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Biomedical subjects

David D Allen

Publications and source records attributed to David D Allen.

At least 19 recordsLinked to original sources
PubMed2006

Knockdown of amyloid precursor protein normalizes cholinergic function in a cell line derived from the cerebral cortex of a trisomy 16 mouse: An animal model of down syndrome.

We have generated immortal neuronal cell lines from normal and trisomy 16 (Ts16) mice, a model for Down syndrome (DS). Ts16 lines overexpress DS-related genes (App, amyloid precursor protein; Sod1, Cu/Zn superoxide dismutase) and show altered cholinergic function (reduced choline uptake, ChAT expression and fractional choline release after stimulation). As previous evidence has related amyloid to cholinergic dysfunction, we reduced APP expression using specific mRNA antisense sequences in our neuronal cell line named CTb, derived from Ts16 cerebral cortex, compared to a cell line derived from a normal animal, named CNh. After transfection, Western blot studies showed APP expression knockdown in CTb cells of 36% (24 hr), 40.4% (48 hr), and 50.2% (72 hr) compared to CNh. Under these reduced APP levels, we studied 3H-choline uptake in CTb and CNh cells. CTb, as reported previously, expressed reduced choline uptake compared to CNh cells (75%, 90%, and 69% reduction at 1, 2, and 5 min incubation, respectively). At 72 hr of APP knockdown, choline uptake levels were essentially similar in both cell types. Further, fractional release of 3H-choline in response to glutamate, nicotine, and depolarization with KCl showed a progressive increase after APP knockdown, reaching values similar to those of CNh after 72 hr of transfection. The results suggest that APP overexpression in CTb cells contributes to impaired cholinergic function, and that gene knockdown in CTb cells is a relevant tool to study DS-related dysfunction.

Acetylcholine↗
PubMed2006

Predictors of academic success in a doctor of pharmacy program.

OBJECTIVES: To evaluate the correlation between specific prepharmacy college variables and academic success in the Texas Tech doctor of pharmacy degree program. METHODS: Undergraduate and pharmacy school transcripts for 424 students admitted to the Texas Tech doctor of pharmacy degree program between May 1996 and May 2001 were reviewed in August of 2005. Statistical analyses were performed using SPSS Release 11.5. The undergraduate college variables included prepharmacy grade point-average (GPA), organic chemistry school type (2- or 4-year institution), chemistry, biology, and math courses beyond required prerequisites, and attainment of a bachelor of science (BS), bachelor of arts (BA), or master of science (MS) degree. Measurements of academic success in pharmacy school included cumulative first-professional year (P1) GPA, cumulative GPA (grade point average of all coursework finished to date), and graduation without academic delay or suspension. RESULTS: Completing advanced biology courses and obtaining a BS degree prior to pharmacy school were each significantly correlated with a higher mean P1 GPA. Furthermore, the mean cumulative GPA of students with a BS degree was 86.4 versus cumulative GPAs of those without a BS degree which were 84.9, respectively (p = 0.039). Matriculates with advanced prerequisite biology coursework or a BS degree prior to pharmacy school were significantly more likely to graduate from the doctor of pharmacy program without academic delay or suspension (p = 0.021 and p = 0.027, respectively). Furthermore, advanced biology coursework was significantly and independently associated with graduating on time (p = 0.044). CONCLUSIONS: Advanced biology coursework and a science baccalaureate degree were significantly associated with academic success in pharmacy school. On multivariate analysis, only advanced biology coursework remained a significant predictor of success.

Achievement↗
PubMed2006

Nicotine exposure does not alter plasma to brain choline transfer.

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(-) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.

Animals↗
PubMed2006

Optimizing the use of open-source software applications in drug discovery.

Drug discovery is a time consuming and costly process. Recently, a trend towards the use of in silico computational chemistry and molecular modeling for computer-aided drug design has gained significant momentum. This review investigates the application of free and/or open-source software in the drug discovery process. Among the reviewed software programs are applications programmed in JAVA, Perl and Python, as well as resources including software libraries. These programs might be useful for cheminformatics approaches to drug discovery, including QSAR studies, energy minimization and docking studies in drug design endeavors. Furthermore, this review explores options for integrating available computer modeling open-source software applications in drug discovery programs.

Computer-Aided Design↗
PubMed2005

Tobacco smoke chemicals attenuate brain-to-blood potassium transport mediated by the Na,K,2Cl-cotransporter during hypoxia-reoxygenation.

Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and cotinine attenuate abluminal (brain facing) K(+) uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K(+) movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCC-mediated K(+) uptake and paracellular integrity were measured with (86)Rb and [(14)C]sucrose, respectively. In addition, K(+) concentrations in brain extracellular fluid were estimated in (86)Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K(+) concentration measured as the brain extracellular fluid (86)Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.

Animals↗
PubMed2005

Molecular modeling of blood-brain barrier nutrient transporters: in silico basis for evaluation of potential drug delivery to the central nervous system.

For drugs that act in the brain, the blood-brain barrier (BBB) is a considerable physical barrier which influences the distribution of drugs to the brain. The BBB is essentially impermeable for hydrophilic and/or charged compounds. Nutrient membrane transporters have an important physiological role in the transport of essential substances across the BBB required for normal brain function. We and others have shown that these transporters may have utility as drug delivery vectors, thereby increasing brain distribution of these compounds via these systems. In this review, we evaluate molecular (in silico) models of BBB transport proteins. Few BBB membrane transporters have been crystallized, but their crystal structures have a possibility for use in homology modeling. Other techniques commonly used are 2D quantitative structure-activity relationships (QSAR), as well as 3D-QSAR techniques including comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA). Each of these models provides valuable information for ascertaining their potential basis for BBB transport and brain drug delivery.

Animals↗
PubMed2005

3D-QSAR study of bis-azaaromatic quaternary ammonium analogs at the blood-brain barrier choline transporter.

Previously, we have developed 3D-QSAR models of the blood-brain barrier (BBB) choline transporter, a transport system that may have utility as a vector for central nervous system drug delivery. In this study, we extended the model by evaluating five bis-azaaromatic quaternary ammonium compounds for their affinity for the choline binding site on the BBB-choline transporter. The compounds, and their affinities for the transporter, were then incorporated into our existing molecular model, in order to update our knowledge on the molecular recognition factors associated with interaction of ligands at the choline binding site. The current compounds are structurally related to previous substrates that we have evaluated, but offer additional three dimensional aspects compared to the series of compounds previously utilized to define the original models. The compounds showed good affinity for the BBB-choline transporter, exhibiting inhibition constants ranging from 10 to 68 microM, as determined by the in situ rat brain perfusion method. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the new 3D QSAR models. When the new bis-azaaromatic quaternary ammonium compounds were included in the model, the best cross-validated CoMFA q2 was found to be 0.536 and the non-cross-validated r2 was 0.818. CoMSIA hydrophobic cross-validated q2 was 0.506 and the non-cross-validated r2 was 0.804. This new model was able to better predict BBB-choline transporter affinity of hemicholinium-3 (predicted 65 microM, actual 54 microM), when compared to an earlier model (predicted 316 microM).

Animals↗
PubMed2005

Novel D-penicillamine carrying nanoparticles for metal chelation therapy in Alzheimer's and other CNS diseases.

Metal ions accumulate in the brain with aging and in several neurodegenerative diseases. Aside from the copper storage disease, Wilson's disease, recent attention has focused on the accumulation of zinc, copper and iron in the Alzheimer's disease (AD) brain and the accumulation of iron in Parkinson's disease. In particular, the parenchymal deposition of beta-amyloid (Abeta) and its interaction with metal ions has been postulated to play a role in the progression of AD. Thus, the strategy of lowering brain metal ions and targeting the interaction of Abeta peptide and metal ions through the administration of chelators has merit. Our recent finding that nanoparticle delivery systems can cross the blood-brain barrier has led us to investigate whether chelators delivered conjugated to nanoparticles could act to reverse metal ion induced protein precipitation. In the present studies, the Cu (I) chelator D-penicillamine was covalently conjugated to nanoparticles via a disulfide bond or a thioether bond. Nanoparticle-chelator conjugates were stable between pH 6-8 in aqueous suspension if stored at 4 degrees C, and did not aggregate when challenged with salts and serum. Release of D-penicillamine from the nanoparticles was achieved using reducing agents such as dithiothreitol (as a model for glutathione). Nanoparticles treated only under reducing conditions that released the conjugated D-penicillamine were able to effectively resolubilize copper-Abeta (1-42) aggregates. These results indicate that nanoparticles have potential to deliver D-penicillamine to the brain for the prevention of Abeta (1-42) accumulation, as well as to reduce metal ion accumulation in other CNS diseases.

Alzheimer Disease↗
PubMed2005

Cell lines as in vitro models for drug screening and toxicity studies.

