Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported. In this study, the kinetics of central KYNA-lowering effect in rats and nonhuman primates (NHPs, Cynomolgus macaques) was investigated using multiple KAT II irreversible inhibitors as pharmacologic probes. Mechanistic pharmacokinetic-pharmacodynamic analysis of in vivo responses to irreversible inhibition quantitatively revealed that 1) KAT II turnover is relatively slow [16-76 hours' half-life (t1/2)], whereas KYNA is cleared more rapidly from the brain (<1 hour t1/2) in both rats and NHPs, 2) KAT II turnover is slower in NHPs than in rats (76 hours vs. 16 hours t1/2, respectively), and 3) the percent contribution of KAT II to KYNA formation is constant (∼80%) across rats and NHPs. Additionally, modeling results enabled establishment of in vitro-in vivo correlation for both enzyme turnover rates and drug potencies. In summary, quantitative translational analysis confirmed the feasibility of central KYNA modulation in humans. Model-based analysis, where system-specific properties and drug-specific properties are mechanistically separated from in vivo responses, enabled quantitative understanding of the KAT II-KYNA pathway, as well as assisted development of promising candidates to test KYNA hypothesis in humans.

Reduction of brain kynurenic acid improves cognitive function.

The elevation of kynurenic acid (KYNA) observed in schizophrenic patients may contribute to core symptoms arising from glutamate hypofunction, including cognitive impairments. Although increased KYNA levels reduce excitatory neurotransmission, KYNA has been proposed to act as an endogenous antagonist at the glycine site of the glutamate NMDA receptor (NMDAR) and as a negative allosteric modulator at the α7 nicotinic acetylcholine receptor. Levels of KYNA are elevated in CSF and the postmortem brain of schizophrenia patients, and these elevated levels of KYNA could contribute to NMDAR hypofunction and the cognitive deficits and negative symptoms associated with this disease. However, the impact of endogenously produced KYNA on brain function and behavior is less well understood due to a paucity of pharmacological tools. To address this issue, we identified PF-04859989, a brain-penetrable inhibitor of kynurenine aminotransferase II (KAT II), the enzyme responsible for most brain KYNA synthesis. In rats, systemic administration of PF-04859989 dose-dependently reduced brain KYNA to as little as 28% of basal levels, and prevented amphetamine- and ketamine-induced disruption of auditory gating and improved performance in a sustained attention task. It also prevented ketamine-induced disruption of performance in a working memory task and a spatial memory task in rodents and nonhuman primates, respectively. Together, these findings support the hypotheses that endogenous KYNA impacts cognitive function and that inhibition of KAT II, and consequent lowering of endogenous brain KYNA levels, improves cognitive performance under conditions considered relevant for schizophrenia.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions.

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

Pharmacokinetics of oral D-serine in D-amino acid oxidase knockout mice.

D-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of D-amino acids including D-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral D-serine, plasma D-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although D-serine levels were rapidly diminished in wild-type mice (t(½) = 1.2 h), sustained drug levels over the course of 4 h (t(½) > 10 h) were observed in mice lacking DAAO. Coadministration of D-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma D-serine levels, although CBIO seems to have only temporary effects on the plasma D-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of D-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma D-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.

Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Alterations in gene regulation following inhibition of the striatum-enriched phosphodiesterase, PDE10A.

PDE10A is a member of the phosphodiesterase superfamily highly enriched within medium spiny neurons (MSN) in mammalian striatum. We have used inhibitors of PDE10A and quantitative measures of mRNA to demonstrate that PDE10A controls striatal gene expression by regulating MSN cyclic nucleotide signaling pathways. Acute treatment with PDE10A inhibitors produces rapid and transient transcription of the immediate early gene cfos in rat striatum. Although inhibition of PDE10A causes accumulation of both cAMP and cGMP, the increase in striatal cfos expression appears to depend on changes in cAMP, since the increase is present in mice deficient in nNOS which fail to increase cGMP in response to PDE10A inhibition. Consistent with its expression in a majority of striatal MSN, PDE10A inhibition significantly induces expression of both substance P and enkephalin, neuropeptide markers for the direct and indirect striatal output pathways, respectively. These findings support the hypothesis that PDE10A modulates signal transduction in both striatal output pathways and suggest that PDE10A inhibitors may offer a unique approach to the treatment of schizophrenia.

Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent D-amino acid oxidase (DAAO) inhibitors.

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Phosphodiesterase 10A inhibitors as a novel therapeutic approach for schizophrenia.

