Search

A novel ketone ester, bis hexanoyl (R)-1,3-butanediol (BH-BD), has been developed as a means to elevate blood ketones, for use as an energy substrate and a signaling metabolite. The metabolism of BH-BD and its effects on blood beta-hydroxybutyrate (BHB) levels was evaluated in various in vitro matrices and through analysis of plasma collected from Sprague Dawley rats and C57/BL6 mice in two oral gavage studies. A well-characterized ketone ester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (HB–BHB), was used as an active control throughout. In vitro assay results demonstrated that BH-BD likely remains intact in the stomach and is hydrolyzed in the small intestine into hexanoate and (R)-1,3-butanediol. If absorbed intact, BH-BD is subject to hydrolysis by non-CYP enzymes in liver and esterases in plasma. If BH-BD reaches the lower intestine it is metabolized by gut flora. Plasma BHB delivery increased in a dose-dependent manner in rats and mice following oral administration of BH-BD. All doses of BH-BD were well tolerated. At doses over 3 g/kg, BHB delivery was similar between BH-BD and HB-BHB. The results of these studies support the hydrolysis of BH-BD into hexanoate and (R)-1,3-butanediol which are metabolized into BHB, delivering a well-tolerated, sustained and dose-dependent increase in plasma BHB in rodents.

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, confirmin...

The discovery of two histamine H3 antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, “best-in-class” molecules.

Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.

Mealey, K.L., Waiting, D., Raunig, D.L., Schmidt, K.R., Nelson, F.R. Oral bioavailability of P-glycoprotein substrate drugs do not differ between ABCB1-1Δ and ABCB1 wild type dogs. J. vet. Pharmacol. Therap. 33, 453–460. Previous studies have indicated that intestinal P-glycoprotein (P-gp) limits the oral bioavailability of substrate drugs and alters systemic pharmacokinetics. In this study, dogs lacking functional P-gp were used to determine the contribution of P-gp to the oral bioavailability and systemic pharmacokinetics of several P-gp substrate drugs. The P-gp substrates quinidine, loperamide, nelfinavir, cyclosporin and the control (non P-gp substrate) drug diazepam were individually administered intravenously and per os to ABCB1-1Δ dogs, which have a P-gp null phenotype and ABCB1 wildtype dogs. ABCB1-1Δ dogs have been shown to have greater brain penetration of P-gp substrates, but limited information is available regarding oral bioavailability of P-gp substrate drugs in this animal model. Plasma drug concentration vs. time curves were generated and pharmacokinetic parameters were calculated for each drug. There were no differences in oral bioavailability between ABCB1-1Δ dogs and ABCB1 wildtype dogs for any of the drugs studied, suggesting that intestinal P-gp does not significantly affect intestinal absorption of these particular substrate drugs in ABCB1-1Δ dogs. However, small sample sizes and individual variability in CYP enzyme activity may have affected the power of the study to detect the impact of P-gp on oral bioavailability.

By use of chemical enablement and prospective design, a novel series of selective, brain penetrant PDE9A inhibitors have been identified that are capable of producing in vivo elevations of brain cGMP.

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.

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.

Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimer's 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.

The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, "best-in-class" molecules.

Increasing beta-amyloid (Abeta) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Abeta clearance by facilitating transport of Abeta out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Abeta acutely, with reduction in plaque pathology after chronic treatment. Thus Abeta levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Abeta levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Abeta. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Abeta were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Abeta (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Abeta levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

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.

The central nervous system (CNS) distribution and transport mechanisms of the investigational drug candidate CP-615,003 (N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide) and its active metabolite CP-900,725 have been characterized. Brain distribution of CP-615,003 and CP-900,725 was low in rats and mice (brain-to-serum ratio0.2). Cerebrospinal fluid (CSF)-to-serum ratios of CP-615,003 were 6- to 8-fold lower than the plasma unbound fraction in rats and dogs. In vitro, CP-615,003 displayed quinidine-like efflux in MDR1-expressing Madin-Darby canine kidney II cells. The brain-to-serum ratio of CP-615,003 in mdr1a/1b (-/-) mice was approximately 7 times that in their wild-type counterparts, confirming that impaired CNS distribution was explained by P-gp efflux transport. In contrast, P-gp efflux did not explain the impaired CNS penetration of CP-900,725. Intracerebral microdialysis was used to characterize rat brain extracellular fluid (ECF) distribution. Interestingly, the ECF-to-serum ratio of the P-gp substrate CP-615,003 was 7-fold below the CSF-to-serum ratio, whereas this disequilibrium was not observed for CP-900,725. In a clinical study, steady-state CSF exposures were measured after administration of 100 mg of CP-615,003 b.i.d. The human CSF-to-plasma ratios of CP-615,003 and CP-900,725 were both approximately 10-fold below their ex vivo plasma unbound fractions, confirming impaired human CNS penetration. Preliminary estimates of CNS receptor occupancy from human CSF concentrations were sensitive to assumptions regarding the magnitude of the CSF-ECF gradient for CP-615,003 in humans. In summary, this case provides an example of intersite differences in CNS pharmacokinetics of a P-gp substrate and potential implications for projection of human CNS receptor occupancy of transporter substrates from CSF pharmacokinetic data when direct imaging-based approaches are not feasible.

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.

This study was designed 1) to examine the effects of blood-brain barrier (BBB) permeability [quantified as permeability-surface 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-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide], fluoxetine, NFPS [N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine], propranolol, 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) = 0.83) and in vitro log f(u,brain) (R(2) = 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 half-life [t(1/2eq,in) = V(b)ln2/(PS . f(u,brain))], 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) < 0.01) but correlates inversely with log(PS . f(u,brain)) (R(2) = 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.

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.

Estradiol-17β (E 2 ) enhances the response of rat pituitary gonadotrophs to gonadotrophin-releasing hormone (GnRH) in vitro . This effect of E 2 on rat gonadotrophs in vitro was applied as a model to study the estrogenic or anti-estrogenic activity of chlordecone in gonadotrophs. Rat pituitary cell cultures were treated with E 2 (10 −10 m ), chlordecone (10 −7 to 10 −5 m ), or both before a 6-hr challenge of d -Lys 6 -GnRH (GnRH-A). Secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the 6-hr of GnRH-A treatment was measured. Pretreatment of cells with E 2 for 2 days increased the GnRH-A-stimulated LH and FSH secretion approximately twofold. This effect of E 2 was antagonized by the coexistence of chlordecone in a dose-dependent manner. The effect of E 2 (10 −10 m ) could be totally abolished by 10 −6 m chlordecone. Further, chlordecone alone had little or no effect on basal secretion of LH and FSH, but it significantly suppressed the response of gonadotrophs to GnRH-A. [Mirex, a compound with a similar chemical structure to chlordecone, did not suppress GnRH-A-stimulated secretion of LH or FSH under any condition tested.] The present study demonstrated that chlordecone had two specific effects on rat pituitary gonadotrophs: (1) to antagonize the effects of estrogen, and (2) to reduce the response of gonadotrophs to GnRH-A. Thus, chlordecone is not estrogenic in rat pituitary cells in vitro .