Kathryn Rogers

Differential and synergistic effects of selective norepinephrine and serotonin reuptake inhibitors in rodent models of pain.

There is increasing recognition that norepinephrine (NE) and serotonin (5-HT) reuptake inhibitors (NRIs and SRIs) are efficacious in treating some types of pain. To date, studies have not systematically evaluated the relative activity at the NE and/or 5-HT transporter required for maximal efficacy in rodent pain models. Known selective NE and 5-HT reuptake inhibitors reboxetine, desipramine, fluoxetine, and paroxetine were evaluated in both in vitro and in vivo assays. Using the spinal nerve ligation model of neuropathic pain, the compounds differentially reversed tactile allodynia. Evaluation of a broader spectrum of reuptake inhibitors in the para-phenylquinone (PPQ)-induced abdominal constriction model, a model of acute visceral pain, demonstrated that both the SRIs and the NRIs significantly blocked abdominal constrictions. However, the magnitude of this effect was greater following treatment with compounds having greater affinity for NRI compared with SRI affinity. In addition, isobolographic analyses indicated significant synergistic effects for all combinations of desipramine and fluoxetine in the PPQ model of visceral pain. Collectively, the present results support the hypothesis that compounds with greater NRI activity should be more effective for the treatment of pain than compounds having only SRI activity, and this hypothesis is also supported by clinical data. These studies also suggest that the potency and effectiveness of NRIs might be enhanced by the presence of 5-HT activity.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice

BackgroundA hallmark of Alzheimer’s disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aβ42 and Aβ40. Many drug discovery efforts have focused on decreasing the production of Aβ42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aβ production to favor shorter, less amyloidogenic peptides than Aβ42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer’s disease.ResultsHere we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aβ42 in H4 cells (IC50 = 67 nM) and increased the shorter Aβ38 by 1.7 fold at the IC50 for lowering of Aβ42. AβTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aβ42 and did not alter AβTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aβ deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aβ aggregates, amyloid plaques, inflammatory markers, and cognitive deficits.ConclusionsEVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aβ42, attenuated memory deficits, and reduced Aβ plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer’s disease.

Reduced GAP-43 message levels are associated with increased neurofibrillary tangle density in the frontal association cortex (area 9) in Alzheimer's disease.

We previously suggested the hypothesis that defective neuronal plasticity is a major neurobiological deficit causing the dementia of Alzheimers disease (AD). We used message levels of the growth-associated protein, GAP-43, as a marker of axonal plasticity to examine the hypothesis of defective neuronal plasticity in AD. When all AD cases are combined, the average level of GAP-43 message in area 9 of the AD frontal association cortex was not significantly different from the level in the comparably aged control cortex. Differentiation of AD cases on the basis of neurofibrillary tangle (NFT) density revealed that in AD cases with high tangle density average GAP-43 message level was reduced fivefold relative to levels in AD cases with low NFT density. AD cases with low neurofibrillary tangle density had levels of GAP-43 message that were not significantly different from the levels of normal controls. Differentiation of AD cases on the basis of neuritic plaque density did not indicate as strong a relationship to GAP-43 message level. The association between neurofibrillary tangle density and GAP-43 message level suggests the hypothesis that neurofibrillary tangles may reduce GAP-43 expression. Data of others show a relationship between high NFT density and reduced levels of synaptophysin-like immunoreactivity and reduced cerebral glucose metabolism. These data combine to suggest a set of AD cases with high NFT density, reduced axonal plasticity, reduced synaptic density, and reduced cerebral glucose metabolism--all variables that may be directly related to the functioning of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs

Regulatory recommendations for providing bioanalytical support for biological therapeutics have co-evolved with the increasing success of these unique pharmaceuticals. Immunoassays have been used to quantify biological macromolecules for more than 50 years. These assays rely on the use of antigen-specific antibodies. More recently, LC-MS methods have being adapted to quantitate biologics. LC-MS has attributes that complement the limitations encountered by immunoassays. Whether employing immunoassay or LC-MS methods, compared with traditional chemical-based therapeutics, biological therapeutics present unique analytical challenges to analysts. In this article, we review bioanalytical strategies for supporting biologics and discuss the regulatory and analytical challenges that must be met.