Phosphodiesterase 10A (PDE10A) is a recently identified member of the cyclic nucleotide phosphodiesterase family. PDE10A is notable in that it has a relatively restricted distribution: PDE10A mRNA and protein are expressed primarily in the dopaminoreceptive medium spiny neurons of the striatum. Potent and selective PDE10A inhibitors have recently been described in the scientific literature and their in vivo effects suggest the potential uses of PDE10A inhibitors in CNS disorders, particularly schizophrenia. This article provides a brief overview of the presently available scientific literature as well as recent meeting presentations.

Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species.

A monoclonal antibody directed against the amino terminal of rat phosphodiesterase 10A (PDE10A) was used to localize PDE10A in multiple central nervous system (CNS) and peripheral tissues from mouse, rat, dog, cynomolgus macaque, and human. PDE10A immunoreactivity is strongly expressed in the CNS of these species with limited expression in peripheral tissues. Within the brain, strong immunoreactivity is present in both neuronal cell bodies and neuropil of the striatum, in striatonigral and striatopallidal white matter tracks, and in the substantia nigra and globus pallidus. Outside the brain, PDE10A immunoreactivity is less intense, and distribution is limited to few tissues such as the testis, epididymal sperm, and enteric ganglia. These data demonstrate that PDE10A is an evolutionarily conserved phosphodiesterase highly expressed in the brain but with restricted distribution in the periphery in multiple mammalian species.

Genetic deletion of the striatum-enriched phosphodiesterase PDE10A: evidence for altered striatal function.

PDE10A is a newly identified phosphodiesterase that is highly expressed by the medium spiny projection neurons of the striatum. In order to investigate the physiological role of PDE10A in the central nervous system, PDE10A knockout mice (PDE10A(-/-)) were characterized both behaviorally and neurochemically. PDE10A(-/-) mice showed decreased exploratory activity and a significant delay in the acquisition of conditioned avoidance behavior when compared to wild-type (PDE10A(+/+)) mice. However, in a variety of other well-characterized behavioral tasks, including the elevated plus maze (anxiety), forced swim test (depression), hot plate (nociception) and two memory models (passive avoidance and Morris water maze), PDE10A(-/-) mice performed similarly to wild-type mice. When challenged with PCP or MK-801, PDE10A(-/-) mice showed a blunted locomotor response in comparison to PDE10A(+/+) mice. In contrast, PDE10A(-/-) and PDE10A(+/+) mice responded similarly to the locomotor stimulating effects of amphetamine and methamphetamine. Our findings suggest that PDE10A is involved in regulating striatal output, possibly by reducing the sensitivity of medium spiny neurons to glutamatergic excitation. These results are discussed in relationship to the hypothesis that PDE10A inhibition presents a novel treatment for psychosis.

Expression of rat I-TAC/CXCL11/SCYA11 during central nervous system inflammation: comparison with other CXCR3 ligands.

The chemokines are a large gene superfamily with critical roles in development and immunity. The chemokine receptor CXCR3 appears to play a major role in the trafficking of activated Th1 lymphocytes. There are at least three major ligands for CXCR3: mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, and of these three ligands, CXCL11 is the least well-characterized. In this study, we have cloned a rat ortholog of CXCL11, evaluated its function, and examined its expression in the Th-1-mediated disease, experimental autoimmune encephalomyelitis (EAE) in the rat. Based on its predicted primary amino-acid sequence, rat I-TAC/CXCL11 was synthesized and shown to induce chemotaxis of activated rat T lymphocytes in vitro and the in vivo migration of T lymphocytes when injected into the skin. I-TAC/CXCL11 expression, as determined by RT-PCR, increased in lymph node and spinal cord tissue collected from rats in which EAE had been actively induced, and in spinal cord tissue from rats in which EAE had been passively induced. The kinetics of expression were similar to that of CXCR3 and IP-10/CXCL10, although expression of both CXCR3 and IP-10/CXCL10 was more intense than that of I-TAC/CXCL11 and increased more rapidly in both lymph nodes and the spinal cord. Only minor levels of expression of the related chemokine mig/CXCL9 were observed. Immunohistochemistry revealed that the major cellular source of I-TAC/CXCL11 in the central nervous system (CNS) during EAE is likely to be the astrocyte. Together, these data indicate that I-TAC/CXCL11 is expressed in the CNS during the clinical phase of EAE. However, the observation that I-TAC/CXCL11 is expressed after receptor expression is detected suggests that it is not essential for the initial migration of CXCR3-bearing cells into the CNS.

Immunohistochemical localization of PDE10A in the rat brain.

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.

Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain.

Matrix metalloproteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several central nervous system disorders. Increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer's disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis. This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity. To allow us to evaluate the effect of increased levels of active MMP-9 in the central nervous system, mutant forms of the enzyme were designed to autocatalytically remove the pro domain, yielding active enzyme. This was accomplished by modifying residues in the cysteine switch autoinhibitor region of the propeptide. Stable cell lines and transgenic mice that express G100L and D103N autoactive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.