Cell culture is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. The use of tissue culture is a valuable tool to study problems of clinical relevance, especially those related to diseases, screening, and studies of cell toxicity mechanisms. Ready access to the cells provides the possibility for easy studies of cellular mechanisms that may suggest new potential drug targets and, in the case of pathological-derived tissue, it has an interesting application in the evaluation of therapeutic agents that potentially may treat the dysfunction. However, special considerations must be addressed to establish stable in vitro function. In primary culture, these factors are primarily linked to greater demands of tissue to adequately survive and develop differentiated conditions in vitro. Additional requirements include the use of special substrates (collagen, laminin, extracellular matrix preparations, etc.), growth factors and soluble media supplements, some of which can be quite complex in their composition. These demands, along with difficulties in obtaining adequate tissue amounts, have prompted interest in developing immortalized cell lines which can provide unlimited tissue amounts. However, cell lines tend to exhibit problems in stability and/or viability, though they serve as a feasible alternative, especially regarding new potential applications in cell transplant therapy. In this regard, stem cells may also be a source for the generation of various cell types in vitro. This review will address aspects of cell culture system application, with focus on immortalized cell lines, in studying cell function and dysfunction with the primary aim being to identify cell targets for drug screening.

Animals↗
PubMed2005

Inhibition of choline uptake by N-cyclohexylcholine, a high affinity ligand for the choline transporter at the blood-brain barrier.

The blood-brain barrier (BBB) choline transporter (CHT) may have utility as a drug delivery vector for drugs that act in the central nervous system. Previous studies suggested the importance of hydrophobic moieties on the cationic nitrogen of choline for improved affinity for this transporter. In a pilot study, we therefore designed five novel N-cycloalkyl derivatives of choline, one of which showed promising inhibition properties. This choline analogue had a cyclohexyl (UMBB-5) moiety substituting one of the methyl groups attached to the cationic nitrogen in choline. In situ experimental data were obtained from in situ rat brain perfusion studies. The binding affinity for the BBB-choline transporter found for UMBB-5 was K(i)=1.9 microM. Comparative molecular field analysis (CoMFA) suggested that the cyclohexyl moiety orientates towards a steric favourable area. Taken together, the results of these in situ and in silico studies provide further evidence or restrictions that occur with binding to this brain drug delivery vector.

Animals↗
PubMed2004

Paclitaxel nanoparticles for the potential treatment of brain tumors.

Despite the advances in tumor therapy, patients with primary brain tumors and brain metastases have a very poor prognosis. Low responses to chemotherapy are mainly attributed to impermeability of the blood-brain barrier to cytotoxic agents. Paclitaxel has been shown to be active against gliomas and various brain metastases. However, its use in treatment of brain tumors is limited due to low blood-brain barrier permeability and serious side effects associated with administration of the paclitaxel solvent, Cremophor EL. Lack of paclitaxel brain uptake is thought to be associated with the p-glycoprotein (p-gp) efflux transporter. In this work, paclitaxel (PX) was entrapped in novel cetyl alcohol/polysorbate nanoparticles. Paclitaxel nanoparticles (PX NPs) were characterized by means of size, short-term stability, drug entrapment efficiency, and release profile. The PX NP cytotoxicity profile was monitored using two different cell lines, U-118 and HCT-15. Brain uptake of PX NPs was evaluated using an in situ rat brain perfusion model. The results suggest that entrapment of paclitaxel in nanoparticles significantly increases the drug brain uptake and its toxicity toward p-glycoprotein expressing tumor cells. It was hypothesized that PX NPs could mask paclitaxel characteristics and thus limit its binding to p-gp, which consequently would lead to higher brain and tumor cell uptake of the otherwise effluxed drug.

Adjuvants, Pharmaceutic↗
PubMed2004

Molecular modeling studies on the active binding site of the blood-brain barrier choline transporter.

The blood-brain barrier choline transporter may have utility as a drug delivery vector to the central nervous system. Surprisingly, this transporter has as yet not been cloned and expressed. We therefore initiated a 3D-QSAR study to develop predictive models for compound binding and identify structural features important for binding to this transporter. In vivo experimental data were obtained from in situ rat brain perfusion studies. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the models. The best cross-validated CoMFA q(2) was found to be 0.47 and the non-cross-validated r(2) was 0.95. CoMSIA hydrophobic cross-validated q(2) was 0.37 and the non-cross-validated r(2) was 0.85. These models rendered a useful approximation for binding requirements in the BBB-choline transporter and, until such time as the cloned transporter becomes available, may have significant utility in developing a predictive model for the rational design of drugs targeted to the brain.

