Altering distribution profile of palbociclib by its prodrugs.

Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, is currently used clinically for treating hormone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Additionally, it has the potential to be utilized in the treatment of various tumors, including malignant glioblastoma. Previous research has indicated that palbociclib is a substrate for two efflux transporters, P-glycoprotein (P-gp; MDR1) and breast cancer-resistant protein (BCRP), which restrict the brain exposure of palbociclib. In the present study, our objective was to alter the brain distribution pattern of palbociclib by creating and assessing two novel prodrugs through in vitro, in situ, and in vivo evaluations. To this end, we synthesized two prodrugs of palbociclib by attaching it to the tyrosine promoiety at the para- (PD1) and meta-(PD2) position via a carbamate bond. We hypothesized that the prodrugs could bypass efflux transporter-mediated drug resistance by leveraging the l-type amino acid transporter (LAT1) to facilitate their transport across the blood-brain barrier (BBB) and into cancer cells, such as glioma cells that express LAT1. The compounds PD1 and PD2 did not show selective binding and had limited inhibitory effects on LAT1 in three cell lines (MCF-7, U87-MG, HEK-hLAT1). However, PD1 and PD2 demonstrated the ability to evade efflux mechanisms, and their in vitro uptake profiles were comparable to that of palbociclib, indicating their potential for effective cellular transport. In in situ and in vivo studies, brain uptake was not significantly improved compared to palbociclib, but the pharmacokinetic profiles showed encouraging enhancements. PD1 exhibited a higher AUCbrain/plasma ratio, suggesting safer dosing, while PD2 showed favorable long-acting pharmacokinetics. Although our prodrug design did not significantly improve palbociclib brain delivery due to the potential size limitation of the prodrugs, the study provides valuable insights for future prodrug development and drug delivery strategies targeting specific transporters.

Targeting Glial Cells by Organic Anion-Transporting Polypeptide 1C1 (OATP1C1)-Utilizing l-Thyroxine-Derived Prodrugs.

OATP1C1 (organic anion-transporting polypeptide 1C1) transports thyroid hormones, particularly thyroxine (T4), into human astrocytes. In this study, we investigated the potential of utilizing OATP1C1 to improve the delivery of anti-inflammatory drugs into glial cells. We designed and synthesized eight novel prodrugs by incorporating T4 and 3,5-diiodo-l-tyrosine (DIT) as promoieties to selected anti-inflammatory drugs. The prodrug uptake in OATP1C1-expressing human U-87MG glioma cells demonstrated higher accumulation with T4 promoiety compared to those with DIT promoiety or the parent drugs themselves. In silico models of OATP1C1 suggested dynamic binding for the prodrugs, wherein the pose changed from vertical to horizontal. The predicted binding energies correlated with the transport profiles, with T4 derivatives exhibiting higher binding energies when compared to prodrugs with a DIT promoiety. Interestingly, the prodrugs also showed utilization of oatp1a4/1a5/1a6 in mouse primary astrocytes, which was further supported by docking studies and a great potential for improved brain drug delivery.

Nigrostriatal 6-hydroxydopamine lesions increase alpha-synuclein levels and permeability in rat colon.

Increasing evidence suggests that the gut-brain axis plays a crucial role in Parkinson's disease (PD). The abnormal accumulation of aggregated alpha-synuclein (aSyn) in the brain is a key pathological feature of PD. Intracerebral 6-hydroxydopamine (6-OHDA) is a widely used dopaminergic lesion model of PD. It exerts no aSyn pathology in the brain, but changes in the gut have not been assessed. Here, 6-OHDA was administered unilaterally either to the rat medial forebrain bundle (MFB) or striatum. Increased levels of glial fibrillary acidic protein in the ileum and colon were detected at 5 weeks postlesion. 6-OHDA decreased the Zonula occludens protein 1 barrier integrity score, suggesting increased colonic permeability. The total aSyn and Ser129 phosphorylated aSyn levels were elevated in the colon after the MFB lesion. Both lesions generally increased the total aSyn, pS129 aSyn, and ionized calcium-binding adapter molecule 1 (Iba1) levels in the lesioned striatum. In conclusion, 6-OHDA-induced nigrostriatal dopaminergic damage leads to increased aSyn levels and glial cell activation particularly in the colon, suggesting that the gut-brain axis interactions in PD are bidirectional and the detrimental process may start in the brain.

