Fast Surface Crystallization of Amorphous Griseofulvin Below <Emphasis Type="Italic">T</Emphasis><Subscript>g</Subscript>

Purpose  

To study crystal growth rates of amorphous griseofulvin (GSF) below its glass transition temperature (T _g) and the effect of surface crystallization on the overall crystallization kinetics of amorphous GSF.     

ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion

AMPA-type glutamate receptor (AMPAR) trafficking is essential for modulating synaptic transmission strength. Prior studies that have characterized signaling pathways underlying AMPAR trafficking have identified the cAMP/PKA-mediated phosphorylation of GluA1, an AMPAR subunit, as a key step in the membrane insertion of AMPAR. Inhibition of ERK impairs AMPAR membrane insertion, but the mechanism by which ERK exerts its effect is unknown. Dopamine, an activator of both PKA and ERK, induces AMPAR insertion, but the relationship between the two protein kinases in the process is not understood. We used a combination of computational modeling and live cell imaging to determine the relationship between ERK and PKA in AMPAR insertion. We developed a dynamical model to study the effects of phosphodiesterase 4 (PDE4), a cAMP phosphodiesterase that is phosphorylated and inhibited by ERK, on the membrane insertion of AMPAR. The model predicted that PKA could be a downstream effector of ERK in regulating AMPAR insertion. We experimentally tested the model predictions and found that dopamine-induced ERK phosphorylates and inhibits PDE4. This regulation results in increased cAMP levels and PKA-mediated phosphorylation of DARPP-32 and GluA1, leading to increased GluA1 trafficking to the membrane. These findings provide unique insight into an unanticipated network topology in which ERK uses PDE4 to regulate PKA output during dopamine signaling. The combination of dynamical models and experiments has helped us unravel the complex interactions between two protein kinase pathways in regulating a fundamental molecular process underlying synaptic plasticity.The strength of synaptic transmission depends on the number of AMPA-type glutamate receptors (AMPARs) localized to the synaptic membrane. The regulated trafficking of AMPARs in and out of the postsynaptic membrane controls the number of synaptic AMPARs and is thought to underlie synaptic plasticity (1). AMPARs are composed of four subunits (GluA1–4), which assemble as homo- or hetero-tetramers to mediate excitatory transmissions in the brain. There are a number of intracellular pathways that regulate signal-initiated trafficking of GluA1-containing AMPARs. For instance, PKA and PKG, the cyclic nucleotide-activated kinases, phosphorylate GluA1 at S845 (2, 3). Phosphorylation of S845 is required for GluA1 synaptic insertion because mutation to A845 prevents GluA1 exocytosis (4).Dopamine, a modulatory neurotransmitter that increases cAMP/PKA levels, promotes GluA1 phosphorylation at S845 and AMPAR insertion into the plasma membrane (3, 5, 6). Additional signaling pathways influence this process, but the role they play in dopamine-mediated AMPAR trafficking is not known. ERK, a downstream effector of dopamine, promotes AMPAR membrane insertion even though ERK does not directly phosphorylate GluA1 (7, 8). The objective of this study was to identify the mechanism by which ERK regulates dopamine-mediated GluA1 membrane insertion. Based on our observation that ERK inhibition decreases dopamine-mediated GluA1 phosphorylation at S845, we looked for ERK substrates that could affect cAMP levels. One possibility was that ERK phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) could increase PKC activity, leading to activation of AC5, the main adenylyl cyclase in the striatum (9). Another substrate of ERK that could affect GluA1 trafficking is phosphodiesterase 4 (PDE4), a phosphodiesterase phosphorylated and inhibited by ERK (10). We tested for the involvement of both ERK substrates on GluA1 membrane insertion. We developed a computational model to explore the ERK regulation of dopamine-induced GluA1 membrane insertion. The model predictions were validated experimentally by monitoring dopamine-stimulated cAMP levels and GluA1 trafficking by live cell imaging in striatal primary neurons. The data presented here show that dopamine-activated ERK increases cAMP levels by phosphorylation and inhibition of PDE4 and results in the elevation of PKA mediated GluA1 phosphorylation and membrane insertion. Our approach allowed us to unravel the complex interaction between PKA and ERK pathways within the dopamine-signaling network.     

The Role of Noninvasive Imaging in Coronary Artery Disease Detection,Prognosis, and Clinical Decision Making

A vast array of noninvasive imaging modalities is available for the evaluation of the presence and severity of coronary artery disease (CAD). Choosing the right test can be challenging but is critical for proper patient diagnosis and management. Presently available imaging tests for CAD include: (1) nuclear myocardial perfusion imaging procedures (single-photon emission tomography) and positron emission tomography, (2) stress echocardiography, (3) computed tomography coronary angiography, and (4) cardiac magnetic resonance imaging. Exercise treadmill testing electrocardiography is another alternative that we will discuss briefly. Selection of the most appropriate imaging modality requires knowledge of the clinical question being addressed, patient characteristics (pretest probability and prevalence of disease), the strengths, limitations, risks, costs, and availability of each procedure. To assist with test selection, we review the relevant literature in detail to consider the relative merits of cardiac imaging modalities for: (1) detection of CAD, (2) risk stratification and prognostication, and (3) guiding clinical decision making.     