Animals↗
PubMed2004

Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release.

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood-brain barrier choline transporter.

Aconitine↗
PubMed2004

Cation transport specificity at the blood-brain barrier.

UNLABELLED: The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. METHOD: Cross-inhibition of [3H]choline and [3H]thiamine brain uptake by in situ rat brain perfusion. RESULTS: [3H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 microM inhibited [3H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. CONCLUSION: (1) The molecular family of the blood-brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.

Algorithms↗
PubMed2004

Nanoparticle surface charges alter blood-brain barrier integrity and permeability.

PURPOSE: The blood-brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability. METHODS: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion. RESULTS: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations. CONCLUSIONS: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.

Animals↗
PubMed2003

Brain uptake of thiamine-coated nanoparticles.

Recently, a novel nanoparticle (NP) comprised of emulsifying wax and Brij 78 was shown to have significant brain uptake using the in-situ rat brain perfusion technique. To further these studies and to specifically target brain, we have incorporated thiamine as a surface ligand on the nanoparticles. Solid nanoparticles were prepared from oil-in-water microemulsion precursors. Nanoparticles were radiolabeled and a thiamine ligand (thiamine linked to distearoylphosphatidylethanolamine via a polyethylene glycol spacer) was coated on the surface of the nanoparticles. Initial experiments focused on assessing uptake of [3H]nanoparticles with and without thiamine surface ligands. Biodistribution nanoparticle studies were also carried out in BALB/c mice. The results showed: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of nanoparticles with and without the thiamine surface ligands, and (3) initial data suggesting mechanisms for nanoparticle brain entry. Comparison of NP brain uptake demonstrated that the thiamine-coated nanoparticle associated with the blood-brain barrier (BBB) thiamine transporter and had an increased K(in) between 45 and 120 s (thiamine coated NP 9.8 +/- 1.1 x 10(-3) ml/s/g versus uncoated NPs; 7.0 +/- 0.3 x 10(-3) ml/s/g). It was concluded that the thiamine ligand facilitated binding and/or association with blood-brain barrier thiamine transporters, which may be a viable mechanism for nanoparticle mediated brain drug delivery.

Animals↗
PubMed2003

Manganese distribution across the blood-brain barrier. I. Evidence for carrier-mediated influx of managanese citrate as well as manganese and manganese transferrin.

Manganese (Mn) is an essential element and a neurotoxicant. Regulation of Mn movement across the blood-brain barrier (BBB) contributes to whether the brain Mn concentration is functional or toxic. In plasma, Mn associates with water, small molecular weight ligands and proteins. Mn speciation may influence the kinetics of its movement through the BBB. In the present work, the brain influx rates of 54Mn2+, 54Mn citrate and 54Mn transferrin (54Mn Tf) were determined using the in situ brain perfusion technique. The influx rates were compared to their predicted diffusion rates, which were determined from their octanol/aqueous partitioning coefficients and molecular weights. The in situ brain perfusion fluid contained 54Mn2+, 54Mn citrate or 54Mn Tf and a vascular volume/extracellular space marker, 14C-sucrose, which did not appreciably cross the BBB during these short experiments (15-180 s). The influx transfer coefficient (Kin) was determined from four perfusion durations for each Mn species in nine brain regions and the lateral ventricular choroid plexus. The brain Kin was (5-13) x 10(-5), (3-51) x 10(-5), and (2-13) x 10(-5) ml/s/g for 54Mn2+, 54Mn citrate, and 54Mn Tf, respectively. Brain Kin values for any one of the three Mn species generally did not significantly differ among the nine brain regions and the choroid plexus. However, the brain Kin for Mn citrate was greater than Mn2+ and Mn Tf Kin values in a number of brain regions. When compared to calculated diffusion rates, brain Kin values suggest carrier-mediated brain influx of 54Mn2+, 54Mn citrate and 54Mn Tf. 55Mn citrate inhibited 54Mn citrate uptake, and 55Mn2+ inhibited 54Mn2+ uptake, supporting the conclusion of carrier-mediated brain Mn influx. The greater Kin values for Mn citrate than Mn2+ and its presence as a major non-protein-bound Mn species in blood plasma suggest Mn citrate may be a major Mn species entering the brain.

Animals↗