Challenges in Permeability Assessment for Oral Drug Product Development.

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed.

Analysis of the Bile Acid Composition in a Fibroblast Growth Factor 19-Expressing Liver-Humanized Mouse Model and Its Use for CYP3A4-Mediated Drug-Drug Interaction Studies.

Numerous biomedical applications have been described for liver-humanized mouse models, such as in drug metabolism or drug-drug interaction (DDI) studies. However, the strong enlargement of the bile acid (BA) pool due to lack of recognition of murine intestine-derived fibroblast growth factor-15 by human hepatocytes and a resulting upregulation in the rate-controlling enzyme for BA synthesis, cytochrome P450 (CYP) 7A1, may pose a challenge in interpreting the results obtained from such mice. To address this challenge, the human fibroblast growth factor-19 (FGF19) gene was inserted into the Fah-/- , Rag2-/- , Il2rg-/- NOD (FRGN) mouse model, allowing repopulation with human hepatocytes capable of responding to FGF19. While a decrease in CYP7A1 expression in human hepatocytes from humanized FRGN19 mice (huFRGN19) and a concomitant reduction in BA production was previously shown, a detailed analysis of the BA pool in these animals has not been elucidated. Furthermore, there are sparse data on the use of this model to assess potential clinical DDI. In the present work, the change in BA composition in huFRGN19 compared with huFRGN control animals was systematically evaluated, and the ability of the model to recapitulate a clinically described CYP3A4-mediated DDI was assessed. In addition to a massive reduction in the total amount of BA, FGF19 expression in huFRGN19 mice resulted in significant changes in the profile of various primary, secondary, and sulfated BAs in serum and feces. Moreover, as observed clinically, administration of the pregnane X receptor agonist rifampicin reduced the oral exposure of the CYP3A4 substrate triazolam. SIGNIFICANCE STATEMENT: Transgenic expression of FGF19 normalizes the unphysiologically high level of bile acids in a chimeric liver-humanized mouse model and leads to massive changes in bile acid composition. These adaptations could overcome one of the potential impediments in the use of these mouse models for drug-drug interaction studies.

Novel Developments to Enable Treatment of CNS Diseases with Targeted Drug Delivery.

The blood-brain barrier (BBB) is a major hurdle for the development of systemically delivered drugs against diseases of the central nervous system (CNS). Because of this barrier there is still a huge unmet need for the treatment of these diseases, despite years of research efforts across the pharmaceutical industry. Novel therapeutic entities, such as gene therapy and degradomers, have become increasingly popular in recent years, but have not been the focus for CNS indications so far. To unfold their full potential for the treatment of CNS diseases, these therapeutic entities will most likely have to rely on innovative delivery technologies. Here we will describe and assess approaches, both invasive and non-invasive, that can enable, or at least increase, the probability of a successful drug development of such novel therapeutics for CNS indications.

Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

OBJECTIVE: In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. METHODS: To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. RESULTS: Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. CONCLUSION: Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

PBPK model for antibody disposition in mouse brain: validation using large-pore microdialysis data.

The objective of this manuscript was to validate a physiologically-based pharmacokinetic (PBPK) model developed to characterize brain pharmacokinetics (PK) of monoclonal antibodies (mAbs) using novel large-pore microdialysis data generated in mice. To support this objective, brain, CSF, and ISF PK of a human anti-tetanus toxin (TeTx) antibody was measured in mice following intraperitoneal (IP) administration. This antibody has no binding in mice. In addition, our recently published mouse brain PK data generated following intravenous (IV) and IP administration of trastuzumab in mice, and other published PK data for brain disposition of antibody in mice, were used to evaluate the PBPK model. All the model parameters were obtained from literature or kept the same as in our previously published manuscript. The revised PBPK model was able to characterize the PK of antibodies in mice brain, CSF, and ISF reasonably well (i.e., within a three-fold error). However, a priori selected parameters led to underprediction of ISF PK during the initial phase of the profile. A local sensitivity analysis suggested that minor changes in several brain-related parameters can help overcome this discrepancy, where an increase in the convective flow of antibodies across BBB was found to be the most parsimonious way to capture all the PK profiles well. However, the presence of this pathway needs further validation. As such, here we have presented an improved PBPK model to characterize and predict the PK of mAbs in different regions of the mouse brain following systemic administration. This model can serve as a quantitative tool to facilitate the discovery, preclinical evaluation, and preclinical-to-clinical translation of novel antibodies targeted against CNS disorders.

