6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

Cleavage of the amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) is the first proteolytic step in the production of amyloid-β (Aβ), the principal component of senile plaques in Alzheimer's disease (AD). While inhibition of BACE1 represents one of the promising therapeutic strategies for AD, development of centrally active, small molecule inhibitors have been challenging. Here we describe in vitro and in vivo characterization of a novel, brain-penetrant BACE1 inhibitor. Potency of the compound was evaluated in various cell-free and whole-cell assays. In vivo Aβ-lowering was assessed in wild-type (WT), Tg2576 transgenic and P-glycoprotein knockout (Pgp KO) mice at 3 hours following subcutaneous injection of the compound (1, 3, 10, 30, 100, 300 mg/kg doses) or vehicle. Brain, plasma and CSF samples were harvested for measurement of Aβx-40 and Aβx-42 by sandwich ELISAs. Drug exposures were determined in brain and plasma samples by LC-MS. The compound potently inhibited total Aβ and sAPPβ production in whole-cell assays (H4 cells) overexpressing human WT APP with IC50 values of 30 and 56 nM, respectively. In WT mice, subcutaneous administration of the BACE inhibitor resulted in dose-dependent brain Aβ lowering at 3h post-drug, with significant effects observed at 100 and 300 mg/kg doses (Aβx-40 reduction of 39% and 59%, and Aβx-42 reduction of 44% and 57%, respectively). These doses also significantly lowered CSF Aβx-40 in the same animals (43% and 54%, respectively). In line with other reports, we found that equivalent doses of the compound were less efficacious at reducing Aβ in the Tg2576 mice compared to WT mice. Aβ-lowering was most robust, although still not complete, in the Pgp KO mice, with maximum effect of 65% Aβx-40 reduction at 300 mg/kg dose. At this dose, free drug brain exposures were greatly in excess of in vitro IC50 values. Present studies demonstrate robust central Aβ reduction in wild-type mice by acute administration of a novel brain penetrant BACE1 inhibitor. Analysis of pharmacokinetic-pharmacodynamic relationships suggest that, unlike the effect of γ-secretase, in vivo BACE1 inhibition may results in an incomplete reduction of Aβ.

BACE1 is responsible for the first step of amyloidogenic cleavage of the amyloid precursor protein (APP), leading to production of amyloid-β (Aβ), the principal component of senile plaques in Alzheimer's disease. Several in vitro assays have been employed to measure compound potency in inhibiting BACE1, such as enzyme activity assay (EAA) and whole-cell assays (WCA) with cells overexpressing wild-type (WT) or mutant APP. To expedite early discovery of BACE1 inhibitors, it is critical to address two issues: which in vitro potency measure best predicts in vivo efficacy and how the relevant in vitro potency translates to in vivo efficacy. A series of compounds were profiled for IC50's for BACE1 inhibition in the EAA, WT-WCA, and mutant-WCA using ELISAs. A subset of the series was tested in WT and Pgp knockout mice for the effects on brain Aβ40 and Aβ42 and the associated drug exposures at 3 hrs after a single dose. We then (1) examined the correlation between any two in vitro potency measures of the series, (2) inspected the relationships of the brain Aβ lowering and the respective exposures normalized by various potency measures (in vitro-in vivo correlation, IVIVC) and identified the best IVIVC, and (3) analyzed the best IVIVC quantitatively to understand in vitro-in vivo translation. Within the potency range of up to 2500 nM, the WT-WCA and mutant-WCA IC50's are modestly correlated (R2 = 0.8); both of them are only weakly correlated with EAA IC50 (R2 < 0.45). The IVIVC inspection found that free brain drug exposure (Cb,u) is more relevant than total brain exposure and that the EAA IC50 best predicts in vivo effect of brain Aβ reduction. Further quantitative analyses of the brain Aβ-Cb,u/EAA IC50 correlation suggested that the maximum brain Aβ reduction under those experimental settings was about 70% and that 50% of reduction from baseline was achieved at Cb,u equivalent to EAA IC50. An IVIVC has been established among brain Aβ reduction, free brain drug exposure, and enzyme activity assay IC50. This correlation is instrumental in expediting early discovery of BACE1 inhibitors.

By utilizing structure-based drug design (SBDD) knowledge, a novel class of phosphodiesterase (PDE) 10A inhibitors was identified. The structure-based drug design efforts identified a unique “selectivity pocket” for PDE10A inhibitors, and interactions within this pocket allowed the design of highly selective and potent PDE10A inhibitors. Further optimization of brain penetration and drug-like properties led to the discovery of 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-2545920). This PDE10A inhibitor is the first reported clinical entry for this mechanism in the treatment of schizophrenia.

