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.

Sustained peripheral depletion of amyloid-ß with a novel form of neprilysin does not affect central levels of amyloid-ß.

Alzheimer's disease is characterized by the accumulation of amyloid deposits in the brain and the progressive loss of cognitive functions. Although the precise role of amyloid-ß in disease progression remains somewhat controversial, many efforts to halt or reverse disease progression have focussed on reducing its synthesis or enhancing its removal. It is believed that brain and peripheral soluble amyloid-ß are in equilibrium and it has previously been hypothesized that a reduction in peripheral amyloid-ß can lower brain amyloid-ß, thereby reducing formation of plaques predominantly composed of insoluble amyloid-ß; the so-called peripheral sink hypothesis. Here we describe the use of an amyloid-ß degrading enzyme, the endogenous metallopeptidase neprilysin, which is fused to albumin to extend plasma half-life and has been engineered to confer increased amyloid-ß degradation activity. We used this molecule to investigate the effect of degradation of peripheral amyloid-ß on amyloid-ß levels in the brain and cerebrospinal fluid after repeated intravenous dosing for up to 4 months in Tg2576 transgenic mice, and 1 month in rats and monkeys. This molecule proved highly effective at degradation of amyloid-ß in the periphery but did not alter brain or cerebrospinal fluid amyloid-ß levels, suggesting that the peripheral sink hypothesis is not valid and is the first time that this has been demonstrated in non-human primates.

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.

Design and synthesis of ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of ß-amyloid peptides.

The evaluation of a series of aminoisoindoles as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aß40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 µM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of ß-amyloid peptides in mouse brain following oral dosing.

An ancient duplication of exon 5 in the Snap25 gene is required for complex neuronal development/function.

Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

Tomosyn is expressed in beta-cells and negatively regulates insulin exocytosis.

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic beta-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting beta-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in beta-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse beta-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta-cell, tomosyn negatively regulates insulin exocytosis.

Increased risk of type 2 diabetes in Alzheimer disease.

Alzheimer disease and type 2 diabetes are characterized by increased prevalence with aging, a genetic predisposition, and comparable pathological features in the islet and brain (amyloid derived from amyloid beta protein in the brain in Alzheimer disease and islet amyloid derived from islet amyloid polypeptide in the pancreas in type 2 diabetes). Evidence is growing to link precursors of amyloid deposition in the brain and pancreas with the pathogenesis of Alzheimer disease and type 2 diabetes, respectively. Given these similarities, we questioned whether there may be a common underlying mechanism predisposing to islet and cerebral amyloid. To address this, we first examined the prevalence of type 2 diabetes in a community-based controlled study, the Mayo Clinic Alzheimer Disease Patient Registry (ADPR), which follows patients with Alzheimer disease versus control subjects without Alzheimer disease. In addition to this clinical study, we performed a pathological study of autopsy cases from this same community to determine whether there is an increased prevalence of islet amyloid in patients with Alzheimer disease and increased prevalence of cerebral amyloid in patients with type 2 diabetes. Patients who were enrolled in the ADPR (Alzheimer disease n = 100, non-Alzheimer disease control subjects n = 138) were classified according to fasting glucose concentration (FPG) as nondiabetic (FPG <110 mg/dl), impaired fasting glucose (IFG, FPG 110-125 mg/dl), and type 2 diabetes (FPG >126 mg/dl). The mean slope of FPG over 10 years in each case was also compared between Alzheimer disease and non-Alzheimer disease control subjects. Pancreas and brain were examined from autopsy specimens obtained from 105 humans (first, 28 cases of Alzheimer disease disease vs. 21 non-Alzheimer disease control subjects and, second, 35 subjects with type 2 diabetes vs. 21 non-type 2 diabetes control subjects) for the presence of islet and brain amyloid. Both type 2 diabetes (35% vs. 18%; P < 0.05) and IFG (46% vs. 24%; P < 0.01) were more prevalent in Alzheimer disease versus non-Alzheimer disease control subjects, so 81% of cases of Alzheimer disease had either type 2 diabetes or IFG. The slope of increase of FPG with age over 10 years was also greater in Alzheimer disease than non-Alzheimer disease control subjects (P < 0.01). Islet amyloid was more frequent (P < 0.05) and extensive (P < 0.05) in patients with Alzheimer disease than in non-Alzheimer disease control subjects. However, diffuse and neuritic plaques were not more common in type 2 diabetes than in control subjects. In cases of type 2 diabetes when they were present, the duration of type 2 diabetes correlated with the density of diffuse (P < 0.001) and neuritic plaques (P < 0.01). In this community cohort from southeast Minnesota, type 2 diabetes and IFG are more common in patients with Alzheimer disease than in control subjects, as is the pathological hallmark of type 2 diabetes, islet amyloid. However, there was no increase in brain plaque formation in cases of type 2 diabetes, although when it was present, it correlated in extent with duration of diabetes. These data support the hypothesis that patients with Alzheimer disease are more vulnerable to type 2 diabetes and the possibility of linkage between the processes responsible for loss of brain cells and beta-cells in these diseases.

Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid.

Nondiabetic obese humans adapt to insulin resistance by increasing beta-cell mass. In contrast, obese humans with type 2 diabetes have an approximately 60% deficit in beta-cell mass. Recent studies in rodents reveal that beta-cell mass is regulated, increasing in response to insulin resistance through increased beta-cell supply (islet neogenesis and beta-cell replication) and/or decreased beta-cell loss (beta-cell apoptosis). Prospective studies of islet turnover are not possible in humans. In an attempt to establish the mechanism for the deficit in beta-cell mass in type 2 diabetes, we used an obese versus lean murine transgenic model for human islet amyloid polypeptide (IAPP) that develops islet pathology comparable to that in humans with type 2 diabetes. By 40 weeks of age, obese nontransgenic mice did not develop diabetes and adapted to insulin resistance by a 9-fold increase (P < 0.001) in beta-cell mass accomplished by a 1.7-fold increase in islet neogenesis (P < 0.05) and a 5-fold increase in beta-cell replication per islet (P < 0.001). Obese transgenic mice developed midlife diabetes with islet amyloid and an 80% (P < 0.001) deficit in beta-cell mass that was due to failure to adaptively increase beta-cell mass. The mechanism subserving this failed expansion was a 10-fold increase in beta-cell apoptosis (P < 0.001). There was no relationship between the extent of islet amyloid or the blood glucose concentration and the frequency of beta-cell apoptosis. However, the frequency of beta-cell apoptosis was related to the rate of increase of islet amyloid. These prospective studies suggest that the formation of islet amyloid rather than the islet amyloid per se is related to increased beta-cell apoptosis in this murine model of type 2 diabetes. This finding is consistent with the hypothesis that soluble IAPP oligomers but not islet amyloid are responsible for increased beta-cell apoptosis. The current studies also support the concept that replicating beta-cells are more vulnerable to apoptosis, possibly accounting for the failure of beta-cell mass to expand appropriately in response to obesity in type 2 diabetes.