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif

Neurons produce polypeptides which can bind the calcium-poor or pre-activated form of calmodulin. It is expected that this class of peptide will serve an important role in maintaining cellular homeostasis since it would modulate calcium-dependent target regulation and redirect intracellular signaling. The lack of conserved sequence has made the identification of these peptides difficult, consequently leading us to exploit their property of binding calcium-poor calmodulin as a means of finding new species. A new peptide termed Calmodulin-Associated Peptide-19 (CAP-19) was purified and characterized. The protein-sequence information was employed in order to recover a cDNA clone from rat which included the entire reading frame for the peptide. Like its counterparts, neuromodulin (GAP-43), neurogranin (RC3) and PEP-19, it contains an IQ motif although the remainder of the peptide is quite different. Northern blot analysis of ribonucleic acid (RNA) from animals of differing ages indicated that the message appears at birth and then persists into adulthood. Antibodies to synthetic peptide were employed for localizing CAP-19. The results indicated that the peptide was localized to neurons in several brain regions. CAP-19 is similar to other calmodulin-binding proteins in that the domain spanning the IQ motif was demonstrated to participate in binding to calmodulin. Database searching showed CAP-19 to be homologous to the silkworm protein, multiprotein bridging factor 1 (MBF1). This homology suggests a potential new role for calmodulin-associated proteins in cellular homeostasis.

The human brain homeogene, DLX-2 : cDNA sequence and alignment with the murine homologue

A novel homeobox-containing cDNA from the developing human brain has been cloned and sequenced. The transcript is most closely related to the Distal-less (Dll) homeogene of Drosophila melanogaster and to the Dlx genes in the mouse, specifically to Dlx-2. As such, it is the first report of a human Dll-like gene.

Chapter 17: The neuropil and GAP-43/B-50 in normally aging and Alzheimer's disease human brain

Publisher Summary Morphological studies of dendritic extent in normal aging and Alzheimers disease (AD) have suggested the hypotheses that normally aging neurons are able to mount plastic, compensatory responses to the death of their neighbors. In AD, dendritic extent has been found to not increase with increasing age, even in regions where there is normally an age-related increase in dendritic extent, suggesting the hypothesis that one of the neurobiological deficits of AD is reduced neuronal plasticity. This chapter examines GAP-43/B50/F1 message levels as a potential marker of neuronal plasticity. Prevalence of this message as a function of normal aging also shows regional differences, with area 9 showing a decrease in GAP message level in normal aging, whereas data obtained from area 11 suggest no significant change of message level per cell in normal aging. Any decreases in GAP-43 message level found in AD brain may be accounted for on the basis of excess neuron loss in AD. However, preliminary data suggest that there may be defective posttranslational processing of GAP-43 in AD.

GSM treatment after the onset of Aβ deposition in mice reduces total Aβ load

Background:Alzheimer’s disease (AD) is a common, debilitating neurodegenerative disorder afflicting the aging population. Synaptic accumulation of Aß-protein oligomers and tau might be responsible for the neurological damage in AD. Thus, special interest has emerged in developing treatments that reduce the formation or facilitate the clearance of these oligomers. Utilizing molecular modeling and structure based design techniques we generated a series of novel organic heterocyclic compounds that are capable of recognizing the aggregated Aß-protein molecule and promoting clearance. Using cell-free and cell-based assays, a compound denominated NPT-400 with activity in the high nanomolar range was identified for pharmacokinetic (PK) and preliminary efficacy studies in APP transgenic (tg) mice. Methods: The mThy1-hAPP751 transgenic mouse was utilized for efficacy studies. This mouse combines the Swedish (K670M/N671L) and London (V717I) APPmutations, and these animals develop AD-like neuropathology and behavioral deficits starting at 3-4 months of age. Female non-tg and tg mice (w5 mo of age) received a single injection of vehicle or NPT-400-3 (IP, 10 mg/kg) three times per week for 6.5 weeks. Behavioral assessments were conducted during week 4 of treatment including locomotor activity and water maze. At the completion of treatment, brains were hemisected and tissue sections were processed for immunolabeling and confocal analysis, and homogenates were analyzed for levels of Abeta-protein by ELISA and immunoblot. For PK studies, wildtype C57Bl6 mice received IVor PO NPT-400-3 compound at 10 mg/kg and blood and plasma levels were analyzed at 0-24 hrs. Results: Treatment with NPT-400-3 resulted in a statistically significant normalization of locomotor activity accompanied by amelioration of the synaptic and dendritic pathology in the neocortex and hippocampus. By immunoblot and ELISA the levels of monomeric and oligomeric Aß-protein were statistically significantly reduced in the brains of the APP tg mice. PK studies showed that NPT400-3 is stable in plasma, is orally bioavailable and penetrates the brain with a B/P ratio of 0.8. Conclusions:NPT-400-3 is an orally bioavailable, brain penetrating Aß-protein stabilizing organic compound that reduces the accumulation of Aß-protein oligomers and ameliorates behavioral deficits and neuropathology in APP tg mice without overt adverse effects.