The Prolyl Oligopeptidase Inhibitor KYP-2047 Is Cytoprotective and Anti-Inflammatory in Human Retinal Pigment Epithelial Cells with Defective Proteasomal Clearance.

Increased oxidative stress, dysfunctional cellular clearance, and chronic inflammation are associated with age-related macular degeneration (AMD). Prolyl oligopeptidase (PREP) is a serine protease that has numerous cellular functions, including the regulation of oxidative stress, protein aggregation, and inflammation. PREP inhibition by KYP-2047 (4-phenylbutanoyl-L-prolyl1(S)-cyanopyrrolidine) has been associated with clearance of cellular protein aggregates and reduced oxidative stress and inflammation. Here, we studied the effects of KYP-2047 on inflammation, oxidative stress, cell viability, and autophagy in human retinal pigment epithelium (RPE) cells with reduced proteasomal clearance. MG-132-mediated proteasomal inhibition in ARPE-19 cells was used to model declined proteasomal clearance in the RPEs of AMD patients. Cell viability was assessed using LDH and MTT assays. The amounts of reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescin diacetate (H2DCFDA). ELISA was used to determine the levels of cytokines and activated mitogen-activated protein kinases. The autophagy markers p62/SQSTM1 and LC3 were measured with the western blot method. MG-132 induced LDH leakage and increased ROS production in the ARPE-19 cells, and KYP-2047 reduced MG-132-induced LDH leakage. Production of the proinflammatory cytokine IL-6 was concurrently alleviated by KYP-2047 when compared with cells treated only with MG-132. KYP-2047 had no effect on autophagy in the RPE cells, but the phosphorylation levels of p38 and ERK1/2 were elevated upon KYP-2047 exposure, and the inhibition of p38 prevented the anti-inflammatory actions of KYP-2047. KYP-2047 showed cytoprotective and anti-inflammatory effects on RPE cells suffering from MG-132-induced proteasomal inhibition.

Enhanced drug delivery by a prodrug approach effectively relieves neuroinflammation in mice.

AIMS: Neuroinflammation is a prominent hallmark in several neurodegenerative diseases (NDs). Halting neuroinflammation can slow down the progression of NDs. Improving the efficacy of clinically available non-steroidal anti-inflammatory drugs (NSAIDs) is a promising approach that may lead to fast-track and effective disease-modifying therapies for NDs. Here, we aimed to utilize the L-type amino acid transporter 1 (LAT1) to improve the efficacy of salicylic acid as an example of an NSAID prodrug, for which brain uptake and intracellular localization have been reported earlier. MAIN METHODS: Firstly, we confirmed the improved LAT1 utilization of the salicylic acid prodrug (SA-AA) in freshly isolated primary mouse microglial cells. Secondly, we performed behavioural rotarod, open field, and four-limb hanging tests in mice, and a whole-brain proteome analysis. KEY FINDINGS: The SA-AA prodrug alleviated the lipopolysaccharide (LPS)-induced inflammation in the rotarod and hanging tests. The proteome analysis indicated decreased neuroinflammation at the molecular level. We identified 399 proteins linked to neuroinflammation out of 7416 proteins detected in the mouse brain. Among them, Gps2, Vamp8, Slc6a3, Slc18a2, Slc5a7, Rgs9, Lrrc1, Ppp1r1b, Gnal, and Adcy5/6 were associated with the drug's effects. The SA-AA prodrug attenuated the LPS-induced neuroinflammation through the regulation of critical pathways of neuroinflammation such as the cellular response to stress and transmission across chemical synapses. SIGNIFICANCE: The efficacy of NSAIDs can be improved via the utilization of LAT1 and repurposed for the treatment of neuroinflammation. This improved brain delivery and microglia localisation can be applied to other inflammatory modulators to achieve effective and targeted CNS therapies.

Alcohol Co-Administration Changes Mephedrone-Induced Alterations of Neuronal Activity.

