Effects of LRRK2 Inhibitors in Nonhuman Primates.

Toxicology studies in nonhuman primates were conducted to evaluate selective, brain penetrant inhibitors of LRRK2. GNE 7915 was limited to 7-day administration in cynomolgus monkeys at 65 mg/kg/day or limited to 14 days in rhesus at 22.5 mg/kg b.i.d. due to physical signs. Compound 25 demonstrated acceptable tolerability at 50 and 225 mg/kg b.i.d. for 7 days in rhesus monkeys. MK-1468 was tolerated during 7-day administration at 100, 200 or 800 mg/kg/day or for 30-day administration at 30, 100, or 500 mg/kg b.i.d. in rhesus monkeys. The lungs revealed hypertrophy of type 2 pneumocytes, with accumulation of intra-alveolar macrophages. Transmission electron microscopy confirmed increased lamellar structures within hypertrophic type 2 pneumocytes. Hypertrophy and hyperplasia of type 2 pneumocytes with accumulation of intra-alveolar macrophages admixed with neutrophils were prominent at peripheral lungs of animals receiving compound 25 or MK-1468. Affected type 2 pneumocytes were immuno-positive for pro-surfactant C, but negative for CD11c, a marker for intra-alveolar macrophages. Accumulation of collagen within alveolar walls, confirmed by histochemical trichrome stain, accompanied changes described for compound 25 and MK-1468. Following a 12-week treatment-free interval, animals previously receiving MK-1468 for 30 days exhibited remodeling of alveolar structure and interstitial components that did not demonstrate reversibility.

Discovery of quinuclidine modulators of cellular progranulin.

Phenotypic screening of an annotated small molecule library identified the quinuclidine tetrahydroisoquinoline solifenacin (1) as a robust enhancer of progranulin secretion with single digit micromolar potency in a murine microglial (BV-2) cell line. Subsequent SAR development led to the identification of 29 with a 38-fold decrease in muscarinic receptor antagonist activity and a 10-fold improvement in BV-2 potency.

Local Effects Following Single and Repeat Intra-Articular Injections of Triamcinolone Acetonide Extended-Release: Results from Three Nonclinical Toxicity Studies in Dogs.

INTRODUCTION: Single intra-articular (IA) injections of poly(lactic-co-glycolic acid) (PLGA) microsphere-based triamcinolone acetonide extended-release (TA-ER; formerly FX006) demonstrated sustained, clinically relevant benefits in patients with knee osteoarthritis. The local effects of TA-ER were assessed in normal canine knees in three nonclinical studies. METHODS: Knees were evaluated for up to 6 weeks or 9 months after a single injection of TA-ER (2.1/6.25/18.75 mg TA), or TA crystalline suspension (TAcs, 18.75 mg TA), and for up to 6 months after three injections (every 1 or 3 months) of TA-ER (6.25/18.75 mg TA) or TAcs (18.75 mg). Vehicle-diluent, blank microspheres, and untreated knees were used as controls. Plasma and synovial fluid (SF) TA concentrations and standard histopathological assessment of the synovium were conducted. Articular cartilage morphology was assessed via modified Mankin scoring. RESULTS: Plasma and SF concentrations indicated prolonged dose-dependent TA joint residency with TA-ER compared with TAcs. Effects in articular cartilage were dose- and time-dependent and consistent with known effects of corticosteroids in the normal knee. Loss of Safranin O staining occurred, indicative of a reduction in cartilage matrix proteoglycan, and recovered in a similar manner for TA-ER and TAcs across all studies. Structural lesions were infrequent and generally comparable in severity between TA-ER and TAcs but slightly higher in incidence for TA-ER. Focal/multifocal foreign-body responses (FBR) to PLGA were observed in the superficial layer of the synovium, peaking after 4-6 weeks, with significant recovery or complete resolution by month 6. CONCLUSIONS: These findings suggest that the effects of IA injections of TA-ER on cartilage are predominantly transient, and comparable to those observed with TAcs in the normal canine knee joint. These mild effects in the normal joint differ from the beneficial effects observed with TA-ER and other corticosteroids in disease models. The synovial FBR to PLGA microspheres was focal and transient. FUNDING: Flexion Therapeutics, Inc. Plain language summary available for this article.

