Multi-modal molecular imaging maps the correlation between tumor microenvironments and nanomedicine distribution.

Gaining insight into the heterogeneity of nanoparticle drug distribution within tumors would improve both design and clinical translation of nanomedicines. There is little data showing the spatio-temporal behavior of nanomedicines in tissues as current methods are not able to provide a comprehensive view of the nanomedicine distribution, released drug or its effects in the context of a complex tissue microenvironment. Methods: A new experimental approach which integrates the molecular imaging and bioanalytical technologies MSI and IMC was developed to determine the biodistribution of total drug and drug metabolite delivered via PLA-PEG nanoparticles and to overlay this with imaging of the nanomedicine in the context of detailed tumor microenvironment markers. This was used to assess the nanomedicine AZD2811 in animals bearing three different pre-clinical PDX tumors. Results: This new approach delivered new insights into the nanoparticle/drug biodistribution. Mass spectrometry imaging was able to differentiate the tumor distribution of co-dosed deuterated non-nanoparticle-formulated free drug alongside the nanoparticle-formulated drug by directly visualizing both delivery approaches within the same animal or tissue. While the IV delivered free drug was uniformly distributed, the nanomedicine delivered drug was heterogeneous. By staining for multiple biomarkers of the tumor microenvironment on the same tumor sections using imaging mass cytometry, co-registering and integrating data from both imaging modalities it was possible to determine the features in regions with highest nanomedicine distribution. Nanomedicine delivered drug was associated with regions higher in macrophages, as well as more stromal regions of the tumor. Such a comparison of complementary molecular data allows delineation of drug abundance in individual cell types and in stroma. Conclusions: This multi-modal imaging solution offers researchers a better understanding of drug and nanocarrier distribution in complex tissues and enables data-driven drug carrier design.

BACE-1 hydroxyethylamine inhibitors using novel edge-to-face interaction with Arg-296.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.

Low dose prenatal testosterone exposure decreases the corticosterone response to stress in adult male, but not female, mice.

Gonadal steroid hormones affect the organization of the brain during sensitive periods of development, resulting in sex differences in the neuroendocrine function and behaviour of the offspring. Although alterations in developmental testosterone exposure have been hypothesized to play a role in male-biased neurodevelopmental disorders, the underlying mechanisms remain unknown. The present study investigated the hypothesis that early prenatal exposure to low concentrations of testosterone might affect the control of stress responses in later life. Pregnant CD1 mice were treated with 10 µg of testosterone propionate or sesame oil control on embryonic days 12, 14, and 16. Effects on development were assessed by measuring litter size, composition and weight, first appearance of hair, eye and ear opening, and adult body weight. Reproductive development was assessed by measuring testosterone levels in neonatal and adult males, gonad weights in both sexes and reproductive cyclicity in females. The function of the hypothalamic-pituitary-adrenal axis was determined by measuring corticosterone in hair samples from juvenile animals, as well as in plasma following restraint stress in adulthood. Prenatal testosterone treatment had no significant effects on any of the overall developmental or reproductive endpoints assessed. However, in adulthood, corticosterone responses to restraint stress were reduced in the male but not the female offspring, with no significant effect on basal corticosterone levels in either sex. Thus, a small prenatal increase in maternal testosterone may be sufficient to produce a lasting sex-specific alteration in the sensitivity of the male HPA axis to stress.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics.

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

Second generation of hydroxyethylamine BACE-1 inhibitors: optimizing potency and oral bioavailability.

BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.

Selumetinib-based therapy in uveal melanoma patient-derived xenografts.

The prognosis of metastatic uveal melanoma (UM) is among the worst of all human cancers. The identification of near-ubiquitous GNAQ/GNA11 mutations and the activation of MAPK signaling in UM have raised hopes of more effective, targeted therapies, based on MEK inhibition, for example. We evaluated the potential of drug combinations to increase the efficacy of the MEK inhibitor selumetinib (AZD6244, ARRY-142886), in UM cell lines and Patient-Derived Xenografts. We first evaluated the combination of selumetinib and DTIC. We found that DTIC did not improve the in vitro or in vivo antitumor efficacy of selumetinib, consistent with the outcome of the SUMIT clinical trial assessing the efficacy of this combination in UM. We then tested additional selumetinib combinations with the chemotherapy agent docetaxel, the ERK inhibitor AZ6197, and the mTORC1/2 inhibitor, vistusertib (AZD2014). Combinations of selumetinib with ERK and mTORC1/2 inhibitors appeared to be the most effective in UM PDX models.

Development of a P-glycoprotein knockout model in rodents to define species differences in its functional effect at the blood-brain barrier.

The objective of this study was to establish the optimal blood concentrations of the potent P-glycoprotein (P-gp) inhibitor GF120918 (Elacridar) required to achieve maximal knockout of this efflux transporter in the blood-brain barrier (BBB) of mice, rats, and guinea pigs. Genetic mdr1a/b(-/-) knockout mice and "chemical" P-gp knockout mice, rats, and guinea pigs, generated by 24 h continuous infusion of GF120918, were used to investigate the effects of P-gp modulation on the brain penetration of SB-487946. Genetic mdr1a/b(-/-) knockout mice demonstrated a >70-fold increase in brain:blood ratio of SB-487946 compared to mdr1a/b(+/+) wild-type mice. There was a similar increase in SB-487946 brain:blood ratio in GF120918-treated mice (ca. >67-fold) and rats (ca. 95-fold) but a significantly smaller increase (ca. 10-fold) in guinea pigs treated with GF120918. An appreciable difference was found in the BBB functional effect of P-gp efflux in rodents. GF120918 blood EC90 in mice and rats were similar however, the EC90 in guinea pigs was ca. 10-fold higher, suggesting a species difference in the activity of P-gp at the BBB in some rodents. This study establishes the optimal blood concentrations of GF120918 in relation to SB-487946, to achieve chemically induced P-gp knockout in rodents.