Mephedrone (4-MMC), despite its illegal status, is still a widely used psychoactive substance. Its effects closely mimic those of the classical stimulant drug methamphetamine (METH). Recent research suggests that unlike METH, 4-MMC is not neurotoxic on its own. However, the neurotoxic effects of 4-MMC may be precipitated under certain circumstances, such as administration at high ambient temperatures. Common use of 4-MMC in conjunction with alcohol raises the question whether this co-consumption could also precipitate neurotoxicity. A total of six groups of adolescent rats were treated twice daily for four consecutive days with vehicle, METH (5 mg/kg) or 4-MMC (30 mg/kg), with or without ethanol (1.5 g/kg). To investigate persistent delayed effects of the administrations at two weeks after the final treatments, manganese-enhanced magnetic resonance imaging brain scans were performed. Following the scans, brains were collected for Golgi staining and spine analysis. 4-MMC alone had only subtle effects on neuronal activity. When administered with ethanol, it produced a widespread pattern of deactivation, similar to what was seen with METH-treated rats. These effects were most profound in brain regions which are known to have high dopamine and serotonin activities including hippocampus, nucleus accumbens and caudate-putamen. In the regions showing the strongest activation changes, no morphological changes were observed in spine analysis. By itself 4-MMC showed few long-term effects. However, when co-administered with ethanol, the apparent functional adaptations were profound and comparable to those of neurotoxic METH.

Species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1) -utilizing compound between mice and rats.

The prodrug approach targeting influx transporters has been extensively studied as a means of central nervous system drug delivery. Transporter and enzyme expression, localization and activity may contribute to significant species differences in preclinical studies. However, data about the possible species differences in the intra-brain distribution of transporter utilizing compounds is scarce. Here, we investigated the species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1)-utilizing L-lysine analogue of ketoprofen (KPF) (compound 1) and KPF itself by the whole tissue and brain microdialysis methods in mice, and compared the results to those previously reported in rats. Their pharmacodynamic responses in both species were assessed by measuring the brain prostaglandin E2 (PGE2) levels by LC-MS/MS. The intracellular delivery of compound 1 was much lower in mice than in rats. Higher target site concentrations of compound 1 and released KPF were reflected on a more pronounced effect on PGE2 levels in the rat brain. In conclusion, these results highlight the need for cross-species characterization of prodrug pharmacokinetics and pharmacodynamics in preclinical studies.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone.

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

L-Type Amino Acid Transporter 1 Enables the Efficient Brain Delivery of Small-Sized Prodrug across the Blood-Brain Barrier and into Human and Mouse Brain Parenchymal Cells.

Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorption. In the brain, however, the transporter-mediated drug delivery has not yet been fully achieved due to the complexity of the blood-brain barrier (BBB). Because L-type amino acid transporter 1 (LAT1) is a good candidate to improve the brain delivery, we developed here four novel LAT1-utilizing prodrugs of four nonsteroidal anti-inflammatory drugs. As a result, all the prodrugs were able to cross the BBB and localize into the brain cells. The brain uptake of salicylic acid (SA) was improved five times, not only across the mouse BBB but also into the cultured mouse and human brain cells. The naproxen prodrug was also transported efficiently into the mouse brain achieving less peripheral exposure, but the brain release of naproxen from the prodrug was not improved. Contrarily, the high plasma protein binding of the flurbiprofen prodrug and the premature bioconversion of the ibuprofen prodrug in the mouse blood hindered the efficient brain delivery. Thus, the structure of the parent drug affects the successful brain delivery of the LAT1-utilizing prodrugs, and the small-sized LAT1-utilizing prodrug of SA constituted a successful model to specifically deliver its parent drug across the mouse BBB and into the cultured mouse and human brain cells.

L-type amino acid transporter 1 (LAT1)-utilizing efflux transporter inhibitors can improve the brain uptake and apoptosis-inducing effects of vinblastine in cancer cells.

Efflux transporter-mediated multidrug resistance (MDR) prevents chemotherapeutics to achieve therapeutically relevant concentrations within the cancer cells. Therefore, inhibitors of efflux transporters have been in demand. However, to be safe, these inhibitors are needed to be targeted into the cancer cells. For this purpose, L-type amino acid transporter 1 (LAT1), overexpressed in many different cancer cell types, can be utilized. In the present study, two LAT1-utilizing derivatives of probenecid (PRB) that can inhibit e.g., multiresistance proteins (MRPs) and organic anion transporters (OATs), were studied for their apoptosis-inducing effects in cancer cells alone and a combination with another chemotherapeutic, vinblastine (VBL). Also, their hemocompatibility and off-target toxicity were evaluated. Moreover, the brain uptake rate and extent of VBL together with the most promising LAT1-utilizing efflux inhibitor was studied after in situ rat brain perfusion and after intraperitoneal administration to mice. As a result, these targeted inhibitors increased significantly the apoptosis-inducing effects of VBL and more effectively than PRB itself. They also were hemocompatible and non-toxic in healthy cells with a concentration below 100 µM. Interestingly, the most promising compound doubled the penetration rate of VBL across the rat blood-brain barrier (BBB). This makes it a promising candidate for further studies to improve efflux transporter-related MDR of brain-targeted anti-cancer agents.

