Structure-activity relationship around PI-2620 highlights the importance of the nitrogen atom position in the tricyclic core.

Tau aggregates represent a critical pathology in Alzheimer's disease (AD) and other forms of dementia. The extent of Tau neurofibrillary tangles across defined brain regions corresponds well to the observed level of cognitive decline in AD. Compound 1 (PI-2620) was recently identified as a promising Tau positron emission tomography tracer for AD and non-AD tauopathies. To evaluate the impact of the N-atom position with respect to Tau- and off-target binding, tricyclic core analogs of PI-2620 with nitrogen atoms at different positions were prepared. Affinity to aggregated Tau was evaluated using human AD brain homogenates, and their off-target binding was evaluated in a monoamine oxidase A (MAO-A) competition assay. The novel tricyclic core derivatives all displayed inferior Tau binding or MAO-A off-target selectivity, indicating PI-2620 to be the optimal design for high affinity binding to Tau and high MAO-A selectivity.

Discovery and preclinical characterization of [18F]PI-2620, a next-generation tau PET tracer for the assessment of tau pathology in Alzheimer's disease and other tauopathies.

PURPOSE: Tau deposition is a key pathological feature of Alzheimer's disease (AD) and other neurodegenerative disorders. The spreading of tau neurofibrillary tangles across defined brain regions corresponds to the observed level of cognitive decline in AD. Positron-emission tomography (PET) has proved to be an important tool for the detection of amyloid-beta (Aß) aggregates in the brain, and is currently being explored for detection of pathological misfolded tau in AD and other non-AD tauopathies. Several PET tracers targeting tau deposits have been discovered and tested in humans. Limitations have been reported, especially regarding their selectivity. METHODS: In our screening campaign we identified pyrrolo[2,3-b:4,5-c']dipyridine core structures with high affinity for aggregated tau. Further characterization showed that compounds containing this moiety had significantly reduced monoamine oxidase A (MAO-A) binding compared to pyrido[4,3-b]indole derivatives such as AV-1451. RESULTS: Here we present preclinical data of all ten fluoropyridine regioisomers attached to the pyrrolo[2,3-b:4,5-c']dipyridine scaffold, revealing compounds 4 and 7 with superior properties. The lead candidate [18F]PI-2620 (compound 7) displayed high affinity for tau deposits in AD brain homogenate competition assays. Specific binding to pathological misfolded tau was further demonstrated by autoradiography on AD brain sections (Braak I-VI), Pick's disease and progressive supranuclear palsy (PSP) pathology, whereas no specific tracer binding was detected on brain slices from non-demented donors. In addition to its high affinity binding to tau aggregates, the compound showed excellent selectivity with no off-target binding to Aß or MAO-A/B. Good brain uptake and fast washout were observed in healthy mice and non-human primates. CONCLUSIONS: Therefore, [18F]PI-2620 was selected for clinical validation.

Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of ß-amyloid-42 aggregation for the treatment of Alzheimer's disease.

The aggregation of amyloid-ß peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-ß. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

PI-2620 Lead Optimization Highlights the Importance of Off-Target Assays to Develop a PET Tracer for the Detection of Pathological Aggregated Tau in Alzheimer's Disease and Other Tauopathies.

The first candidate PI-2014 was tested in healthy controls and subjects with Alzheimer's disease (AD). As PI-2014 displayed off-target binding to monoamine oxidase A (MAO-A), a new lead with improved binding to Tau and decreased MAO-A binding was required. For compound optimization, Tau binding assays based on both human AD brain homogenate and Tau-paired helical filaments were employed. Furthermore, two MAO-A screening assays based on (1) human-recombinant MAO-A and (2) displacement of 2-fluoro-ethyl-harmine from mouse brain homogenate were employed. Removing the N-methyl group from the tricyclic core resulted in compounds displaying improved Tau binding. For the final round of optimization, the cyclic amine substituents were replaced by pyridine derivatives. PI-2620 (2-(2-fluoropyridin-4-yl)-9H-pyrrolo[2,3-b:4,5-c']dipyridine) emerged as a best candidate displaying high Tau binding, low MAO-A binding, high brain uptake, and fast and complete brain washout. Furthermore, PI-2620 showed Tau binding on brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease.