A selective prostaglandin E2 receptor subtype 2 (EP2) antagonist increases the macrophage-mediated clearance of amyloid-beta plaques.

A high-throughput screen resulted in the discovery of benzoxazepine 1, an EP2 antagonist possessing low microsomal stability and potent CYP3A4 inhibition. Modular optimization of lead compound 1 resulted in the discovery of benzoxazepine 52, a molecule with single-digit nM binding affinity for the EP2 receptor and significantly improved microsomal stability. It was devoid of CYP inhibition and was ∼4000-fold selective against the other EP receptors. Compound 52 was shown to have good PK properties in CD-1 mice and high CNS permeability in C57Bl/6s mice and Sprague-Dawley rats. In an ex vivo assay, it demonstrated the ability to increase the macrophage-mediated clearance of amyloid-beta plaques from brain slices in a dose-dependent manner.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Discovery of 2-methylpyridine-based biaryl amides as γ-secretase modulators for the treatment of Alzheimer's disease.

γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.

Establishing the relationship between in vitro potency, pharmacokinetic, and pharmacodynamic parameters in a series of orally available, hydroxyethylamine-derived ß-secretase inhibitors.

Sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex produces the amyloid-ß peptide (Aß), which is believed to play a critical role in the pathology of Alzheimer's disease (AD). The aspartyl protease BACE1 catalyzes the rate-limiting step in the production of Aß, and as such it is considered to be an important target for drug development in AD. The development of a BACE1 inhibitor therapeutic has proven to be difficult. The active site of BACE1 is relatively large. Consequently, to achieve sufficient potency, many BACE1 inhibitors have required unfavorable physicochemical properties such as high molecular weight and polar surface area that are detrimental to efficient passage across the blood-brain barrier. Using a rational drug design approach we have designed and developed a new series of hydroxyethylamine-based inhibitors of BACE1 capable of lowering Aß levels in the brains of rats after oral administration. Herein we describe the in vitro and in vivo characterization of two of these molecules and the overall relationship of compound properties [e.g., in vitro permeability, P-glycoprotein (P-gp) efflux, metabolic stability, and pharmacological potency] to the in vivo pharmacodynamic effect with more than 100 compounds across the chemical series. We demonstrate that high in vitro potency for BACE1 was not sufficient to provide central efficacy. A combination of potency, high permeability, low P-gp-mediated efflux, and low clearance was required for compounds to produce robust central Aß reduction after oral dosing.

Design and preparation of a potent series of hydroxyethylamine containing ß-secretase inhibitors that demonstrate robust reduction of central ß-amyloid.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of ß-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS ß-amyloid (Aß) in Sprague-Dawley rats following oral administration.

Design and synthesis of potent, orally efficacious hydroxyethylamine derived ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-ß peptide (Aß) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aß levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

A Potent and Orally Efficacious, Hydroxyethylamine-Based Inhibitor of ß-Secretase.

ß-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against ß-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aß40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

A novel statistical algorithm for gene expression analysis helps differentiate pregnane X receptor-dependent and independent mechanisms of toxicity.

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution. Our method for determining differential expression consists of two steps: 1) We apply a nominal threshold on fold change and platform p-value to designate whether a gene is differentially expressed in each treated and control sample relative to the averaged control pool, and 2) We compared the number of samples satisfying criteria in step 1 between the treated and control groups to estimate the statistical significance based on a null distribution established by sample permutations. The method captures group effect without being too sensitive to anomalies as it allows tolerance for potential non-responders in the treatment group and outliers in the control group. Performance and results of this method were compared with the Significant Analysis of Microarrays (SAM) method. These two methods were applied to investigate hepatic transcriptional responses of wild-type (PXR(+/+)) and pregnane X receptor-knockout (PXR(-/-)) mice after 96 h exposure to CMP013, an inhibitor of ß-secretase (ß-site of amyloid precursor protein cleaving enzyme 1 or BACE1). Our results showed that CMP013 led to transcriptional changes in hallmark PXR-regulated genes and induced a cascade of gene expression changes that explained the hepatomegaly observed only in PXR(+/+) animals. Comparison of concordant expression changes between PXR(+/+) and PXR(-/-) mice also suggested a PXR-independent association between CMP013 and perturbations to cellular stress, lipid metabolism, and biliary transport.