Mucin-Protected Caco-2 Assay to Study Drug Permeation in the Presence of Complex Biorelevant Media.

The poor solubility and permeability of compounds beyond Lipinski's Rule of Five (bRo5) are major challenges for cell-based permeability assays. Due to their incompatibility with gastrointestinal components in biorelevant media, the exploration of important questions addressing food effects is limited. Thus, we established a robust mucin-protected Caco-2 assay to allow the assessment of drug permeation in complex biorelevant media. To do that, the assay conditions were first optimized with dependence of the concentration of porcine mucin added to the cells. Mucin-specific effects on drug permeability were evaluated by analyzing cell permeability values for 15 reference drugs (BCS class I-IV). Secondly, a sigmoidal relationship between mucin-dependent permeability and fraction absorbed in human (fa) was established. A case study with venetoclax (BCS class IV) was performed to investigate the impact of medium complexity and the prandial state on drug permeation. Luminal fluids obtained from the tiny-TIM system showed a higher solubilization capacity for venetoclax, and a better read-out for the drug permeability, as compared to FaSSIF or FeSSIF media. In conclusion, the mucin-protected Caco-2 assay combined with biorelevant media improves the mechanistic understanding of drug permeation and addresses complex biopharmaceutical questions, such as food effects on oral drug absorption.

Tau in the brain interstitial fluid is fragmented and seeding-competent.

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood-brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.

Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Discovery of Novel Aminotetralines and Aminochromanes as Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors.

The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7 S,8 R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7 S,8 R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7 S,8 R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.

De Novo Design, Synthesis, and Biological Evaluation of 3,4-Disubstituted Pyrrolidine Sulfonamides as Potent and Selective Glycine Transporter 1 Competitive Inhibitors.

The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate (NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug-drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure-activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

Evaluation of organic anion transporting polypeptide 1B1 and 1B3 humanized mice as a translational model to study the pharmacokinetics of statins.

Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography-tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.

The two faces of the pharmacological interaction of mGlu2 and 5-HT2A - relevance of receptor heterocomplexes and interaction through functional brain pathways.

Important functional interactions between the metabotropic glutamate 2 (mGlu2) and 5-hydroxytryptamine2A (5-HT2A) neurotransmitter receptors have been established based on electrophysiological, biochemical and behavioral evidence. Over the last several years, dimerization between 5-HT2A and mGlu2 receptors has been proposed to account for the functional cross-talk between these two receptors in the prefrontal cortex. The pros and cons for the existence of a heteromeric complex between 5-HT2A and mGlu2 receptors will be reviewed here. First, the fundamental criteria needing to establish evidence for heteromeric complexes will be reviewed. Then, the in vitro evidence for and against heteromeric complexes between 5-HT2A and mGlu2 receptors will be discussed in regard to physical and functional interactions. Finally, the data with native in situ mGlu2 and 5-HT2A receptors will be discussed with respect to whether heteromeric complexes or a simple functional interaction between two distinct GPCRs based on brain network activity is the more simple explanation for a range of in vivo data.

Synthetic Aß oligomers (Aß(1-42) globulomer) modulate presynaptic calcium currents: prevention of Aß-induced synaptic deficits by calcium channel blockers.