Survival From In-hospital Cardiac Arrest on the Internal Medicine Clinical Teaching Unit

Background

There is a paucity of data on patient outcomes following in-hospital cardiac arrest (IHCA) on the Internal Medicine clinical teaching unit (CTU). Accurate outcome data enhances discussions between patients, surrogates, and physicians, and assists in their management.

Methods

We performed a retrospective cohort study of consecutive “Code Blue” calls on 2 medical CTUs in a Canadian tertiary centre from January 1, 2003 to June 30, 2007. The medical records of identified patients were screened for eligibility and patient-specific and arrest-specific data were collected for eligible events. Primary outcome was survival to hospital discharge.

Results

Our cohort comprised 83 patients; including 54 (65.1%) men with a mean age of 75 years (range, 38-97). Infection (34.9%) was the principal reason for admission and over half of patients had 3 or more comorbid illnesses. Forty-three (51.8%) of the IHCA events were witnessed. In all, 39 (90.7%) of the witnessed and 36 (90%) of the unwitnessed arrests were pulseless electrical activity (PEA) or asystole (P = not significant). Return of spontaneous circulation occurred in 29 patients (34.9%) and 2 (2.4%) survived to hospital discharge. No patients survived to discharge after unwitnessed arrest.

Conclusions

IHCA in Internal Medicine CTU patients is characterized by a high rate of PEA/asystole and a minimal chance of survival to hospital discharge. Moreover, no patient with an unwitnessed arrest survived to hospital discharge. While these findings require confirmation in a larger study, they merit consideration in the context of code status discussions, particularly with respect to the response to unwitnessed arrests.     

Population pharmacokinetics of lamotrigine in Indian epileptic patients

Purpose

The aim of this analysis was to describe the pharmacokinetics of oral lamotrigine (LTG) in Indian epileptic patients using a population pharmacokinetic (PPK) modeling approach to confirm that the PK is similar to that of the Caucasian population, and to evaluate and confirm the impact of covariates predictive of inter-individual variability using a simulation platform.

Methods

Blood samples were obtained from 95 patients, and LTG plasma concentrations were determined. Population PK modeling was performed using NONMEM. A one-compartment PK model with first-order absorption and elimination was used to describe the LTG PK. Log-likelihood profiling and normalized prediction distribution errors (NPDE) were used for model evaluation. A simulation study was performed to investigate dose regimens.

Results

Clearance (CL) was estimated to be 2.27 L/h with inter-individual variability (IIV) of 29 CV%. Volume of distribution (V) was estimated to be 53.6 L (31 CV% IIV). Body weight and concurrent use of carbamazepine and valproate were identified as significant covariates on clearance. Log-likelihood profiling indicated that parameters could be estimated with adequate precision, and NPDE indicated that the model adequately described the data observed. The simulation study illustrated the impact of carbamazepine and valproate on LTG PK, and negligible differences in PK between Indian and Caucasian patients.

Conclusions

This is the first PK analysis of LTG in Indian patients. The population PK model developed adequately described the data observed. Comparison of identified PK parameters with previous PK analyses in Caucasian patients indicates that CL of LTG is similar, while V is somewhat lower compared with Caucasian patients, but this is not expected to lead to relevant differences in PK profiles during steady state.     

Effect of pH and Excipients on Structure, Dynamics, and Long-Term Stability of a Model IgG1 Monoclonal Antibody upon Freeze-Drying

Purpose

To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.

Methods

IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR ¹H relaxation was used to monitor glassy state dynamics.

Results

IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.

Conclusions

Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.     

Evaluating new therapies in gastrointestinal stromal tumor using in vivo molecular optical imaging

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the US. The majority (~85%) of GISTs possess gain-of-function mutations in KIT or PDGFRA, causing constitutive activation of the kinase receptor. GIST management has been transformed by the identification of tumor driver mutations leading to unprecedented disease control of advanced GIST with the introduction of imatinib mesylate (IM). Despite IM’s efficacy, most patients experience primary and/or secondary resistance within 2 y of treatment. Additional therapies and methods to optimize screening of novel approaches in preclinical studies are warranted. Clinically, treatment efficacy is typically assessed using Response Evaluation Criteria In Solid Tumors (RECIST) guidelines or Choi criteria. Both require a period of time on therapy before changes indicative of response can be observed. In addition, neither informs directly about cell death. We evaluated the use of molecular imaging technology in an animal model using near-infrared (NIR) imaging probes together with three-dimensional fluorescence molecular tomography (FMT) for assessing therapeutic response and ultimately optimizing our understanding of the biologic effects of these agents. We determined the potential of NIR probes (PSVue^TM794 and cell-penetrating KcapQ647) for detecting distinct markers of apoptosis and compare this to tumor size measured by MRI in response to IM treatment in GIST-T1 xenografts. Our studies revealed statistically significant increases in apoptosis due to IM treatment using both probes as early as 24 h post IM treatment which was confirmed by IHC. Molecular imaging will allow for faster and more effective screening of novel therapies in preclinical GIST models.     

PGD2 and CRTH2 counteract Type 2 cytokine–elicited intestinal epithelial responses during helminth infection

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D₂ (PGD₂) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD₂ and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine–mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD₂–CRTH2 pathway negatively regulates the Type 2 cytokine–driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.