The geminal dimethyl analogue of Flurbiprofen as a novel Abeta42 inhibitor and potential Alzheimer's disease modifying agent.

The subtle modification of a selection of Abeta42 inhibiting non-steroidal anti-inflammatory drugs (NSAIDs), through synthesis of the geminal dimethyl analogues, was anticipated to ablate their cyclooxygenase activity whilst maintaining Abeta42 inhibition. Methylflurbiprofen 6 exhibited similar in vitro Abeta42 inhibition to its parent NSAID Flurbiprofen and was further evaluated in the Tg2576 mouse model of Alzheimer's disease and an animal model of gastro-intestinal (GI) impairment, but proved unviable for further clinical development.

The effects of AMPA receptor antagonists in models of stroke and neurodegeneration.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists have been shown to have neuroprotective effects in stroke models and although clinical trials with some agents are still ongoing, published results have not been favourable. We therefore wished to compare the effects of GYKI 52466, GYKI 53405, EGIS-8332 and EGIS-10608, non-competitive AMPA receptor antagonists with homophthalazine chemical structures, in standard animal stroke models with effects in a neurodegenerative model--excitoxicity in newborn mice. All compounds inhibited the S-AMPA-induced spreading depression in the chicken retina, in vitro, and were potent anticonvulsants against maximal electroshock in mice, in vivo. The AMPA receptor antagonists prevented domoate-induced cell death of motoneurons, in vitro, and reduced infarct size in a dose-dependent manner in the permanent middle cerebral artery occlusion model in mice, in vivo. In newborn mice (P5, histopathology at P10), local injection of the AMPA receptor agonist S-bromo-willardiine at day 5 after birth induced cortical damage and white matter damage, which was reduced in a dose-dependent manner by the AMPA receptor antagonists. EGIS 10608 was a very powerful receptor antagonist of white matter damage. In contrast, GYKI 52466 did not antagonize cortical and white matter damage induced by ibotenic acid. These models allow quantification of the effects of AMPA receptor antagonists in vitro and in vivo.

Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide].

The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (Abeta) peptide thereby allowing the detection of changes in Abeta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect Abeta(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of the gamma-secretase inhibitor LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF Abeta(40) levels were observed with ID(50) values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice. Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF Abeta(40), further suggesting these two pools of Abeta are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF Abeta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

Role of spin trapping and P2Y receptor antagonism in the neuroprotective effects of 2,2'-pyridylisatogen tosylate and related compounds.

2,2'-Pyridylisatogen tosylate (PIT) is both an allosteric modulator of P2Y receptors, and an immine oxide, acting as a spin trap for free radicals. PIT (10 mg kg(-1), i.p.) was found to be a powerful neuroprotective agent in protecting against the lesions induced by 15 micro g S-bromo-willardiine injected into the cortex or white matter of 5-day-old mice pups. As the multiple effects of PIT may induce both beneficial and deleterious effects, a reanalysis of the structure-activity relationship was undertaken. PIT (50 micro M) and 2,3'-pyridylisatogen were potent antagonists of responses to ATP in the taenia preparation of the guinea-pig caecum, but 2,3'-nitrophenylisatogen was not. The reactive immine oxide group could be substituted by a keto moiety (N-(2'-pyridyl)phthalide) while maintaining antagonism of responses to ATP, equivalent to PIT. Thus, antagonism of P2Y receptors was not restricted to the isatogen nucleus. Other spin traps did not antagonise P2Y receptors, although dimethyl-pyrroline-N-oxide (DMPO) increased the sensitivity of responses to ATP. Both N-(2'-pyridyl)phthalide and 2,3'-nitrophenylisatogen was less neuroprotective than PIT (10 mg kg(-1), i.p.) in protecting against the S-bromo-willardiine-induced lesions in mice, implying that both antagonism of P2Y receptors and the immine oxide moiety may be important for the neuroprotective effects of PIT. However, the usefulness of the neuroprotection was limited because, in motoneurones obtained from rat embryos, PIT (10-100 micro M) exacerbated cell death.