Alzheimer's disease is accompanied by increased brain levels of soluble amyloid-ß (Aß) oligomers. It has been suggested that oligomers directly impair synaptic function, thereby causing cognitive deficits in Alzheimer's disease patients. Recently, it has been shown that synthetic Aß oligomers directly modulate P/Q-type calcium channels, possibly leading to excitotoxic cascades and subsequent synaptic decline. Using whole-cell recordings we studied the modulation of recombinant presynaptic calcium channels in HEK293 cells after application of a stable Aß oligomer preparation (Aß1-42 globulomer). Aß globulomer shifted the half-activation voltage of P/Q-type and N-type calcium channels to more hyperpolarized values (by 11.5 and 7.5 mV). Application of non-aggregated Aß peptides had no effect. We then analyzed the potential of calcium channel blockers to prevent Aß globulomer-induced synaptic decline in hippocampal slice cultures. Specific block of P/Q-type or N-type calcium channels with peptide toxins completely reversed Aß globulomer-induced deficits in glutamatergic neurotransmission. Two state-dependent low molecular weight P/Q-type and N-type calcium channel blockers also protected neurons from Aß-induced alterations. On the contrary, inhibition of L-type calcium channels failed to reverse the deficit. Our data show that Aß globulomer directly modulates recombinant P/Q-type and N-type calcium channels in HEK293 cells. Block of presynaptic calcium channels with both state-dependent and state-independent modulators can reverse Aß-induced functional deficits in synaptic transmission. These findings indicate that presynaptic calcium channel blockers may be a therapeutic strategy for the treatment of Alzheimer's disease.

Establishment of a secondary screening assay for P/Q-type calcium channel blockers.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steady-state inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and--if necessary--a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

Heterocomplex formation of 5-HT2A-mGlu2 and its relevance for cellular signaling cascades.

Dopamine, serotonin and glutamate play a role in the pathophysiology of schizophrenia. In the brain a functional crosstalk between the serotonin receptor 5-HT(2A) and the metabotropic glutamate receptor mGlu(2) has been demonstrated. Such a crosstalk may be mediated indirectly through neuronal networks or directly by receptor oligomerization. A direct link of the 5-HT(2A)-mGlu(2) heterocomplex formation to receptor function, i.e. to intracellular signaling, has not been fully demonstrated yet. Here we confirm the formation of 5-HT(2A)-mGlu(2) heterocomplexes using quantitative Snap/Clip-tag based HTRF methods. Additionally, mGlu(2) formed complexes with 5-HT(2B) and mGlu(5) but not 5-HT(2C) indicating that complex formation is not specific to the 5-HT(2A)-mGlu(2) pair. We studied the functional consequences of the 5-HT(2A)-mGlu(2) heterocomplex addressing cellular signaling pathways. Co-expression of receptors in HEK-293 cells had no relevant effects on signaling mediated by the individual receptors when mGlu(2) agonists, antagonists and PAMs, or 5-HT(2A) hallucinogenic and non-hallucinogenic agonists and antagonists were used. Hallucinogenic 5-HT(2A) agonists induced signaling through G(q/11), but not G(i) and thus did not lead to modulation of intracellular cAMP levels. In membranes of the medial prefrontal cortex [(3)H]-LY341495 binding competition of mGlu(2/3) agonist LY354740 was not influenced by 2,5-dimethoxy-4-iodoamphetamine (DOI). Taken together, the formation of GPCR heterocomplexes does not necessarily translate into second messenger effects. These results do not put into question the well-documented functional cross-talk of the two receptors in the brain, but do challenge the biological relevance of the 5-HT(2A)-mGlu(2) heterocomplex.

NOGO-66 receptor deficient mice show slow acquisition of spatial memory task performance.

The Nogo-66 receptor (NgR1) is part of a co-receptor complex on neurons that transmits a signal for inhibition of neurite outgrowth. In addition, NgR1 function has also been related to other disorders such as schizophrenia and Alzheimer's disease. Here, we studied the effect of life-long deletion of NgR1 (ngr(-/-)) in tests for cognition and positive symptoms of schizophrenia. In the water maze, ngr(-/-) mice learned to locate the hidden platform as well as wild type mice, although with slower acquisition. Deletion of NgR1 did not affect amphetamine- or phencyclidine (PCP)-induced hyperactivity, two models of positive symptoms of schizophrenia. Taken together, ngr(-/-) animals show slower acquisition of a spatial learning and memory task.