Discovery of 2-(4-(2-fluoroethoxy)piperidin-1-yl)-9-methyl-9H-pyrrolo[2,3-b:4,5-c']dipyridine ([18F]PI-2014) as PET tracer for the detection of pathological aggregated tau in Alzheimer's disease and other tauopathies.

The compound screening was initiated with a direct staining assay to identify compounds binding to Tau aggregates and not Abeta plaques using human brain sections derived from late stage Alzheimer's disease donors. The binding of Tau aggregate selective compounds was then quantitatively assessed with human brain derived paired helical filaments utilizing the label-free Back Scattering Interferometry assay. In vivo biodistribution experiments of selected fluorine-18 labeled compounds were performed in mice to assess brain uptake, brain washout, and defluorination. Compound 11 emerged as the most promising candidate, displaying high in vitro binding affinity and selectivity to neurofibrillary tangles. Fluorine-18 labeled compound 11 showed high brain uptake and rapid washout from the mouse brain with no observed bone uptake. Furthermore, compound 11 was able to detect Tau aggregates in tauopathy brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease donors. Thus, 2-(4-(2-fluoroethoxy)piperidin-1-yl)-9-methyl-9H-pyrrolo[2,3-b:4,5-c']dipyridine (PI-2014, compound 11) was selected for characterization in a first-in-human study.

THYROID PHANTOM MEASUREMENTS IN JOINT EURADOS-LLNL INTERCOMPARISON EXERCISE.

The European Radiation Dosimetry Group (EURADOS), in collaboration with Lawrence Livermore National Laboratory's (LLNL's) Thyroid Intercomparison Program (TRIP), conducted an intercomparison exercise consistent with the goals of EURADOS. In total, 35 in vivo radiobioassay facilities from 18 countries participated to evaluate the differences between the neck and thyroid phantoms specified in two standards issued by the American National Standards Institute. Radioiodine (125I and 131I) measurement results were compared to the traceable standard activity levels added to each phantom. Measurement data showed no statistically significant differences between normalized activity measurements of the thyroid phantom types (20 and 30 ml). Differences were noted between the laboratories that routinely participate in the radioiodine thyroid intercomparison program (TRIP participants) and laboratories that have not previously participated in TRIP. Evaluation of the reasons for these differences will require additional EURADOS-LLNL collaborations. Finally, the measurement data from this intercomparison was used with a designed intake scenario for intercomparison of dose evaluations. Results from the dose intercomparison will be presented in a subsequent article.

Immunization with liposome-anchored pegylated peptides modulates doxorubicin sensitivity in P-glycoprotein-expressing P388 cells.

The clinical use of chemotherapy in cancer treatment is limited by the occurrence of multidrug resistance (MDR) associated with the overexpression of membrane transporters, one of the best known is P-glycoprotein (Pgp), that actively expels drugs out of tumor cells. To overcome Pgp-mediated MDR, synthetic peptides corresponding to fragments from extracellular loops 1, 2 and 4 of the murine Pgp were coupled to polyethylene glycol-distearoylphosphatidylethanolamine and inserted into empty or monophosphoryl lipid A-containing liposomes. This formulation elicited specific antibodies which blocked Pgp-mediated efflux of doxorubicin, resulting in increased intracellular drug accumulation and subsequent potentiation of the cytotoxic effect of doxorubicin on multidrug-resistant P388 (P388R) cells. Previous immunizations with MDR1 peptides improved the efficiency of chemotherapy against P388R cells in vivo, with an increase of 83% of mice survival time. Overall, these results suggest that this approach can modulate Pgp activity by blocking drug efflux and may have clinical relevance as an alternative strategy to toxic chemosensitizers in drug-resistant cancer therapy.