Sleep-State Dependent Alterations in Brain Functional Connectivity under Urethane Anesthesia in a Rat Model of Early-Stage Parkinson's Disease.

Parkinson's disease (PD) is characterized by the gradual degeneration of dopaminergic neurons in the substantia nigra, leading to striatal dopamine depletion. A partial unilateral striatal 6-hydroxydopamine (6-OHDA) lesion causes 40-60% dopamine depletion in the lesioned rat striatum, modeling the early stage of PD. In this study, we explored the connectivity between the brain regions in partially 6-OHDA lesioned male Wistar rats under urethane anesthesia using functional magnetic resonance imaging (fMRI) at 5 weeks after the 6-OHDA infusion. Under urethane anesthesia, the brain fluctuates between the two states, resembling rapid eye movement (REM) and non-REM sleep states. We observed clear urethane-induced sleep-like states in 8/19 lesioned animals and 8/18 control animals. 6-OHDA lesioned animals exhibited significantly lower functional connectivity between the brain regions. However, we observed these differences only during the REM-like sleep state, suggesting the involvement of the nigrostriatal dopaminergic pathway in REM sleep regulation. Corticocortical and corticostriatal connections were decreased in both hemispheres, reflecting the global effect of the lesion. Overall, this study describes a promising model to study PD-related sleep disorders in rats using fMRI.

Extracellular prolyl oligopeptidase derived from activated microglia is a potential neuroprotection target.

Prolyl oligopeptidase (PREP) is an abundant peptidase in the brain and periphery, but its physiological functions are still largely unknown. Recent findings point to a role for PREP in inflammatory processes. This study assessed the cellular and extracellular PREP activities in cultures of mouse primary cortical neurons, microglial cells and astrocytes, and immortalized microglial BV-2 cells under neuroinflammatory conditions induced by lipopolysaccharide (LPS) and interferon gamma (IFNγ). Furthermore, we evaluated the neuroprotective effect of a specific PREP inhibitor, KYP-2047, in a neuroinflammation model based on a coculture of primary cortical neurons and activated BV-2 cells. The inflammatory insult reduced intracellular and increased extracellular PREP activity specifically in microglial cells, suggesting that activated microglia excretes active PREP. A targeted proteomics approach revealed up-regulation in PREP protein levels in BV-2 cell growth medium but down-regulation in crude membrane-bound PREP after LPS+IFNγ. In the coculture of BV-2 cells and primary neurons, an increase in extracellular PREP activity was also detected after inflammation. KYP-2047 (10 µmol/L) significantly protected neurons against microglial toxicity and reduced the levels of the pro-inflammatory cytokine tumour necrosis factor alpha. In conclusion, these data point to an extracellular role for microglial PREP in the inflammatory process. Inhibition of PREP during neuroinflammation is a potential target for neuroprotection. Thus, PREP inhibitors may offer a novel therapeutic approach for the treatment of neurodegenerative disorders with an inflammatory component including Parkinson's and Alzheimer's diseases.

Selective adrenergic alpha2C receptor antagonist ameliorates acute phencyclidine-induced schizophrenia-like social interaction deficits in rats.

