Surface-mediated spontaneous emulsification of the acylated peptide, semaglutide.

Acylated peptides composed of glucagon-like peptide-1 receptor agonists modified with a fatty acid side chain are an important class of therapeutics for type 2 diabetes and obesity but are susceptible to an unusual physical instability in the presence of hydrophobic surfaces, i.e., spontaneous emulsification, also known as ouzo formation in practice. In this work, light scattering, small-angle X-ray scattering, and circular dichroism measurements are used to characterize the physical properties of the semaglutide colloidal phase, including size distribution, shape, secondary structure, internal structure, and internal composition, as a function of solution physico-chemical conditions. The existence and size of the colloids formed are successfully predicted by a classical Rayleigh model, which identifies the parameters controlling their size and formation. Colloid formation is found to be catalyzed by hydrophobic surfaces, and formation rates are modeled as an autocatalytic reaction, enabling the formation of a master curve for various surfaces that elucidates the mechanism. Surfaces differ due to differences in surface wettability, which can be correlated with Hansen solubility parameters. This work provides insights into this unusual colloidal phenomenon and guides the peptide synthesis process and drug product formulation in the pharmaceutical industry.

Micelle Formation and Phase Separation of Poloxamer 188 and Preservative Molecules in Aqueous Solutions Studied by Small Angle X-ray Scattering.

Multi-injection pharmaceutical products such as insulin must be formulated to prevent aggregation and microbial contamination. Small-molecule preservatives and nonionic surfactants such as poloxamer 188 (P188) are thus often employed in protein drug formulations. However, mixtures of preservatives and surfactants can induce aggregation and even phase separation over time, despite the fact that all components are well dissolvable when used alone in aqueous solution. A systematic study is conducted here to understand the phase behavior and morphological causes of aggregation of P188 in the presence of the preservatives phenol and benzyl alcohol, primarily using small-angle x-ray scattering (SAXS). Based on SAXS results, P188 remains as unimers in solution when below a certain phenol concentration. Upon increasing the phenol concentration, a regime of micelle formation is observed due to the interaction between P188 and phenol. Further increasing the phenol concentration causes mixtures to become turbid and phase-separate over time. The effect of benzyl alcohol on the phase behavior is also investigated.

Aggregation Kinetics of Polysorbate 80/m-Cresol Solutions: A Small-Angle Neutron Scattering Study.

Polysorbate 80 (PS80), a nonionic surfactant used in pharmaceutical formulation, is known to be incompatible with m-cresol, an antimicrobial agent for multi-dose injectable formulations. This incompatibility results in increased turbidity caused by micelle aggregation progressing over weeks or longer, where storage temperature, ionic strength, and component concentration influence the aggregation kinetics. Small-angle neutron scattering (SANS) analysis of PS80/m-cresol solutions over a pharmaceutically relevant concentration range of each component reveals the cause of aggregation, the coalescence mechanism, and aggregate structure. PS80 solutions containing m-cresol concentrations below ≈2.0 mg/mL and above ≈4.5 mg/mL are kinetically stable and do not aggregate over a 50 h period. At 5 mg/mL of m-cresol, the mixture forms a kinetically stable microemulsion phase, despite being well below the aqueous solubility limit of m-cresol. Solutions containing intermediate m-cresol concentrations (2.0-4.5 mg/mL) are unstable, resulting in aggregation, coalescence, and eventual phase separation. In unstable solutions, two stages of aggregate growth (nucleation and power-law growth) are observed at m-cresol concentrations at or below ≈3.6 mg/mL. At higher m-cresol concentrations, aggregates experience a third stage of exponential growth. A single kinetic model is developed to explain the stages of aggregate growth observed in both kinetic mechanisms. This work establishes the phase diagram of PS80/m-cresol solution stability and identifies component concentrations necessary for producing stable formulations.

Preservative Induced Polysorbate 80 Micelle Aggregation.

Small angle neutron scattering (SANS) studies of a model pharmaceutical formulation reveal how formulation stability depends on the compatibility of individual components. Solutions of two common protein formulation excipients, polysorbate 80 (PS80), a nonionic surfactant that prevents aggregation, and m-cresol, an antimicrobial agent for multi-dose injectable formulations, are investigated. The addition of m-cresol to PS80 solutions leads to solution turbidity and irreversibly alters PS80 micelle morphology. This slow preservative-induced destabilization of PS80 micelles progresses over days or even weeks, which highlights the essential role that aggregation kinetics plays in preservative-surfactant interactions. The temperature-dependence of PS80 micelle growth kinetics is quantified by SANS in the presence of m-cresol. Aggregation is a two-step process, where initial formation of small aggregates is followed by a period of monotonic power-law growth, providing evidence for the mechanism. Total aggregate mass stays constant after initial aggregate formation, and addition of a pH-regulating citrate buffer dramatically accelerates aggregation kinetics.

Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool.