Mapping mandibular rest in humans utilizing electromyographic patterns from masticatory muscles.

As the mandible assumes its resting position in space, antagonistic muscles should assume their resting lengths as is demonstrated by resting and isometric electromyography. This zone of mandibular rest can be mapped using these physiologic parameters of muscle activity. Three positions were evaluated: a maximum physiologic open position, a maximum physiologic closed position, and a physiologic rest position. Additionally, each subject's maximum intercuspation position was evaluated. Within the physiologic zone of rest, formed by the maximum physiologic open position and maximum physiologic closed position, muscle recruitment was the greatest in a physiologic group. Results indicated that muscle function was significantly greater within the zone of mandibular rest than at the intercuspal position.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope.

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer's disease.

An Anti-ß-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome.

In Down syndrome (DS) or trisomy of chromosome 21, the ß-amyloid (Aß) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aß as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aß is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer's disease (AD). At present, no treatment targets Aß-related pathogenesis in people with DS. Herein we used a vaccine containing the Aß 1-15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aß vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aß IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aß without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aß levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aß as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aß immunotherapeutic approach may act to target Aß-related pathology in a mouse model of DS.

Nucleic acid binding properties of thyminyl and adeninyl pyrrolidine-amide oligonucleotide mimics (POM).

Adeninyl POM was prepared using solid-phase peptide chemistry and shown to exhibit higher affinity for complementary DNA and RNA than the corresponding adeninyl PNA.

Financial risk relationships and adoption of management strategies in physician groups for self-administered injectable drugs.

OBJECTIVE: To consider the extent, nature, and range of risk arrangements between physician groups and health maintenance organizations (HMOs) for self-administered injectable (SAI) drugs; to examine types and frequencies of SAI drug-use management strategies adopted by physician groups; and to explore the relationship between locus and level of financial risk for SAIs and physician group strategy adoption. METHODS: We used a multiple case-study design to select physician groups and their health maintenance organization (HMO) contractual partners in 4 markets in the United States (Northwest, Northeast, Midwest, Southwest). Physician groups in these markets were chosen based on size (e50 physicians) and experience with drug risk (e1 year). Physician groups were asked to identify their 3 major HMO contractual partners in each market. Telephone interviews were conducted from January 2000 to June 2001, with the resulting purposive sample of 37 individuals representing 20 physician groups. RESULTS: We found that the level and locus of SAI financial risk were related to the adoption of management strategies. Physician groups with higher financial risk for SAIs adopted more strategies than lower-risk groups. Groups with SAI financial risk in the medical services capitation (MSC) adopted 9.2 strategies per group. In contrast, groups with SAI financial risk in the pharmacy-risk budget (PRB) averaged 1.5 strategies per group. Groups with SAI financial risk in both the MSC and PRB fell in-between, averaging 4.5 strategies per group. The most frequently adopted strategy was designing evidenced-based therapeutic guidelines, i.e., protocols based on evidence from the peer-reviewed literature used to guide physicians in the treatment of typically chronic conditions (9 groups, 45% of sample). The second most common strategy involved adapting the existing utilization management system to process SAIs (7 groups, 35%) and the establishment of office procedures for internal authorization (5 groups, 25%). The least frequently used strategies were determining amount paid to out-of-group physician providers (1 group, 5%) and hiring personnel (e.g., pharmacists) in claims or utilization management departments to implement and manage SAI programs (1 group, 5%). We also identified potential factors that increased the likelihood of strategy adoption and that could slow the rate of SAI cost increases. CONCLUSION: Our findings suggest that adoption of SAI drug-use management strategies may be more likely to occur when there is a minimum level of risk for SAI drug costs. Likewise, both the adoption of strategies and the opportunity to slow the rate of SAI cost increases may be more likely to occur when 3 additional factors are present: a contractual environment conducive to controlling SAI drug costs, the ability to implement SAI drug-use management strategies, and power in negotiations with drug manufacturers to reduce SAI prices. A sustainable and affordable SAI financial risk management program maximizing these factors while minimizing the financial burden for patients will require collaboration among all stakeholders, payers, providers, drug manufacturers, and patients.

