AZ-4217: a high potency BACE inhibitor displaying acute central efficacy in different in vivo models and reduced amyloid deposition in Tg2576 mice.

Aß, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimer's disease (AD). ß-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aß peptides. Small molecule BACE1 inhibitors are expected to decrease Aß-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pM in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aß production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

Prediction of in vivo rat biliary drug clearance from an in vitro hepatocyte efflux model.

Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

Decreased axonal transport rates in the Tg2576 APP transgenic mouse: improvement with the gamma-secretase inhibitor MRK-560 as detected by manganese-enhanced MRI.

Neuropil deposition of beta-amyloid (Aß) peptides is believed to be a key event in the neurodegenerative process of Alzheimer's disease (AD). An early and consistent clinical finding in AD is olfactory dysfunction with associated pathology. Interestingly, transgenic amyloid precursor protein (Tg2576) mice also show early amyloid pathology in olfactory regions. Moreover, a recent study indicates that axonal transport is compromised in the olfactory system of Tg2576 mice, as measured by manganese-enhanced magnetic resonance imaging (MEMRI). Here we tested whether the putative axonal transport deficit in the Tg2576 mouse model improves in response to a selective gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560). Tg2576 mice or wild-type (WT) littermates were treated daily with MRK-560 (30 µmol/kg) or vehicle for 4 (acute) or 29 days (chronic). The subsequent MEMRI analysis revealed a distinct axonal transport dysfunction in the Tg2576 mice compared with its littermate controls. Interestingly, the impairment of axonal transport could be fully reversed by chronic administration of MRK-560, in line with the significantly lowered levels of both soluble and insoluble forms of Aß found in the brain and olfactory bulbs (OBs) following treatment. However, no improvement of axonal transport was observed after acute treatment with MRK-560, where soluble but not insoluble forms of Aß were reduced in the brain and OBs. The present results show that axonal transport is impaired in Tg2576 mice compared with WT controls, as measured by MEMRI. Chronic treatment in vivo with a gamma-secretase inhibitor, MRK-560, significantly reduces soluble and insoluble forms of Aß, and fully reverses the axonal transport dysfunction.

Optimizing DMPK Properties: Experiences from a Big Pharma DMPK Department.

BACKGROUND: The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Södertälje, Sweden. METHODS: A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man. RESULTS AND CONCLUSIONS: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

High-throughput analysis of standardized pharmacokinetic studies in the rat using sample pooling and UPLC-MS/MS.

As a consequence of a continuous demand for increased throughput of pharmacokinetic (PK) studies, industries have introduced strategies to reduce the number of samples such as cassette analysis (pooling of samples after the in-life phase). Here, we have investigated whether relevant PK parameters change as a consequence of cassette analysis, and whether there are circumstances that disqualify this technique from being used. 22 compounds were intravenously and orally administered to parallel groups of 3 rats. Each compound was administered discretely. Equal volumes of three plasma samples corresponding to each time point of three discretely dosed rats with different compounds were pooled (cassette analysis). Samples were prepared by protein precipitation followed by UPLC-MS/MS analysis using pos/neg switching when required. With cassette analysis, 4 compounds, morphine, phenytoin, rofecoxib and diclofenac, showed high limit of quantification (LOQ) values after pooling, which led to less reliable PK analyses. Of all samples with contents above LOQ, about 5% could not be detected in pool samples compared to single samples. However, an excellent correlation was seen for all PK parameters when comparing the parameters obtained from discrete analysis versus those obtained from cassette analysis, although half life showed somewhat more scatter than the others. When PK parameters were grouped as low-medium-high, clearance, volume of distribution, half life and bioavailability were similar between discrete and cassette analysis for 90%, 86%, 95% and 90% of the total number of compounds tested, respectively. Some additional improvement was achieved if compounds with a low MS response were excluded. In summary, cassette analysis is an effective strategy to reduce samples without affecting the estimated PK parameters that are important for decision-making.

Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies.

In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.

Description and validation of a four-channel staggered LC-MS/MS system for high-throughput in vitro screens.

A parallel four-channel LC-MS/MS system for quantitative high-throughput in vitro screens is described. The system comprises four pumps, a four-valve autosampler equipped with a nine-port stream selector valve, and a triple quadrupole mass spectrometer. Staggered injections across the sample list are performed by four injector valves onto separate columns in such a manner that the chromatographic window of interest is always eluting from one of the columns into the mass spectrometer. To demonstrate the applicability of the system, three different compounds (verapamil, propanolol, dextromethorphan) were analyzed. The results showed that the sample throughput could be increased more than three times in comparison with a conventional single-column LC-MS/MS system. Validation results for the three compounds showed an accuracy of 85.0-108%, a precision (given as relative standard deviation) of 2.60-10.7%, and a carryover of less than 0.1%.