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Susanna Tse,1 Kendall D Powell,2 Stephen MacLennan,3 Allan R Moorman,4 Craig Paterson,5 Rosonald R Bell11Pfizer Inc, Groton, CT, USA; 2Tandem Labs, Durham, NC, USA; 3BioCryst Pharmaceuticals Inc, Durham, NC, USA; 4Alta Vetta Pharmaceutical Consulting LLC, Durham, NC, USA; 5Salix Pharmaceuticals Inc, Raleigh, NC, USAPurpose: This study compared the pharmacokinetic profile, and systemic and local absorption of diclofenac, following dermal patch application and oral administration in Yorkshire- Landrace pigs.Patients and methods: Twelve anesthetized, female, Yorkshire-Landrace pigs were randomized to receive either the dermal patch (FLECTOR® patch, 10 × 14 cm; Alpharma Pharmaceuticals, a subsidiary of Pfizer Inc, New York, NY) or 50 mg oral diclofenac (Voltaren®; Novartis, East Hanover, NJ). Tissue (skin area of 2 × 2 cm and underlying muscles approximately 2–3 cm in depth) and blood (10 mL) samples were collected at timed intervals up to 11.5 hours after initial patch application or oral administration. The concentrations of diclofenac in plasma, skin, and muscle samples were analyzed using validated ultra performance liquid chromatography tandem mass spectrometric methods.Results: Peak systemic exposure of diclofenac was very low by dermal application compared with oral administration (maximum concentration [Cmax] values of 3.5 vs 9640 ng/mL, respectively). Absorption of diclofenac into underlying muscles beneath the dermal patch was sustained, and followed apparently zero-order kinetics, with the skin serving as a depot with elevated concentrations of diclofenac. Concentrations of diclofenac in muscles beneath the patch application site were similar to corresponding tissues after oral administration (Cmax values of 879 and 1160 ng/mL, respectively). In contrast to the wide tissue distribution of diclofenac after oral administration, dermal patch application resulted in high concentrations of diclofenac only on the treated skin and immediate tissue underneath the patch. Low concentrations of diclofenac were observed in the skin and muscles collected from untreated areas contralateral to the site of dermal patch application.Conclusion: Dermal patch application resulted in low systemic absorption and high tissue penetration of diclofenac compared with oral administration.Keywords: NSAIDs, systemic absorption, topical patch, tissue distribution

Aims: Heparan sulfate (HS) accumulates in the central nervous system in mucopolysaccharidosis III type A (MPS IIIA). A validated LC-MS/MS assay was developed to measure HS in human cerebrospinal fluid (CSF)., Methods & Results: HS was extracted and digested and the resultant disaccharides were derivatized with a novel label, 4-butylaniline, enabling isoform separation and isotope-tagged analog introduction as an internal standard for LC-MS/MS. The assay has a LLOQ for disaccharides of 0.1 μM, ±20% accuracy and ≤20% precision. CSF samples from patients with MPS IIIA showed elevated HS levels (mean 4.9 μM) compared with negative controls (0.37 μM)., Conclusion: This assay detected elevated HS levels in the CSF of patients with MPS IIIA and provides a method to assess experimental therapies.

Purpose: To investigate the ocular distribution of 1% azithromycin ophthalmic solution and the effect of polycarbophil-based mucoadhesive formulation on ocular tissue levels of azithromycin after single and multiple topical administrations in the rabbit eye., Methods: Rabbits were treated with either a single administration of 1% azithromycin solution with or without polycarbophil, or with multiple administrations of 1% azithromycin solution in polycarbophil. Drug concentrations were measured using LC/MS/MS. Conjunctiva, cornea, aqueous humor, and tear samples were analyzed over a period of 144 h after a single administration of azithromycin with or without polycarbophil. Eyelid, conjunctiva, cornea, aqueous humor, and tear samples were collected over a period of 288 h during and after multiple administrations of azithromycin., Results: Azithromycin was rapidly absorbed and distributed in the ocular tissues, reaching within 5 min, concentrations of 10,539 microg/mL in tear film, 108 microg/g in conjunctiva, and 40 microg/g in the cornea. The drug demonstrated tissue-specific half-lives of 15, 63, and 67 h, respectively. Following multiple administrations, the drug gradually accumulated. The polycarbophil formulation increased the bioavailability of the drug, producing peak concentrations that were between 5- and 12-fold higher than those without polycarbophil. Azithromycin also distributed rapidly in the eyelids, reaching peak concentrations of 180 mug/g at the end of the 7-day treatment, and was eliminated with a half-life of 125 h. Six days after treatment was discontinued, eyelid levels of azithromycin were above 40 microg/g., Conclusions: Sustained and high concentrations were encountered with 7-day approved administration of 1% azithromycin formulation (AzaSite, Inspire Pharmaceuticals, Inc., Durham, NC) within all ocular surface tissues, particularly the lids. Many ocular surface disorders involving the tear film, eyelids, and adnexal structures are associated with chronic, low-grade bacterial infection and may potentially lead to decreased vision secondary to corneal scarring. Various topical antibiotic and steroid combinations with or without oral tetracyclines are commonly used with variable clinical response and known potential side effects. The clinical relevance of this study is unknown; however, the long-lasting antibacterial and additional anti-inflammatory properties of topical azithromycin might offer an effective alternative treatment option and should be explored further in clinical studies.

Neural activity in the lateral intraparietal area (LIP) has been associated with attention to a location in visual space, and with the intention to make saccadic eye movement. In this study we show that neurons in LIP respond to recently flashed task-irrelevant stimuli and saccade targets brought into the receptive field by a saccade, although they respond much to the same stimuli when they are stable in the environment. LIP neurons respond to the appearance of a flashed distractor even when a monkey is planning a memory-guided delayed saccade elsewhere. We then show that a monkey's attention, as defined by an increase in contrast sensitivity, is pinned to the goal of a memory-guided saccade throughout the delay period, unless a distractor appears, in which case attention transiently moves to the site of the distractor and then returns to the goal of the saccade. LIP neurons respond to both the saccade goal and the distractor, and this activity correlates with the monkey's locus of attention. In particular, the activity of LIP neurons predicts when attention migrates from the distractor back to the saccade goal. We suggest that the activity in LIP provides a salience map that is interpreted by the oculomotor system as a saccade goal when a saccade is appropriate, and simultaneously is used by the visual system to determine the locus of attention.

Two-dimensional arrays of microlenses can be used in wearable display applications as numerical aperture expanders or exit-pupil expanders (EPEs) to increase the size of the display exit pupil. A novel EPE approach that uses two microlens arrays (MLAs) is presented. The approach is based on cascading two identical microlens arrays spaced precisely at one focal-length distance with submicrometer registration tolerances relative to each other. The ideal MLA for this application requires a 100% fillfactor, sharp seams between microlenses, and a perfect spherical profile. We demonstrate a dual-MLA-based EPE that produces excellent exit-pupil uniformity and better than 90% diffraction efficiency for all three wavelengths in a color-display system. Two-MLA registration is performed with submicrometer precision by use of far-field alignment techniques. Fourier optics theory is used to derive the analytical formulas, and physical optics beam propagation is used for numerical computations. Three MLA fabrication technologies, including gray-scale lithography, photoresist reflow, and isotropic etching, are evaluated and compared for an EPE application.

The visual system uses the pattern of motion on the retina to analyze the motion of objects in the world, and the motion of the observer him/herself. Distinguishing between retinal motion evoked by movement of the retina in space and retinal motion evoked by movement of objects in the environment is computationally difficult, and the human visual system frequently misinterprets the meaning of retinal motion. In this study, we demonstrate that the visual system of the Rhesus monkey also misinterprets retinal motion. We show that monkeys erroneously report the trajectories of pursuit targets or their own pursuit eye movements during an epoch of smooth pursuit across an orthogonally moving background. Furthermore, when they make saccades to the spatial location of stimuli that flashed early in an epoch of smooth pursuit or fixation, they make large errors that appear to take into account the erroneous smooth eye movement that they report in the first experiment, and not the eye movement that they actually make.

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA. As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)). The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.

Here, we describe a new protein-ligand binding assay that is amenable to high-throughput screening applications. The assay involves the use of SUPREX (stability of unpurified proteins from rates of H/D exchange), a new H/D exchange and mass spectrometry-based technique we recently developed for the quantitative analysis of protein-ligand binding interactions. As part of this work, we describe a new high-throughput SUPREX protocol, and we demonstrate that this protocol can be used to efficiently screen peptide ligands in a model combinatorial library for binding to a model protein system, the S-protein. The high-throughput SUPREX protocol developed here is generally applicable to a wide variety of protein ligands, including DNA, small molecules, metals, and other proteins. On the basis of the results of the model study in this work, one person with access to one MALDI mass spectrometer should be able to screen approximately 10 000 compounds per 24-h period using the protocol described here. With full automation and the use of a commercially available MALDI mass spectrometer optimized for high-throughput analyses, we estimate that the SUPREX-based assay described here could be used to screen on the order of 100 000 ligands per day.

A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.

A new method that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the hydrogen/deuterium (H/D) exchange properties of proteins was developed for measuring the thermodynamic properties of protein-ligand complexes in solution. Dissociation constants (Kd values) determined by the method for five model protein-ligand complexes that included those with small molecules, nucleic acids, peptides, and other proteins were generally in good agreement with Kd values measured by conventional methods. Important experimental advantages of the described method over existing methods include: the ability to make measurements in a high-throughput and automated fashion, the ability to make measurements using only picomole quantitities of protein, and the ability to analyze either purified or unpurified protein-ligand complexes.

The brain cannot monitor or react towards the entire world at a given time. Instead, using the process of attention, it selects objects in the world for further analysis. Neuronal activity in the monkey intraparietal area has the properties appropriate for a neuronal substrate of attention: instead of all objects being represented in the parietal cortex, only salient objects are. Such objects can be salient because of their physical properties (recently flashed objects or moving objects) or because they can be made important to the animal by virtue of a task. Although lateral intraparietal area (LIP) neurons respond through the delay period of a memory-guided saccade, they also respond in an enhanced manner to distractors flashed during the delay period of a memory-guided saccade being generated to a position outside the receptive field. This activity parallels the monkey's psychophysical attentional process: attention is ordinarily pinned at the goal of a memory-guided saccade, but it shifts briefly to the locus of a task-irrelevant distractor flashed briefly during the delay period and then returns to the goal. Although neurons in LIP have been implicated as being directly involved in the generation of saccadic eye movements, their activity does not predict where, when, or if a saccade will occur. The ensemble of activity in LIP, however, does accurately describe the locus of attention.

We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods.

Recently, we reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique, termed SUPREX, that can be used to measure the thermodynamic stability of a protein (Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296-8301). In the work described here, we report on our efforts to optimize the sensitivity of SUPREX analyses. We describe a new sample handling protocol for SUPREX that involves the use of batch chromatography methods with reversed-phase chromatographic media for the microconcentration and desalting of SUPREX samples. Using ribonuclease A as a model protein system, we demonstrate that our new protocol permits the SUPREX analysis of as little as 10 pmol of protein. This amount of protein is 100-fold less than the amount of material required in our initial SUPREX protocol, and it is 1-2 orders of magnitude less than the amount of material required in conventional spectroscopy-based methods for measuring the thermodynamic stability of a protein.

Recent experiments raised the possibility that the lateral intraparietal area (LIP) might be specialized for saccade planning. If this was true, one would expect a decreased sensitivity to irrelevant visual stimuli appearing late in the delay period of a memory-guided delayed-saccade task to a target outside the neurons' receptive fields. We trained two monkeys to perform a standard memory-guided delayed-saccade task and a distractor task in which a stimulus flashed for 200 ms at a predictable time 300-100 ms before the end of the delay period. We used two locations, one in the most active part of the receptive field and another well outside the receptive field. We used six kinds of trials randomly intermixed: simple delayed-saccade trials into or away from the receptive field and distractor trials with saccade target and distractor both in the receptive field, both out of the receptive field, or one at each location. This enabled us to study the response to the distractor as a function of the monkey's preparation of a memory-guided delayed-saccade task. We had assumed that the preparation of a saccade away from the receptive field would result in an attenuation of the response to the distractor, i.e., a distractor at the location of the saccade goal would evoke a greater response than when it appeared at a location far from the saccade goal. Instead we found that neurons exhibited either a normal or an enhanced visual response to the distractor during the memory period when the target flashed outside the receptive field. When the distractor flashed at the location of the saccade target, the response to the distractor was either unchanged or diminished. The response to a distractor away from the target location of a memory-guided saccade was even greater than the response to the same target when it was the target for the memory-guided saccade task. Immediate presaccadic activity did not distinguish between a saccade to the receptive field when there was no distractor and a distractor in the receptive field when the monkey made a saccade elsewhere. Nonetheless the distractor had no significant effect on the saccade latency, accuracy, or velocity despite the brisk response it evoked immediately before the saccade. We suggest that these results are inconsistent with a role for LIP in the specific generation of saccades, but they are consistent with a role for LIP in the generation of visual attention.

The ability of the vestibulo-ocular reflex (VOR) to alter the phase of the motor output relative to the sensory input is examined. Alert cats were trained for 2 h with 0.25 Hz sinusoidal horizontal vestibular and vertical optokinetic rotational stimuli. In each experiment the optokinetic training stimulus was phase shifted by 0 degree, +45 degrees, -45 degrees, or 90 degrees from the vestibular stimulus. Vertical and horizontal eye movements were measured during horizontal rotations in darkness before and after the training procedure. Phase-advance experiments (+45 degrees) produced an adaptive vertical VOR with a mean phase of +28 degrees. After phase-delay experiments (-45 degrees), the adapted VOR had a mean phase of -19 degrees. The peak adaptive change in VOR gain was at or near the 0.25 Hz training frequency in each experimental group, but the gain depended in a complex manner on the testing frequency and the degree of phase shift of the training stimulus. Training with a 45 degrees phase-delayed optokinetic stimulus produced an adaptive vertical VOR with a gain that was relatively higher at frequencies below the training stimulus than at those that were above. Training with a 45 degrees phase-advanced optokinetic stimulus produced an adaptive vertical VOR with a gain that was higher at frequencies above the training frequency than at those that were below. During training with a phase-shifted optokinetic stimulus, adjustment of the relative efficacies of two neural pathways, a velocity pathway and an integrating pathway, could account for gain dependence on testing frequency and phase shift. This was corroborated by a model of the VOR that incorporates parallel velocity and integrating pathways. Data from 45 degrees phase advances were fit by increasing the gain of the velocity versus integrating pathway, whereas 45 degrees phase delay data were fit by decreasing the gain of the direct versus integrating pathway. The models altered the time constants of either the common oculomotor integrator or the velocity storage mechanism.

Responses of 35 Purkinje cells in decerebrate cats were recorded during 0.5 Hz rotations in 4-11 vertical planes and the horizontal plane to determine their semicircular canal input. Most neurons received convergent input from two canals (21 neurons) or 3 canals (5 neurons). Few Purkinje cells were maximally sensitive to rotations about an axis appropriate to their inferior olivary input as determined by Gerrits and Voogd [15,24,27,49].

Vertical and horizontal vestibulo-ocular reflex (VOR) eye movements were recorded in alert cats during horizontal rotation in total darkness before and after a 2 h vestibulo-ocular reflex direction adaptation procedure. Adaptation stimuli were whole body horizontal vestibular rotation coupled to synchronous vertical optokinetic motion. The waveform of the adaptation stimuli was either a sinusoid at 0.05, 0.1, 0.25, 0.5, or 1 Hz, or a sum of sinusoids containing 0.2, 0.3, 0.5, 0.7, 1.1, and 1.7 Hz. Exposure to single frequency stimuli produced adaptive vertical VOR with a gain that was greatest near the training frequency; adaptive VOR phases were advanced below, accurate at, and lagged above the training frequency. Exposure to the multifrequency waveform produced a uniform modest increase in gain across frequencies, with accurate adaptive VOR phase.