Techniques for reducing anesthetic injection pain: an interdisciplinary survey of knowledge and application.

BACKGROUND: The authors conducted a study that considered family physicians' and dentists' knowledge and application of techniques to reduce the pain associated with anesthetic injections. They also assessed practitioners' discomfort with patients' injection pain and needle anxiety/phobia. METHODS: The authors designed a questionnaire about awareness and use of 10 techniques for reducing pain of anesthetic injection and mailed it to 2,000 randomly selected family physicians and general dentists. They analyzed the data to examine differences between disciplines regarding awareness and use of techniques, reasons for not using techniques, number of injections given per week, and predictive value of certain demographic variables on reported use of individual techniques and on practitioner reactions to patients' pain and anxiety. RESULTS: The response rate was 35 percent. The authors used the chi2 test for differences between disciplines' awareness of and use or nonuse of techniques, Wilcoxon testing to assess differences between disciplines' median values of number of weekly injections and logistic regression to study demographic variables' predictive values (P = .01). General dentists give more injections than do family physicians. Differences existed between disciplines' awareness and use of eight of 10 techniques. Disciplines reported cost and time issues as reasons for not using some techniques. Number of years in practice and age were associated with use of six techniques. Dentists reported feeling greater personal effects of patients' pain and needle anxiety/phobia than did family physicians. CONCLUSIONS: Those not using pain-lessening techniques inaccurately identified time and cost as problems, suggesting that respondents may be less familiar with these techniques than otherwise reported. Further study is recommended. CLINICAL IMPLICATIONS: Pain reduction techniques for anesthetic injection cost little to implement, are not time liabilities, and can lessen avoidable pain and reduce the incidence of needle phobia.

A whole body counting facility in a remote Enewetak Island setting.

The U.S. Department of Energy (DOE) has recently implemented a series of strategic initiatives to address long-term radiological surveillance needs at former U.S. test sites in the Marshall Islands. The plan is to engage local atoll communities in developing shared responsibilities for implementing radiation protection programs for resettled and resettling populations. As part of this new initiative, DOE agreed to design and construct a radiological laboratory on Enewetak Island, and help develop the necessary local resources to maintain and operate the facility. This cooperative effort was formalized in August 2000 between the DOE, the Republic of the Marshall Islands (RMI), and the Enewetak/Ujelang Local Atoll Government (EULGOV). The laboratory facility was completed in May 2001. The laboratory incorporates both a permanent whole body counting system to assess internal exposures to 137Cs, and clean living space for people providing 24-h void urine samples. DOE continues to provide on-going technical assistance, training, and data quality review while EULGOV provides manpower and infrastructure development to sustain facility operations on a full-time basis. This paper will detail the special construction, transportation and installation issues in establishing a whole body counting facility in an isolated, harsh environmental setting.

Historical review of lung counting efficiencies for low energy photon emitters.

This publication reviews the measured efficiency and variability over time of a high purity planar germanium in vivo lung count system for multiple photon energies using increasingly thick overlays with the Lawrence Livermore Torso Phantom. The measured variations in efficiency are compared with the current requirement for in vivo bioassay performance as defined by the American National Standards Institute Standard in ANSI Standard N13.30.

Engineering amyloid-like assemblies from unstructured peptides via site-specific lipid conjugation.

Aggregation of amyloid beta (Aß) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of ß-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of ß-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.

Efficacy and safety of a liposome-based vaccine against protein Tau, assessed in tau.P301L mice that model tauopathy.

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.