ABSTRACT: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 CNSactive 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.Active transport mechanisms as determinants of drug absorption, distribution, and clearance have been the focus of considerable research effort over the past decade. Of the numerous transporter proteins recently investigated, the one for which the greatest amount of knowledge exists is P-glycoprotein (MDR1). Originally described as a transporter involved in imparting drug resistance to tumor cells, P-glycoprotein has been demonstrated to be important in reducing absorption of drugs from the intestinal lumen, in active secretion of drugs into urine and bile, and in extrusion of drugs from vital organs such as the brain and reproductive tissues (Troutman et al., 2002). As such, P-glycoprotein-mediated transport has become an important issue in the discovery and development of new drugs. For example, new compounds that are promising with regard to target receptor/ enzyme activity can be severely hampered in their ability to elicit pharmacological effects in vivo should they be good substrates for P-glycoprotein, especially if the route of administration is intended to be oral or the target tissues is one rich in P-glycoprotein activity. Furthermore, the potential for drug-drug interactions arises in the event that the P-glycoprotein substrate is coadministered with another agent that can inhibit P-glycoprotein.Several models have been developed to assess drugs as P-glycoprotein substrates. In vitro models have included the Caco...

Casein kinase I (CKI) 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 ϭ 7.7 Ϯ 2.2 nM) and selective (Ͼ30-fold with respect to 42 additional kinases) inhibitor of CKI in isolated enzyme preparations; in transfected whole cell assays, it caused a concentrationrelated 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 (Ϫ1.97 Ϯ 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 druginduced 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 ϭ 0.46 Ϯ 0.04 h). Together, these results suggest that inhibition of CKI 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.Circadian behavior is mediated by a timed sequence of intracellular events, genomic in nature, occurring in mammals within so-called pacemaker cells of the suprachiasmatic nucleus (SCN) (Antle and Silver, 2005). Here, rhythmicity relies on a common theme of precisely regulated gene transcription and translation as a means to perpetuate cycling, and in doing so, to dictate the timing of downstream events (Reppert and Weaver, 2001; for review, see Schibler, 2006, among others). Clock-related proteins rise and fall in concentration as a consequence of nuclear and cytosolic feedback loops, adjusted daily to maintain reproducible cycling timed at roughly 24-h intervals. Light is the primary zeitgeber, or "time-giver", in this process, and its actions on loop dynamics are thought to comprise the most important external influence on pacemaker function.In its simplest conception, the oscillations of the clock can run using four genes, Period (Per1, Per2) and Cryptochrome (Cry1, Cry2), that are activated in early circadian day by heterodimeric complexes of CLOCK and BMAL1 (Griffin et al., 1999;Kume et al., 1999;Cermakian and Boivin, 2003). Translated PER partners in the cytoplasm with CRY, the resultant heterodimer, readily gaining nuclear entry and opposing activation by Clock and BMAL1. A feedback loop is...

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.

This study was designed 1) to examine the effects of bloodbrain barrier (BBB) permeability [quantified as permeabilitysurface area product (PS)], unbound fraction in plasma (f u,plasma ), and brain tissue (f u,brain ) on the time to reach equilibrium between brain and plasma and 2) to investigate the drug discovery strategies to design and select compounds that can rapidly penetrate the BBB and distribute to the site of action. The pharmacokinetics of seven model compounds: caffeine, CP-141938 [methoxy-3-[(2-phenyl-piperadinyl-3-aminopropranolol, theobromine, and theophylline in rat brain and plasma after subcutaneous administration were studied. The in vivo log PS and log f u,brain calculated using a physiologically based pharmacokinetic model correlates with in situ log PS (R 2 ϭ 0.83) and in vitro log f u,brain (R 2 ϭ 0.69), where the in situ PS and in vitro f u,brain was determined using in situ brain perfusion and equilibrium dialysis using brain homogenate, respectively. The time to achieve brain equilibrium can be quantitated with a proposed parameter, intrinsic brain equilibrium, where V b is the physiological volume of brain. The in vivo log t 1/2eq,in does not correlate with in situ log PS (R 2 Ͻ 0.01) but correlates inversely with log(PS ⅐ f u,brain ) (R 2 ϭ 0.85). The present study demonstrates that rapid brain equilibration requires a combination of high BBB permeability and low brain tissue binding. A high BBB permeability alone cannot guarantee a rapid equilibration. The strategy to select compounds with rapid brain equilibration in drug discovery should identify compounds with high BBB permeability and low nonspecific binding in brain tissue.The blood-brain barrier (BBB) consists of a continuous layer of endothelial cells joined by tight junctions at the cerebral vasculature. It represents a physical and enzymatic barrier to restrict and regulate the penetration of compounds into and out of the brain and maintain the homeostasis of the brain microenvironment. Brain penetration is essential for compounds where the site of action is within the central nervous system (CNS), whereas BBB penetration needs to be minimized for compounds that target peripheral sites to reduce potential CNS-related side effects. Therefore, it is critical during the drug discovery phase to design and select compounds having appropriate brain penetration properties for drug targets that reside within and outside the CNS (Chen et al., 2003a; Golden and Pollack, 2003).The kinetics of brain penetration consists of the extent of brain equilibrium and the time to achieve brain equilibrium. The extent of brain equilibrium is often quantified by brainplasma partition coefficient (K p ), the ratio of total brain concentration and plasma concentration at steady state. This parameter depends upon drug binding in plasma and brain tissue, the uptake and efflux transporters at BBB, metabolism in the brain, and the bulk flow of cerebrospinal fluid (Hammarlund-Udenaes et al., 1997). If active transporters, brain metabolism and the...