Pharmacokinetic and pharmacodynamic analysis of the gamma-secretase modulator (GSM) EVP-0015962

Background: CAD106 is an active immunotherapy developed for Alzheimer’s disease. It comprises amino acids 1-6 of Ab coupled to a virus-like particle derived from E. coli bacteriophage Qb. In APP overexpressing mice, administration of CAD106 induced Ab antibodies as well as a reduction of amyloid accumulation in the brain. Infusion of antibodies or active immunization against Ab have been shown to increase the level of total Ab in plasma indicating target engagement by the antibodies. In the present study we have investigated the active Ab immunotherapy CAD106 for its effect on plasma Ab. Methods: Cynomolgus monkeys aged 3 to 6 years were administered 600 mg CAD106 per subcutaneous or intramuscular injection at days 1, 14, 28 and every 4 weeks thereafter for a total of 24 weeks. Each dose contained either Alum or MF58 as adjuvant. Controls received adjuvant only. Total plasma Ab was determined at baseline and 12 to 14 days after the injections at week 8 and 24 using two different ELISAs and Western blotting. To minimize an interference of the CAD106-induced antibodies samples were SDSdenatured prior to quantification with an ELISA using a N-terminal capturing antibody. Alternatively, an assay combining C-terminal capture and mid-region detection antibodies was used. In addition, results were verified by direct SDS-PAGE separation of plasma followed by Western blotting. Results: Following active immunotherapy with CAD106, total plasma Ab levels increased compared to baseline and further between the intermediate and final time points of the study. Individual animals showed increases of up to about 50-fold over baseline. Median group increases reached up to 25-fold. Very similar results were obtained with the different assays. Overall the Ab levels in plasma correlated well with the Ab antibody titers. Conclusions: The increase in plasma Ab indicates an interaction of CAD106 induced antibodies with Ab in vivo. Its extent is in line with observations made with Nterminal monoclonal antibodies.

The impact of decreased bead count to determine concentrations of amyloid beta1‐42, total‐tau, and phosphorylated‐tau181 in human cerebrospinal fluid using xMAP technology

Alzheimers disease is the leading cause of human dementia. The lack of diagnostic tests and limited therapeutic options has driven the search for endogenous biomarkers. The INNO-BIA AlzBio3 assay is a multiplex flow-based immunoassay measuring Aβ42, tau, and p-tau in cerebrospinal fluid (CSF). This study assesses assays performance under varying bead count (BC) parameters. Original method validation parameters at 100 BC were acceptable. Reanalyses performed at 3, 10, 25, and 50 BCs were compared to 100 BC data by ANOVA, Bland-Altman analysis, evaluation of concordance correlation coefficients, and frequency distribution of coefficient of variation (CV) ranges. Method validation characteristics were acceptable with 100 BCs. Equivalency for 25 and 50 versus 100 BCs was demonstrated, but not for 3 and 10 BCs. A general trend of decreasing agreement between decreasing BCs and the 100 BC reference resulted in decreases in concordance coefficients ρ(c) . The frequency of CV values greater than 20% increased with decreasing BCs, and CV values of 5% or less decreased with decreased BCs. Statistical analyses demonstrate that BCs of 3 and 10 are not equivalent with the reference and should not be used as a basis for determination of Aβ42, tau, and p-tau concentration in human CSF.