RATIONALE: Social withdrawal is a core feature of the negative symptoms of schizophrenia. Currently available pharmacotherapies have only limited efficacy towards the negative symptoms, i.e., there is a significant unmet medical need in the treatment of these symptoms. OBJECTIVE: We wanted to confirm whether selective adrenergic α2C receptor (AR) antagonist therapy could ameliorate acute phencyclidine (PCP)-induced schizophrenia-like social interaction deficits in rats, and to compare the effects of an α2C AR antagonist to another putative therapeutic alternative, an α7 nicotinic acetylcholine receptor (nAChR) partial agonist, as well against three commonly used atypical antipsychotics. METHODS: Here, we used acute PCP administration and modified a protocol for testing social interaction deficits in male Wistar rats and then used this model to compare the effects of an α2C AR antagonist (ORM-13070 0.3 and 1.0 mg/kg s.c.) with an α7 nAChR partial agonist (EVP-6124 0.3 mg/kg s.c.) and three atypical antipsychotics (clozapine 2.5 mg/kg i.p., risperidone 0.04 and 0.08 mg/kg s.c., olanzapine 0.125 and 0.5 mg/kg s.c.) on social interaction behavior. RESULTS: Acute PCP (1.5 mg/kg s.c.) produced robust and reproducible deficits in social interaction behavior without affecting locomotor activity. The selective α2C AR antagonist significantly ameliorated PCP-induced social interaction deficits. In contrast, neither the partial α7 nAChR agonist nor any of the three atypical antipsychotics were able to reverse the behavioral deficits at the selected doses. CONCLUSION: Our findings confirm that α2C AR antagonism is a potential mechanism for the treatment of the negative symptoms of schizophrenia.

Brief isoflurane anesthesia regulates striatal AKT-GSK3ß signaling and ameliorates motor deficits in a rat model of early-stage Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder primarily affecting the nigrostriatal dopaminergic system. The link between heightened activity of glycogen synthase kinase 3ß (GSK3ß) and neurodegene-rative processes has encouraged investigation into the potential disease-modifying effects of novel GSK3ß inhibitors in experimental models of PD. Therefore, the intriguing ability of several anesthetics to readily inhibit GSK3ß within the cortex and hippocampus led us to investigate the effects of brief isoflurane anesthesia on striatal GSK3ß signaling in naïve rats and in a rat model of early-stage PD. Deep but brief (20-min) isoflurane anesthesia exposure increased the phosphorylation of GSK3ß at the inhibitory Ser9 residue, and induced phosphorylation of AKTThr308 (protein kinase B; negative regulator of GSK3ß) in the striatum of naïve rats and rats with unilateral striatal 6-hydroxydopamine (6-OHDA) lesion. The 6-OHDA protocol produced gradual functional deficiency within the nigrostriatal pathway, reflected as a preference for using the limb ipsilateral to the lesioned striatum at 2 weeks post 6-OHDA. Interestingly, such motor impairment was not observed in animals exposed to four consecutive isoflurane treatments (20-min anesthesia every 48 h; treatments started 7 days after 6-OHDA delivery). However, isoflurane had no effect on striatal or nigral tyrosine hydroxylase (a marker of dopaminergic neurons) protein levels. This brief report provides promising results regarding the therapeutic potential and neurobiological mechanisms of anesthetics in experimental models of PD and guides development of novel disease-modifying therapies.

Combined ipsilateral limb use score as an index of motor deficits and neurorestoration in parkinsonian rats.

Our aim was to apply a robust non-drug induced sensorimotor test battery to assess the efficacy of neurorestorative therapies on the motor deficits caused by partial unilateral 6-OHDA lesion mimicking early stage PD. Since the 6-OHDA lesion protocols to induce partial DA depletion in striatum vary extensively between laboratories, we evaluated the associations between different intrastriatal 6-OHDA doses (1 X 0-20 and 2 X 0-30 µg), striatal DA depletion (HPLC-ECD) and D-amphetamine induced rotation to identify a lesion protocol that would produce 40-60% striatal DA depletion. Doses ≥ 6 µg produced a significant DA depletion (ANOVA, P < 0.0001). 6-OHDA dose range (6-14 µg) causing 40-60% DA depletion induced very variable rotational responses. Next, intrastriatal 1 × 10 and 1 × 14 µg doses were compared with a full lesion (10 µg into the medial forebrain bundle) with regard to their effects on adjusting step, cylinder, and vibrissae test performance. A combined ipsilateral score (average of each test) was found more sensitive in distinguishing between different lesions than any test alone. Finally, five-week treadmill exercise starting two weeks post-lesion was able to restore impaired limb use (combined score; mixed model, P < 0.05) and striatal DA depletion (ANOVA, P < 0.05) in rats with partial lesion (1 × 10 µg). Notably, D-amphetamine induced rotation significantly decreased between weeks one to seven post-lesion (t-test, P < 0.01). In conclusion, intrastriatal 1 × 10 µg of 6-OHDA produces 40-60% striatal DA depletion robustly, and the combined ipsilateral score provides an efficient means for testing of the efficacy of neurorestorative or neuroprotective treatments for PD. © 2017 Wiley Periodicals, Inc.

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.

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.

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.