PDE9A is a cGMP-specific phosphodiesterase widely expressed in the mammalian central nervous system. PDE9A inhibitors are hypothesized to regulate cGMP signaling pathways involved in synaptic plasticity and, therefore, such inhibitors are proposed as a novel approach to remediate the synaptic dysfunction at the core of Alzheimer's disease (AD). PF-04447943 is a novel, selective PDE9A inhibitor being evaluated for the treatment of AD. Here we examine the effect of PDE9 inhibition on cerebrospinal fluid (CSF) cGMP in rats and monkeys and evaluate its potential as a biomarker in humans. Systemically administered PDE9 inhibitors were used to examine the effect of PDE9 inhibition on CSF cGMP in rats and monkeys. Following administration of a single dose of PDE9 inhibitor in rats and monkeys, plasma and CSF were collected and analyzed for drug and cGMP (CSF only). Pharmacokinetic/pharmacodynamic analyses, performed using both drug concentration data in plasma (and/or CSF) and CSF cGMP, were used to predict human exposures associated with potential modulation of CSF cGMP. After safety and tolerability of single-dose PF-04447943 was established in healthy volunteers, a separate cohort of healthy volunteers was dosed to measure the effect of PF-04447943 on CSF cGMP. cGMP was increased in rats, monkeys and humans following drug administration. In rats, PF-04447943 was rapidly distributed to CSF and had similar Tmax values for plasma and CSF drug levels, and CSF cGMP. Maximal increases in CSF cGMP were over 2-fold (change from baseline). In the monkey model, a delay in the increase of CSF cGMP as compared to maximal CSF drug concentrations was observed. In healthy volunteers, PF-04447943 was rapidly absorbed but showed a lag in distribution to CSF and mild hysteresis in the CSF cGMP response, similar to that observed in the monkey. At 6 hours post-dose, CSF cGMP had increased over 3-fold, greater than predicted from the rat model. In humans, PF-04447943 caused a significant increase in CSF cGMP levels, which was slightly greater than predicted from rat models. Based on these findings, PF-04447943 is a functional PDE9A inhibitor in humans with potential as a therapeutic agent for the treatment of AD.

P-glycoprotein is considered to be a major factor impeding effective drug therapy for many diseases of the central nervous system (CNS). Thus, efforts are being made to gain a better understanding of P-glycoprotein's role in drug distribution to brain parenchyma and cerebrospinal fluid (CSF). The goal of this study was to validate and introduce a novel P-glycoprotein-deficient (ABCB1-1Delta) canine model for studying P-glycoprotein-mediated effects of drug distribution to brain tissue and CSF. CSF concentrations of drug are often used to correlate efficacy of CNS drug therapy as a surrogate for determining drug concentration in brain tissue. A secondary goal of this study was to investigate the validity of using CSF concentrations of P-glycoprotein substrates to predict brain tissue concentrations. Loperamide, an opioid that is excluded from the brain by P-glycoprotein, was used to confirm a P-glycoprotein-null phenotype in the dog model. ABCB1-1Delta dogs experienced CNS depression following loperamide administration, whereas ABCB1 wild-type dogs experienced no CNS depression. In summary, we have validated a novel P-glycoprotein-deficient canine model and have used the model to investigate transport of the P-glycoprotein substrate (99m)Tc-sestamibi at the blood-brain barrier and blood-CSF barrier.

Casein kinase Iepsilon (CKIepsilon) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC(50) = 7.7 +/- 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (-1.97 +/- 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.

Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.

The cation-exchange behavior of a number of elements in concentrated HClO4-HCl mixtures has been studied with a Dowex 50 resin. Adsorption data as a function of acid composition and application of the data to column separations are given. Activity coefficients of HCl and HClO4 in the resin phase have been determined for two- and three-component aqueous systems. Some implications of the results for determination of complex constants of species in the aqueous phase are discussed.

An adsorbent based on “filling” the macropores of activated carbon with tin oxide was prepared and characterized. This material, as well as hydrous tin oxide, has an unsually high selectivity for lithium permitting its separation from the other alkali metals. This selectivity is not shown by a number of other hydrous oxides, such as those of Al(III), Fe(III), Zr(IV), and Nb(V).

Results of a systematic study of the cation exchange behavior of elements in HBr solutions are given for a Dowex 50 resin. Many elements show adsorption minima at moderate HBr concentrations folowed by a rapid increase in adsorbability at high HBr concentrations. Marked differences in adsorbabilities of the elements occur, particularly in dilute and concentrated HBr solutions. Implications of the data for separations are discussed and typical separations illustrated.

A procedure is described for separating silver from a number of elements by partition chromatography in nitric acid solutions. A small column of a finely divided polyfluorocarbon impregnated with a highly selective extractant for Ag(I) (triisooctyl thiophosphate in carbon tetrachloride) is used to effect separation.

This chapter presents chromatography as a branch of science that bridges a century of science and discovery. Chromatography represents the premier analytical method of the 20th century for the advancement of a variety of the disciplines of science. Chromatography has grown over the past century to be a vigorous enterprise of scientists; societies with the background of their patrons from government, academia, and research institutions; and the corporate sector and private foundations. The scientific community and public at large has benefited from these interactions. Chromatography as one of the separation sciences has become a common denominator for analytical methods and biological/medical sciences research. They have caused many changes in other sciences, but particularly in analytical chemistry—a change from the determination of one or several components in a sample to the separation, detection, and the quantitative measurement of all the components in a sample.

A cation exchange procedure is described for recovering microcurie amounts of carrier-free UX1 (234Th) from uranyl nitrate solution. Two ion exchange columns are employed, one for initial concentration and the other for final purification.

An anion exhange procedure is described for separating uranium, neptunium and plutonium fro each other and from several non-adsorbed elements.

A cation exchange procedure is described for separating barium and radium. Separation is achieved with EDTA solutions of controlled pH.