Newcastle disease is a devastating disease of poultry caused by Newcastle disease virus (NDV), a virulent form of avian avulavirus 1 (AAvV-1). A rapid, sensitive and specific means for the detection of NDV is fundamental for the control of this notifiable transboundary virus. Although several real-time RT-PCR assays exist for the detection of AAvV-1, diagnostic sensitivity and specificities can be sub-optimal. In this study, we describe a modification to an existing AAvV-1 l-gene RT-PCR screening assay, where the original probe set was replaced with minor groove binding (MGB) probes, to create the MGB l-gene assay. The diagnostic sensitivity and specificity of this assay was evaluated against a broad panel of both Class I and Class II AAvV-1 viruses of diverse and representative lineages/genotypes in both clinical samples and amplified viruses, and compared with a number of previously published real-time RT-PCR screening assays for AAvV-1. The MGB l-gene assay outperformed all other assays in this assessment, with enhanced sensitivity and specificity, detecting isolates from a broad range of virus lineages/genotypes (including contemporaneously-circulating strains). The assay has also proved its value for screening original clinical samples for the presence of AAvV-1, thus providing an improved screening assay for routine detection of this notifiable disease agent.

Altered premotor cortical oscillations during repetitive movement in persons with Parkinson's disease.

Premotor areas play a critical role in the control of repetitive movements. While research has shown that movement-related oscillations are abnormal during repetitive movements in persons with Parkinson's disease (PD), there is limited research examining the contribution of premotor areas, such as the contralateral dorsal premotor area (PMd) and supplementary motor area (SMA), to this impairment. This study compared movement-related oscillations over premotor regions between participants with PD and control participants. Nine participants with PD off and on medication and nine matched control participants were studied. Participants performed cued index finger movements. Spectral power was derived from electroencephalographic recordings from electrodes FC3/FC4 and Cz over the regions of the contralateral PMd and SMA respectively. Movement-related alpha and beta band oscillations were suppressed over electrode FC3/FC4 (contralateral PMd) in participants with PD, particularly at higher movement rates, in both the off and on medication conditions compared to control subjects. The pattern of movement-related oscillations recorded from Cz (SMA) was similar between PD and control groups. This would suggest that the region of the contralateral PMd may be preferentially involved with the control of externally cued repetitive movements and that changes in this activity may contribute to the deterioration of repetitive finger movements at higher rates in persons with PD.

Motor cortical oscillations are abnormally suppressed during repetitive movement in patients with Parkinson's disease.

OBJECTIVE: Impaired repetitive movement in persons with Parkinson's disease (PD) is associated with reduced amplitude, paradoxical hastening and hesitations or arrest at higher movement rates. This study examined the effects of movement rate and medication on movement-related cortical oscillations in persons with PD. METHODS: Nine participants with PD were studied off and on medication and compared to nine control participants. Participants performed index finger movements cued by tones from 1 to 3 Hz. Movement-related oscillations were derived from electroencephalographic recordings over the region of the contralateral sensorimotor cortex (S1/M1) during rest, listening, or synchronized movement. RESULTS: At rest, spectral power recorded over the region of the contralateral S1/M1 was increased in the alpha band and decreased in the beta band in participants with PD relative to controls. During movement, the level of alpha and beta band power relative to baseline was significantly reduced in the PD group, off and on medication, compared to controls. Reduced movement amplitude and hastening at movement rates near 2 Hz was associated with abnormally suppressed and persistent desynchronization of oscillations in alpha and beta bands. CONCLUSION: Motor cortical oscillations in the alpha and beta bands are abnormally suppressed in PD, particularly during higher rate movements. SIGNIFICANCE: These findings contribute to the understanding of mechanisms underlying impaired repetitive movement in PD.

Early and unintentional release of planned motor actions during motor cortical preparation.

Voluntary movements are often preceded by a movement-related potential beginning as much as two seconds prior to the onset of movement. In light of evidence that motor actions can be prepared and initiated in less than 200 ms, the function of this early activity has remained enigmatic. We hypothesized that the movement-related potential reflects the state of preparation of the planned movement. This was tested by delivering a startling acoustic stimulus during the preparation phase of a load-release task. The cue to release the load was presented either 3.5 seconds after a warning cue (PREDICT condition) or randomly between 4-12 seconds (REACT condition). Electroencephalographic, electromyographic and limb and load kinematic signals were recorded. In a subset of trials, a startle stimulus was delivered at -1500, -1000, -500, -250, -100 or 0 ms before the release cue. A contingent-negative variation (CNV) waveform, with a late phase of slow-rising negativity beginning an average of 1459 ms prior to movement, was observed for the PREDICT condition but not the REACT condition. For both conditions, the startle stimulus frequently evoked the early and unintentional release of the load-release sequence. The incidence of release was significantly (p<0.001) correlated with the late phase of the CNV for the PREDICT condition but not the REACT condition. For the REACT condition, the incidence of movement release was subject-specific, constant across the preparation interval, and uncorrelated with cortical activity. The onset of movement release by the startle stimulus was significantly shorter (p<0.001) for the PREDICT compared to the REACT condition. These findings provide evidence that the late phase of the CNV reflects cortical activity mediating the progressive preparation and storage of the forthcoming movement and that during this phase an intense sensory stimulus can evoke early and unintentional release of the planned action.

