PBPK-PD modeling for the preclinical development and clinical translation of tau antibodies for Alzheimer's disease.

Disrupted tau proteostasis and transneuronal spread is a pathological hallmark of Alzheimer's disease. Neurodegenerative diseases remain an unmet medical need and novel disease modifying therapeutics are paramount. Our objective was to develop a mechanistic mathematical model to enhance our understanding of tau antibody pharmacokinetics and pharmacodynamics in animals and humans. A physiologically-based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling approach was employed to support the preclinical development and clinical translation of therapeutic antibodies targeting tau for the treatment of Alzheimer's disease. The pharmacokinetics of a tau antibody was evaluated in rat and non-human primate microdialysis studies. Model validation for humans was performed using publicly available clinical data for gosuranemab. In-silico analyses were performed to predict tau engagement in human brain for a range of tau antibody affinities and various dosing regimens. PBPK-PD modeling enabled a quantitative understanding for the relationship between dose, affinity, and target engagement, which supported lead candidate optimization and predictions of clinically efficacious dosing regimens.

Integrative Pharmacology: Advancing Development of Effective Immunotherapies.

With the recent advances in cancer immunotherapy, it is now evident that the antigen-specific activation of the patients' immune responses can be utilized for achieving significant therapeutic benefits. Novel molecules have been developed and promising advances have been achieved in cancer therapy. The recent success of cancer immunotherapy clearly reflects the novelty of the approach and importance of this class of therapeutics. Due to the nature of immunotherapy, i.e., harnessing the patient's immune system, it becomes critical to evaluate the important variables that can guide preclinical development, translational strategies, patient selection, and effective clinical dosing paradigms following single and combination therapies. To further boost the durability and efficacy profiles of IO (immuno-oncology) drugs following single agent therapy, novel combination therapies are being sought. Combination strategies have become critical for enhancing the anti-tumor immunity in broader cancer indications. Comprehensive methods are being developed to quantify the synergistic combination effect profiles at various development phases. Further evaluation of the signaling and pathway components can potentially establish a unique "signature" characteristic for specific combination therapies following modulation of various immunomodulatory pathways. In this article, critical topics related to preclinical, translational, and clinical development of IO agents are discussed.

Microfluidic fabrication of shape-tunable alginate microgels: effect of size and impact velocity.

We report on a capillary-based microfluidic platform for the fabrication of non-spherical sodium alginate microgels. The sodium alginate droplets were crosslinked off-chip in a mixture of barium acetate and glycerol solution. Novel morphologies such as tear drop, lamp-like, mushroom-like, double-dimpled and bowl-like microgels were fabricated by controlling the size, impact velocity (at the crosslinking solution/oil interface), and concentration of sodium alginate solution. We monitored the microscale deformation process in situ at the interface and proposeed a deformation mechanism resulting in unique morphologies. Additionally, we constructed microgel superstructures by assembling the non-spherical alginate microgels to spherical poly(N-isopropylacrylamide) (pNIPAAm) microgels via electrostatic interaction.

Dilution of protein-surfactant complexes: a fluorescence study.

Dilution of protein-surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta-galactosidase as model proteins. The fluorescent signature of protein-surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein-surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein-SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein-surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics.

Improved antimicrobial potency through synergistic action of chitosan microparticles and low electric field.

Techniques to inhibit gram-negative bacteria such as Shiga toxin-producing Escherichia coli are valuable as the prevalence of large-scale industrial food preparation increases the likelihood of contamination. Chitosan, the deacetylated derivative of chitin, has been demonstrated to inhibit bacteria growth in acidic environments, but is significantly less effective in preventing bacteria grown at pH >7.0. Pulsed electric fields, constituting another method of bacteria inhibition, are difficult to generate at sufficient strength due to the high electric potentials required. This study utilizes adsorption of particulate chitosan in a very low electric field for an increased inhibition of gram-negative bacteria in neutral or alkaline pH conditions. Chitosan microparticles are demonstrated to flocculate E. coli, inhibit growth, and exhibit increased efficacy when combined with a low voltage electric field applied over 2-min intervals. Using sustained pulses of approximately 100 V/cm, it is demonstrated that bacteria viability is reduced by several orders of magnitude. The degree of bacterial inhibition is increased when chitosan microparticles are introduced to the system prior to imposing a small electric field.

Surfactant-induced electroosmotic flow in microfluidic capillaries.

Control of EOF in microfluidic devices is essential in applications such as protein/DNA sizing and high-throughput drug screening. With the growing popularity of poly(methyl methacrylate) (PMMA) as the substrate for polymeric-based microfludics, it is important to understand the effect of surfactants on EOF in these devices. In this article, we present an extensive investigation exploring changes in EOF rate induced by SDS, polyoxyethylene lauryl ether (Brij35) and CTAB in PMMA microfluidic capillaries. In a standard protein buffer (Tris-Glycine), PMMA capillaries exhibited a cathodic EOF with measured mobility of 1.54 ± 0.1 (× 10⁻4 cm²/V.s). In the presence of surfactant below a critical concentration, EOF was independent of surfactant concentration. At high concentrations of surfactants, the electroosmotic mobility was found to linearly increase/decrease as the logarithm of concentration before reaching a constant value. With SDS, the EOF increased by 257% (compared to buffer), while it was decreased by 238% with CTAB. In the case of Brij35, the electroosmotic mobility was reduced by 70%. In a binary surfactant system of SDS/CTAB and SDS/Brij35, addition of oppositely charged CTAB reduced the SDS-induced EOF more effectively compared to nonionic Brij35. We propose possible mechanisms that explain the observed changes in EOF and zeta potential values. Use of neutral polymer coatings in combination with SDS resulted in 50% reduction in the electroosmotic mobility with 0.1% hydroxypropyl methyl cellulose (HPMC), while including 2% poly (N,N-dimethylacrylamide) (PDMA) had no effect. These results will potentially contribute to the development of PMMA-based microfluidic devices.

Rapid detection and quantification of specific proteins by immunodepletion and microfluidic separation.

Conventional immunoblotting techniques are labor intensive, time consuming and rely on the elution of target protein after depletion. Here we describe a new method for detection and quantification of proteins, independent of washing and elution. In this method, the target protein is first captured by immunodepletion with antibody-coated microbeads. In the second step, both the supernatant after immunodepletion and the untreated protein sample are directly analyzed by microfluidic electrophoresis without further processing. Subsequently, the detection and quantification are performed by comparing the electropherograms of these two samples. This method was tested using an Escherichia coli lysate with a FLAG-tagged protein and anti-FLAG magnetic beads. An incubation of as short as one min was sufficient for detectable depletion (66%) by microchip electrophoresis. Longer incubation (up to 60 min) resulted in more depletion of the target band (82%). Our results show that only 19% of the target is recovered after elution from the beads. By eliminating multiple wash and elution steps, our method is faster, less labor intensive, and highly reproducible. The target protein can still be easily identified even in the case of nonspecific binding at low concentrations. This work highlights the advantages of integrating immunodepletion techniques on a microfluidic platform.