Stacey L. Becker

Evaluation of Cerebrospinal Fluid Concentration and Plasma Free Concentration As a Surrogate Measurement for Brain Free Concentration

This study was designed to evaluate the use of cerebrospinal fluid (CSF) drug concentration and plasma unbound concentration (Cu,plasma) to predict brain unbound concentration (Cu,brain). The concentration-time profiles in CSF, plasma, and brain of seven model compounds were determined after subcutaneous administration in rats. The Cu,brain was estimated from the product of total brain concentrations and unbound fractions, which were determined using brain tissue slice and brain homogenate methods. For theobromine, theophylline, caffeine, fluoxetine, and propranolol, which represent rapid brain penetration compounds with a simple diffusion mechanism, the ratios of the area under the curve of Cu,brain/CCSF and Cu,brain/Cu,plasma were 0.27 to 1.5 and 0.29 to 2.1, respectively, using the brain slice method, and were 0.27 to 2.9 and 0.36 to 3.9, respectively, using the brain homogenate method. A P-glycoprotein substrate, CP-141938 (methoxy-3-[(2-phenyl-piperadinyl-3-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide), had Cu,brain/CCSF and Cu,brain/Cu,plasma ratios of 0.57 and 0.066, using the brain slice method, and 1.1 and 0.13, using the brain homogenate method, respectively. The slow brain-penetrating compound, N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl-]sarcosine, had Cu,brain/CCSF and Cu,brain/Cu,plasma ratios of 0.94 and 0.12 using the brain slice method and 0.15 and 0.018 using the brain homogenate method, respectively. Therefore, for quick brain penetration with simple diffusion mechanism compounds, CCSF and Cu,plasma represent Cu,brain equally well; for efflux substrates or slow brain penetration compounds, CCSF appears to be equivalent to or more accurate than Cu,plasma to represent Cu,brain. Thus, we hypothesize that CCSF is equivalent to or better than Cu,plasma to predict Cu,brain. This hypothesis is supported by the literature data.

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.

Evaluation of the Utility of Brain Slice Methods to Study Brain Penetration

The objective of this study was to evaluate the utility of brain tissue slices to determine the effect of plasma and brain tissue nonspecific binding on the brain-to-plasma ratio (Kp). Mouse or rat brain slices (400 μm) were prepared using a McIlwain tissue chopper (Surrey, UK) and incubated with 1 μg/ml of compound at 37°C either in a physiological buffer to determine the buffer-to-slice concentration ratio, i.e., unbound fraction in brain tissue (fu,slice), or in plasma to determine the slice-to-plasma concentration ratio (Cslice/Cplasma). The unbound fraction in plasma, fu,plasma, was determined using equilibrium dialysis. In vitro-in vivo correlation of the brain-to-plasma ratio was examined for 13 and eight model compounds in mice and rats, respectively. Cslice/Cplasma and fu,plasma/fu,slice predicted the Kp in rats, and Cslice/Cplasma predicted the Kp in FVB mice for non-P-glycoprotein substrates within 3-fold but overpredicted Kp for P-glycoprotein substrates by more than 3-fold. However, Cslice/Cplasma predicted the Kp in mdr1a/1b knockout mice for both non-P-glycoprotein and P-glycoprotein substrates. Our present study demonstrates that a brain slice method can be used to differentiate whether a compound having a low Kp is due to the effect of low nonspecific binding to brain tissue relative to plasma proteins or because of efflux transport at the blood-brain barrier.

Metabolism-Directed Design of Oxetane-Containing Arylsulfonamide Derivatives as γ-Secretase Inhibitors

A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aβ in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.

Cerebrospinal Fluid Amyloid-β (Aβ) as an Effect Biomarker for Brain Aβ Lowering Verified by Quantitative Preclinical Analyses

Reducing the generation of amyloid-β (Aβ) in the brain via inhibition of β-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimers disease. For the discovery and development of β-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aβ in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aβ lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aβ lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aβ lowering is related to that for brain Aβ through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aβ as an effect biomarker for brain Aβ lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aβ lowering in the brain.

Application of structure-based drug design and parallel chemistry to identify selective, brain penetrant, in vivo active phosphodiesterase 9A inhibitors.

Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimers disease. Our clinical candidate, PF-04447943 (2), demonstrated acceptable CNS permeability in rats with modest asymmetry between central and peripheral compartments (free brain/free plasma = 0.32; CSF/free plasma = 0.19) yet had physicochemical properties outside the range associated with traditional CNS drugs. To address the potential risk of restricted CNS penetration with 2 in human clinical trials, we sought to identify a preclinical candidate with no asymmetry in rat brain penetration and that could advance into development. Merging the medicinal chemistry strategies of structure-based design with parallel chemistry, a novel series of PDE9A inhibitors was identified that showed improved selectivity over PDE1C. Optimization afforded preclinical candidate 19 that demonstrated free brain/free plasma ≥ 1 in rat and reduced microsomal clearance along with the ability to increase cyclic guanosine monophosphosphate levels in rat CSF.

Pharmacodynamics and Pharmacokinetics of the γ-Secretase Inhibitor PF-3084014

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Aβ. To further characterize Aβ dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

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.

Quantitative Pharmacokinetic/Pharmacodynamic Analyses Suggest That the 129/SVE Mouse Is a Suitable Preclinical Pharmacology Model for Identifying Small-Molecule γ-Secretase Inhibitors

Alzheimers disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of β- or γ-secretase, key enzymes for the production of amyloid β (Aβ), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aβ species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aβ lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aβ across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aβx-42 and CSF Aβx-40 in the 129/SVE mouse is indicative of that for human CSF Aβ. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aβ data are established.

Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Aβ. To further characterize Aβ dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.