpose of this study was to investigate the brain pharmacokinetics and tumor uptake of GCV in the BT4C rat glioma model. GCV’s brain and tumor uptakes were investigated by in vivo microdialysis in rats with orthotopic BT4C glioma. In addition, the ability of GCV to cross the blood-brain barrier and tumor vasculature was assessed with in situ rat brain perfusion. Finally, the extent to which GCV could permeate across the BT4C glioma cell membrane was assessed in vitro. The areas under the concentration curve of unbound GCV in blood, brain extracellular fluid (ECF), and tumor ECF were 6157, 1658, and 4834 μM⋅min, respectively. The apparent maximum unbound concentrations achieved within 60 minutes were 46.9, 11.8, and 25.8 μM in blood, brain, and tumor, respectively. The unbound GCV concentrations in brain and tumor after in situ rat brain perfusion were 0.41 and 1.39 nmol/g, respectively. The highly polar GCV likely crosses the fenestrated tumor vasculature by paracellular diffusion. Thus, GCV is able to reach the extracellular space around the tumor at higher concentrations than that in healthy brain. However, GCV uptake into BT4C cells at 100 μM was only 2.1 pmol/mg of protein, and no active transporter–mediated disposition of GCV could be detected in vitro. In conclusion, the limited efficacy of HSV-tk/GCV gene therapy may be due to the poor cellular uptake and rapid elimination of GCV.

Prolyl oligopeptidase inhibition decreases extracellular acetylcholine levels in rat hippocampus and prefrontal cortex

Several investigative prolyl oligopeptidase (PREP) inhibitors have been shown to improve learning and memory in various preclinical trials but the mechanism of action behind these effects remains unclear. Since hippocampal and cortical acetylcholine (ACh) is known to play an important role in cognitive processes, the effects of two potent PREP inhibitors, JTP-4819 and KYP-2047, on extracellular ACh levels in hippocampus and medial prefrontal cortex were assessed using in vivo microdialysis. Conscious rats were treated with a single dose (15 or 50μmol/kg i.p.) of JTP-4819, KYP-2047 or vehicle, and extracellular ACh levels were monitored for 5h after treatment. In hippocampus, KYP-2047 had no significant effect on the ACh levels, although a trend towards decreased levels was observed at the higher dose. JTP-4819 had no significant effect on the hippocampal ACh levels at the lower dose (15μmol/kg), but the higher dose (50μmol/kg) significantly decreased ACh levels in hippocampus by about 25%. In cortex, the smaller dose (15μmol/kg) of KYP-2047 decreased ACh levels maximally by 25%, and a similar (ns) effect was also observed after the higher dose. JTP-4819 had no effect at the lower dose, but the higher dose decreased ACh levels maximally by about 30%. In conclusion, the present results suggest that the cognition-enhancing effects of investigative PREP inhibitors are not due to enhanced cholinergic transmission in hippocampus or cortex.