Concentration-Dependent Modulation of Amyloid-␤ in Vivo and in Vitro Using the ␥-Secretase Inhibitor, LY-450139

ABSTRACT LY-450139 is a ␥-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-␤ (A␤) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A␤ responses to LY-450139 in the guinea pig, a nontransgenic model that has an A␤ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma A␤ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A␤ levels at early time points, with return to baseline within hours. Higher doses inhibited A␤ levels in all compartments at early time points, but elevated plasma A␤ levels at later time points. To determine whether this phenomenon occurs under steadystate drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma A␤ was significantly inhibited at 10 -30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A␤ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A␤ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the ␥-secretase complex, eliciting A␤ lowering at high concentrations but A␤ elevation at low concentrations. The pathological accumulation of amyloid-␤ peptide into dense core plaques in the brains of Alzheimer's disease patients is the ultimate target of multiple disease-modifying drug discovery efforts. One strategy that has entered the clinic is the use of a ␥-secretase inhibitor to reduce central A␤ production. Preclinically, multiple ␥-secretase inhibitors have demonstrated central and peripheral A␤-lowering activity in transgenic mouse lines overexpressing human mutant amyloid precursor protein The ability of plasma and CSF A␤ to track pharmacological changes in brain A␤ provides a useful method for tracking the efficacy of ␥-secretase inhibitors in the clinic. Because each compartment may have varying degrees of drug exposure and different clearance rates for both drug and A␤, it is important to understand the dynamics of A␤ in each compartment. Dose-response and time course studies with ␥-secretase inhibitors in transgenic mice have revealed difArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.110700. ABBREVIATIONS

Macrolides : chemistry and progress towards a catalytic antibody

As part of a continuing study examining the chemistry and conformational behaviour of keto 13-tetradecanolides, 11 -oxo- 13-tetradecanolide (24) was synthesized. In the preparation of the acyclic precursor, optimal yields were obtained when the C-11 ketone was protected until the macrocyclic ring had been formed. Otherwise, a protected β-hydroxy group was eliminated under both acidic and basic conditions. The cyclization was accomplished via a trichlorobenzoyl chloride intermediate. Reduction of 24 using lithium tri-sec-butylborohydride (L-Selectride) gave the (11R* , 13S*)isomer of 49 in >99.8% diastereoselectivity. The relative stereochemistry of 49 was determined by reduction and conversion of the 1,3-diol to an acetonide for ¹³C NMR analysis. Molecular mechanics calculations were used to determine the possible low energy conformations for compounds 24 and 49. Reduction of 24 from the preferred twist [3434] conformations would be expected to give rise to product 49 as is indeed observed. The calculations also predicted ¹H NMR coupling constants in good agreement with the observed values for both 24 and 49. [chemical compound diagrams] As part of an effort to develop a catalytic antibody for the synthesis of macrocyclic lactones, polyclonal antibodies against the phosphonate hapten 130 were raised. These antibodies demonstrated hapten-specific binding, but they did not catalyze the lactonization of a corresponding hydroxy ester substrate under the conditions examined. [chemical compound diagrams]Science, Faculty ofChemistry, Department ofGraduat

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) Metabolite Bioanalysis Working Group (WG) on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from IND enabling studies, and Phase I, Phase II, and Phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development Under the “Metabolites in Safety Testing” Regulatory Guidances

Since the introduction of MIST guidance by FDA in 2008 there have been major changes in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and non-clinical studies to generate these information. In this cross-industry article we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radiochromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated which has led to more frequent determination of human metabolite profiles from multiple ascending dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. This, together with technological advances, led to a general shift of the focus towards earlier human metabolism studies, using high resolution mass spectrometry, and to a reduction in animal radiolabel ADME studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industr

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm

Non-labelled, Stable Labelled or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: a Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (IV PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as IV PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade