Serlopitant for the treatment of chronic pruritus: Results of a randomized, multicenter, placebo-controlled phase 2 clinical trial.

BACKGROUND: The substance P/neurokinin 1 receptor pathway is critical in chronic pruritus; anecdotal evidence suggests that antagonism of this pathway can reduce chronic itch. OBJECTIVE: To assess the safety and efficacy of the substance P/neurokinin 1 receptor antagonist serlopitant in treating chronic pruritus. METHODS: Eligible patients with severe chronic pruritus who were refractory to antihistamines or topical steroids were randomized to serlopitant, 0.25, 1, or 5 mg, or to placebo, administered once daily for 6 weeks as monotherapy or with midpotency steroids and emollients. The primary efficacy end point was percentage change in visual analog scale pruritus score from baseline. RESULTS: Serlopitant treatment resulted in a dose-dependent decrease in pruritus. The mean percentage decreases from baseline visual analog scale pruritus scores were statistically significantly larger with the 1- and 5-mg doses of serlopitant (P = .022 and P = .013, respectively) than with placebo at week 6. No significant safety or tolerability differences were detected among the groups. LIMITATIONS: The sample size was insufficient for subgroup analyses of the efficacy of serlopitant for chronic pruritus on the basis of underlying conditions. CONCLUSIONS: Serlopitant, 1 mg and 5 mg daily, was associated with a statistically significant reduction in chronic pruritus and was well tolerated (NCT01951274).

Free-solution electrophoretic separations of DNA-drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength.

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.

Early in vitro transcription termination in human H5 influenza viral RNA synthesis.

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5' SP6 promoter and 3' restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

Circulating IgSF proteins inhibit adhesion of antibody targeted microspheres to endothelial inflammatory ligands.

Proposed methods for detecting circulatory system disease include targeting ultrasound contrast agents to inflammatory markers on vascular endothelial cells. For antibody-based therapies, soluble forms of the targeted adhesion proteins of the immunoglobulin superfamily (IgSF) reduce adhesion yet were left unaccounted in prior reports. Microspheres labeled simply with a maximum level of antibodies can reduce the diagnostic sensitivity by adhering to proteins expressed normally at a low level, while sparsely coated particles may be rendered ineffective by circulating soluble forms of the targeted proteins. A new microdevice technique is applied to simultaneously measure the adhesion profile to a series of IgSF-protein-coated surfaces. In this investigation, we quantify the in vitro binding characteristics of 5-microm microspheres to oriented intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein-coated surfaces in the presence of human serum at physiological concentrations. Defined regions of a slide were coated with recombinant chimeric Fc-human ICAM-1 and VCAM-1 in variable ratios but constant total concentration. Monoclonal human anti-ICAM-1 or anti-VCAM-1 antibodies in competition with non-binding mouse anti-rabbit antibodies coat the microsphere surface at a constant surface density with variable yet controlled surface activities. Using multiple slide surface IgSF protein and microsphere antibody concentrations, an adhesion profile was developed for the microspheres with and without IgSF proteins from human serum, which demonstrated that exposure to serum reduced microsphere binding, on average, more than 50% compared to the no-serum condition.. The serum effects were limited to antibodies on the microsphere, since binding inhibition was reversed after rinsing serum from the system and fresh antibody-coated microspheres were introduced. This analysis quantifies the binding effects of soluble IgSF proteins from human serum on antibody-based targeted ultrasound detection and drug delivery methods.

Nanoneedle method for high-sensitivity low-background monitoring of protein activity.

We describe a new method for measuring the activity of protein in miniscule quantities using a carbon nanotube nanoneedle. The unique features of this new method are (a) the immobilization of a few molecules; (b) subsequent translocation and isolation of them near the tip of a position-actuated nanoneedle; and (c) a fixed, defined, and unhindered molecular position to allow rapid real-time sensing and monitoring. The kinetic bioactivity of immobilized alkaline phosphatase (AP) molecules was measured as test model. Results show no decrease in enzymatic activity compared to that of the solution-phase enzyme reaction, suggesting that the immobilization provided unhindered access for ligand binding and minimal conformational modulation caused by undesired surface interactions.

Direct sequence detection of structured h5 influenza viral RNA.

We describe the development of sequence-specific molecular beacons (dual-labeled DNA probes) for identification of the H5 influenza subtype, cleavage motif, and receptor specificity when hybridized directly with in vitro transcribed viral RNA (vRNA). The cloned hemagglutinin segment from a highly pathogenic H5N1 strain, A/Hanoi/30408/2005(H5N1), isolated from humans was used as template for in vitro transcription of sense-strand vRNA. The hybridization behavior of vRNA and a conserved subtype probe was characterized experimentally by varying conditions of time, temperature, and Mg2+ to optimize detection. Comparison of the hybridization rates of probe to DNA and RNA targets indicates that conformational switching of influenza RNA structure is a rate-limiting step and that the secondary structure of vRNA dominates the binding kinetics. The sensitivity and specificity of probe recognition of other H5 strains was calculated from sequence matches to the National Center for Biotechnology Information influenza database. The hybridization specificity of the subtype probes was experimentally verified with point mutations within the probe loop at five locations corresponding to the other human H5 strains. The abundance frequencies of the hemagglutinin cleavage motif and sialic acid recognition sequences were experimentally tested for H5 in all host viral species. Although the detection assay must be coupled with isothermal amplification on the chip, the new probes form the basis of a portable point-of-care diagnostic device for influenza subtyping.

Electrophoretic migration of proteins in semidilute polymer solutions.

We present a systematic study of the electrophoretic migration of 10-200 kDa protein fragments in dilute-polymer solutions using microfluidic chips. The electrophoretic mobility and dispersion of protein samples were measured in a series of monodisperse polydimethylacrylamide (PDMA) polymers of different molecular masses (243, 443, and 764 kDa, polydispersivity index <2) of varying concentration. The polymer solutions were characterized using rheometry. Prior to loading onto the microchip, the polymer solution was mixed with known concentrations of SDS (SDS) surfactant and a staining dye. SDS-denatured protein samples were electrokinetically injected, separated, and detected in the microchip using electric fields ranging from 100 to 300 V/cm. Our results show that the electrophoretic mobility of protein fragments decreases exponentially with the concentration c of the polymer solution. The mobility was found to decrease logarithmically with the molecular weight of the protein fragment. In addition, the mobility was found to be independent of the electric field in the separation channel. The dispersion is relatively independent of polymer concentration and it first increases with protein size and then decreases with a maximum at about 45 kDa. The resolution power of the device decreases with concentration of the PDMA solution but it is always better than 10% of the protein size. The protein migration does not seem to correspond to the Ogston or the reptation models. A semiempirical expression for mobility given by van Winkle fits the data very well.

Measurements of kinetic parameters in a microfluidic reactor.

Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, residence time, and bed resistance were measured with dye pulses. A simple criterion was derived to evaluate the importance of flow-dependent mass-transfer resistances when using microreactors for calculating kinetic rate constants. In the absence of mass-transfer resistances, the Michaelis-Menten kinetic parameters are shown to be flow independent and are appropriately predicted using low substrate conversion data. A comparison of the kinetic parameters with those obtained using solution-phase enzymatic reactions shows a significant decrease in enzyme activity in the immobilized conformation. The immobilized Km of AP is approximately 6 times greater while the kcat is reduced by approximately 28 times. Contradictions found in literature on the evaluation of Michaelis-Menten kinetic parameters for immobilized enzymes in microfluidic reactors are addressed. When product molecules occupy a significant number of enzymatic sites or modify the enzyme activity, the assumed Michaelis-Menten mechanism can no longer be valid. Under these conditions, the calculations of "apparent" kinetic rate constants, based on Michaelis-Menten kinetics, can superficially show a dependence on flow rate conditions even in the absence of mass-transfer resistances. High substrate conversions are shown to depend on flow rate. A kinetic model based on known mechanisms of the alkaline phosphatase enzyme reaction is tested to predict the measurements for high substrate conversion. The study provides a basis for appropriate use of mass-transfer and reaction arguments in successful application of enzymatic microreactors.

Characterization of the in vitro adherence behavior of ultrasound responsive double-shelled microspheres targeted to cellular adhesion molecules.

We have developed novel adhesion molecule-targeted double-shelled microspheres which encapsulate nitrogen. We report in vitro targeting studies utilizing these microspheres conjugated to target-specific antibodies directed towards ICAM-1 and VCAM-1. In static adherence experiments, the adherence patterns of microspheres conjugated to three different monoclonal antibodies (two targeted to ICAM-1 and one to VCAM-1) to their target surfaces were very different. Maximum microsphere adherence at the lowest target and/or ligand densities was observed with the VCAM-1 system. Differences in target-specific adherence were also observed between anti-ICAM-1 and anti-VCAM-1 microsphere conjugates in flow adherence studies. Equilibrium binding studies of the target proteins in solution to the microsphere-bound ligands showed that the affinity constants of two microsphere-bound monoclonal antibodies for their target proteins are similar. Thus, ligand-target affinity is not the only determinant of microsphere adherence to the target surface in our systems. Shear stress was found to have an effect on the mean diameter of adhered microspheres; a decrease in the mean diameter with increasing shear was observed. The magnitude of this effect was dependent on both microsphere-bound ligand and target surface densities, with a more pronounced change at lower densities. Adhered microspheres were readily detectable using ultrasound at the lowest tested surface density of 40 mm(-2).

Kinetic measurements of protein conformation in a microchip.

This paper presents a microchip-based system for collecting kinetic time-based information on protein refolding and unfolding. Dynamic protein conformational change pathways were studied in microchannel flow using a microfluidic device. We present a protein-conserving approach for quantifying refolding by dynamically varying the concentration of the chemical denaturants, guanidine hydrochloride and urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. To validate this protein assay method, folding transition experiments were performed using two well-characterized proteins, human serum albumin (HSA) and bovine carbonic anhydrase (BCA). Transition events were monitored through fluorescence intensity shifts of the protein dye 8-anilino-1-naphthalenesulfonic acid (ANS) during dilutions of protein from urea or guanidine hydrochloride solutions. The enzymatic activity of refolded BCA was measured by UV absorption through the conversion of p-nitrophenyl acetate (p-NPA). The microchip protein refolding transitions using ANS were well-correlated with conventional plate-based experiments. The microfluidic platform enables refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material.

Increase of separation resolution through field enhancement in microchips.

An enhanced ability to separate charged species from neutral compounds in a microfluidic chip is demonstrated using a chip design with low-resistance electrode channels operating with a multiport pressure/voltage controller. A factor of 2.7 improvement in resolution was obtained from chips made using identical mask designs but different etch depth protocols. Greater separation power allows one to cover a wider dynamic range for compounds with different electrophoretic mobilities.

Selective ion extraction: a separation method for microfluidic devices.

A separation concept, selective ion extraction (SIE), is proposed on the basis of the combination of hydrodynamic and electrokinetic flow controls in microfluidic devices. Using a control system with multiple pressure and voltage sources, the hydrodynamic flow and electric field in any section of the microfluidic network can be set to desired values. Mixtures of compounds sent into a T-junction on a chip can be completely separated into different channels on the basis of their electrophoretic mobilities. A simple velocity balance model proved useful for predicting the voltage and pressure settings needed for separation. SIE provides a highly efficient separation with minimal additional dispersion. It is an ideal technique for high-throughput screening systems and demonstrates the power of lab-on-a-chip systems.