Rate-dependent impairments in repetitive finger movements in patients with Parkinson's disease are not due to peripheral fatigue.

Performance of repetitive finger movements is an important clinical measure of disease severity in patients with Parkinson's disease (PD) and is associated with a dramatic deterioration in performance at movement rates near 2 Hz and above. The mechanisms contributing to this rate-dependent movement impairment are poorly understood. Since clinical and experimental testing of these movements involve prolonged repetition of movement, a loss of force-generating capacity due to peripheral fatigue may contribute to performance deterioration. This study examined the contribution of peripheral fatigue to the performance of unconstrained index finger flexion movements by measuring maximum voluntary contractions (MVC) immediately before and after repetitive finger movements in patients with PD (both off- and on-medication) and matched control subjects. Movement performance was quantified using finger kinematics, maximum force production, and electromyography (EMG). The principal finding was that peak force and EMG activity during the MVC did not significantly change from the pre- to post-movement task in patients with PD despite the marked deterioration in movement performance of repetitive finger movements. These findings show that the rate-dependent deterioration of repetitive finger movements in PD cannot be explained by a loss of force-generating capacity due to peripheral fatigue, and further suggest that mechanisms contributing to impaired isometric force production in PD are different from those that mediate impaired performance of high-rate repetitive movements.

Suppression of deep brain stimulation artifacts from the electroencephalogram by frequency-domain Hampel filtering.

OBJECTIVE: Currently, electroencephalography (EEG) cannot be used to record cortical activity during clinically effective DBS due to the presence of large stimulation artifact with components that overlap the useful spectrum of the EEG. A filtering method is presented that removes these artifacts whilst preserving the spectral and temporal fidelity of the underlying EEG. METHODS: The filter is based on the Hampel identifier that treats artifacts as outliers in the frequency domain and replaces them with interpolated values. Performance of the filter was tested with a synthesized DBS signal and actual data recorded during bilateral monopolar DBS. RESULTS: Mean increases in signal-to-noise ratio of 7.8dB for single-frequency stimulation and 13.8dB for dual-frequency stimulation are reported. Correlation analysis between EEG with synthesized artifacts and artifact-free EEG reveals that distortion to the underlying EEG in the filtered signal is negligible (r(2)>0.99). CONCLUSIONS: Frequency-domain Hampel filtering has been shown to remove monopolar DBS artifacts under a number of common stimulation conditions used for the treatment of Parkinson's disease. SIGNIFICANCE: Application of frequency-domain Hampel filtering will allow the measurement of EEG in patients during clinically effective DBS and thus may increase our understanding of the mechanisms of action of this important therapeutic intervention.

Time-frequency analysis of movement-related spectral power in EEG during repetitive movements: a comparison of methods.

During dynamic voluntary movements, power in the alpha- and beta-bands resulting from synchronized neuronal activity is modulated in a manner that is time-locked to movement onset. These signals can be readily recorded from the scalp surface using electroencephalography. Abnormalities in the magnitude and timing of these oscillations are present in a wide variety of movement disorders including Parkinson's disease and dystonia. Most studies have examined movement-related oscillations in the context of single discrete movements, yet marked impairments are often seen during the performance of repetitive movements. For this reason, there is considerable need for analysis methods that can resolve the modulation of these oscillations in both the frequency and time domains. Presently, there is little consensus on which is the most appropriate method for this purpose. In this paper, a comparison of commonly used time-frequency methods is presented for the analysis of movement-related power in the alpha- and beta-bands during repetitive movements. The same principles hold, however, for any form of repetitive or rhythmic input-output processes in the brain. In particular, methods based on band-pass filtering, the short-time Fourier transform (STFT), continuous wavelet transform and reduced interference distributions are discussed. The relative merits and limitations in terms of spectral or temporal resolution of each method are shown with the use of simulated and experimental data. It is shown that the STFT provides the best compromise between spectral and temporal resolution and thus is the most appropriate approach for the analysis and interpretation of repetitive movement-related oscillations in health and disease.

A frequency domain Hampel filter for blind rejection of sinusoidal interference from electromyograms.

A frequency domain filtering method is presented for the removal of power-line and other sinusoidal interference from electromyograms. The presence of such interference results in spikes in the complex-valued fast Fourier transform which can be considered to be outliers. The filter uses the outlier-robust Hampel identifier to detect spikes and replaces them with the median value of local values. The performance of the filter was tested using a set of simulated electromyograms and on experimental data. It was shown that the filter is more effective at rejecting interference than multiple notch filtering and spectral interpolation. The main advantages of using this method are that the frequencies at which the interference occurs need not be known a priori and that signals are not distorted in the absence of interference. In the case of single-frequency interference added to simulated and experimental data, errors in root-mean-squared amplitude and median frequency of <5% are reported.