This study was designed to evaluate the use of cerebrospinal fluid (CSF) drug concentration and plasma unbound concentration (C(u,plasma)) to predict brain unbound concentration (C(u,brain)). The concentration-time profiles in CSF, plasma, and brain of seven model compounds were determined after subcutaneous administration in rats. The C(u,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 C(u,brain)/C(CSF) and C(u,brain)/C(u,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 C(u,brain)/C(CSF) and C(u,brain)/C(u,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 C(u,brain)/C(CSF) and C(u,brain)/C(u,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, C(CSF) and C(u,plasma) represent C(u,brain) equally well; for efflux substrates or slow brain penetration compounds, C(CSF) appears to be equivalent to or more accurate than C(u,plasma) to represent C(u,brain). Thus, we hypothesize that C(CSF) is equivalent to or better than C(u,plasma) to predict C(u,brain). This hypothesis is supported by the literature data.

ABSTRACT:The role of breast cancer resistance protein (Bcrp) and the combined activities of Bcrp and P-glycoprotein (P-gp, Mdr1a/1b) in limiting the brain penetration of drugs at the blood-brain barrier (BBB) were investigated using wild-type FVB, Mdr1a/1b(؊/؊), (؊/؊), Bcrp(؊/؊), and Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice. Four drugs, flavopiridol, imatinib mesylate (Gleevec), PF-407288, and prazosin, with different transport specificity for BCRP/Bcrp and MDR1/ Mdr1a were selected, and the drug levels in plasma, cerebrospinal fluid, and brain of mice were determined. Flavopiridol and prazosin were identified as substrates for both mouse Bcrp and Mdr1a with greater transport associated with Bcrp. The brain/plasma (B/P) ratios at 0.5 and 2 h in Mdr1a/1b(؊/؊), (؊/؊) and Bcrp(؊/؊) mice were 1-to 2-fold for both compounds, whereas the ratios in Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice were more than 5-fold of those observed in FVB mice. For imatinib, a better substrate of P-gp than Bcrp, the B/P ratios in Bcrp(؊/؊) were comparable to those in FVB mice, whereas the B/P ratios in Mdr1a/1b(؊/؊), (؊/؊) and Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice were more than 4-and 28-fold of those in FVB mice at both time points, respectively. Finally, the Bcrp-specific substrate PF-407288 exhibited comparable B/P ratios in Mdr1a/1b(؊/؊), (؊/؊) and Bcrp(؊/؊) mice and slightly but significantly increased B/P ratios in Mdr1a/ 1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice compared with those in FVB mice. The B/P ratios of compounds in Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice compared with those in Mdr1a/1b(؊/؊), (؊/؊) mice clearly demonstrate that Bcrp impairs the brain penetration of its substrates. Moreover, P-gp and Bcrp at BBB function synergistically to limit the brain penetration of shared substrates.It is widely recognized that the tight junctions between adjacent brain endothelial cells forming the blood-brain barrier (BBB) restrict the entry of compounds by paracellular diffusion from the blood to the brain. Moreover, the transcellular diffusion of compounds through the brain endothelial cells can also be impeded by transmembrane efflux transporters, such as P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These efflux transporters can eliminate xenobiotics from the brain against a concentration gradient, thereby limiting central nervous system (CNS) exposure to these compounds. Indeed, the prominent effect of P-gp at the BBB is well established, and P-gp is functionally important in limiting the brain penetration of its substrates (Schinkel et al., 1994;Chen et al., 2003;Scherrmann, 2005). Like P-gp, BCRP is another major member of the ATP-binding cassette family of drug transporters and is highly expressed in the BBB as well. BCRP has been found at the luminal side of human brain capillary endothelial cells (Cooray et al., 2002), and the mRNA level of mouse analog Bcrp was ϳ700 times greater in brain microvessels than in the cortex of the mice (Cisternino et al., 2004), implying that mouse Bcrp may play a key role at the BBB. B...

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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.