Metabolism, Excretion, and Pharmacokinetics of Selumetinib, an MEK1/2 inhibitor, in Healthy Adult Male Subjects.

PURPOSE: Selumetinib (AZD6244, ARRY-142886), an oral mitogen activated kinase 1/2 inhibitor, is in clinical development for the treatment of a variety of different tumor types. Herein, we report a study that determined the distribution, metabolism, and excretion of selumetinib in healthy male volunteers. METHODS: In this open-label, single-center, Phase I clinical trial, 6 subjects received a single 75-mg dose of [14C]-selumetinib. Blood and excreta samples were collected for pharmacokinetic and radiometric analyses. Tolerability monitoring was performed throughout the study. FINDINGS: The Cmax of plasma selumetinib was 1520 ng/mL at 1 hour postdose and declined with a t1/2 of 13.7 hours. Over a 216-hour postdose collection period, total dose recovery was 93% of the radioactive dose, with 59% recovered from feces and 33% from urine. Circulating drug-related material was primarily associated with plasma, with minimal distribution into red blood cells. Selumetinib was the major circulating drug-related component and accounted for 40% of the plasma radioactivity (mean of AUC0-72h pool). The major circulating metabolite (M2; accounting for 22% of the plasma radioactivity) resulted from multiple biotransformation pathways, including loss of the ethanediol moiety in combination with glucuronidation. A further 6 circulating metabolites were identified, each accounting for between 2% and 7% of plasma radioactivity. Selumetinib was a minor component in urine, accounting for ≤1% of the dose. M2 was the most abundant metabolite in urine, accounting for 10% of the dose, and there were 5 other metabolites accounting for between 1% and 10% of the dose. In feces, selumetinib accounted for a mean of 19% of the dose. Also present were 7 metabolites accounting for between 1% and 9% of the dose. The majority of the dose was recovered as metabolites, indicating that the liver is the major route of drug elimination. There were no tolerability concerns. IMPLICATIONS: The findings from this study will inform the label and will contribute to the understanding of the clinical pharmacology of selumetinib. ClinicalTrials.gov identifier: NCT01931761.

Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo.

Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

Stereoselective preparation of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3- (2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide, a potent and orally active dual neurokinin NK(1)/NK(2) receptor antagonist.

In a program aimed at the development of neurokinin antagonists, N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1, DNK333) has been discovered as a potent and balanced neurokinin (tachykinin) NK(1)/NK(2) receptor antagonist. Enantiomerically pure (>99.5% ee) 1 can be prepared in 6 + 1 synthetic steps starting from commercially available optically active BOC-d-3,4-dichlorophenylalanine in an overall yield of ca. 25-30%. 1 showed potent affinities to cloned human NK(1) (pK(i) = 8.38) and NK(2) (pK(i) = 8.02) receptors. When 1 was compared to the other possible three diastereoisomers, it could be demonstrated that only the R,R-isomer (1) exhibits potent and balanced affinity for the cloned human NK(1) and NK(2) receptors. 1 exhibited favorable pharmacokinetic properties in guinea pigs following oral administration and demonstrated in vivo activity in pharmacological models of substance P- and neurokinin A (NKA)-induced bronchoconstriction in guinea pigs after intravenous and in squirrel monkeys after oral application.

The identification of indacaterol as an ultralong-acting inhaled beta2-adrenoceptor agonist.

Following a lipophilicity-based hypothesis, an 8-hydroxyquinolinone 2-aminoindan derived series of beta(2)-adrenoceptor agonists have been prepared and evaluated for their potential as inhaled ultralong-acting bronchodilators. Determination of their activities at the human beta(2)-adrenoceptor receptor showed symmetrical substitution of the 2-aminoindan moiety at the 5- and 6-positions delivered the targeted intermediate potency and intrinsic-efficacy profiles relative to a series of clinical reference beta(2)-adrenoceptor agonists. Further assessment with an in vitro superfused electrically stimulated guinea-pig tracheal-strip assay established the onset and duration of action time courses, which could be rationalized by considering the lipophilicity, potency, and intrinsic efficacy of the compounds. From these studies the 5,6-diethylindan analogue indacaterol 1c was shown to possess a unique profile of combining a rapid onset of action with a long duration of action. Further in vivo profiling of 1c supported the long duration of action and a wide therapeutic index following administration to the lung, which led to the compound being selected as a development candidate.

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo.

Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.

Potent and selective xanthine-based inhibitors of phosphodiesterase 5.

Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.

Synthesis and biological properties of novel glucocorticoid androstene C-17 furoate esters.

A series of novel corticosteroid derivatives featuring C-17 furoate ester functionality have been synthesised. Profiling in vitro and in vivo has resulted in the identification of a compound with a longer duration of action and a lower oral side effect profile in rodents compared to budesonide.