Clinical Pharmacokinetics of Ponesimod, a Selective S1P1 Receptor Modulator, in the Treatment of Multiple Sclerosis.

Ponesimod, a selective, rapidly reversible, and orally active, sphingosine-1 phosphate receptor (S1P) modulator, is indicated for the treatment of relapsing-remitting multiple sclerosis (RRMS). The clinical pharmacokinetics (PK) and pharmacodynamics (PD) of ponesimod was studied in 16 phase I, one phase II, and one phase III clinical studies. Ponesimod population PK was characterized by an open two-compartment disposition model with a terminal half-life of 33 h (accumulation factor of 2- to 2.6-fold), and fast and almost complete oral absorption (absolute oral bioavailability: 84%), reaching peak plasma and blood concentrations within 2-4 h. Ponesimod is highly metabolized, and the parent compound along with its two major (non-clinically active) metabolites are mainly excreted in the feces (recovery: 57.3-79.6%) and to a lesser extent in the urine (recovery: 10.3-18.4%). Additionally, the population PKPD model characterized the ponesimod effects on heart rate: a transient, dose-dependent decrease in heart rate in the first days of dosing, that is mitigated by administering the first doses of ponesimod treatment using a gradual up-titration schedule, before reaching the daily maintenance dose of 20 mg. This selected maintenance dose has been shown to be superior in reducing annualized relapse rate (ARR) when compared with teriflunomide in a pivotal phase III study. Furthermore, a dose-dependent reduction of peripheral lymphocyte counts that is sustained with continued daily oral dosing of ponesimod and is rapidly (4-7 days) reversible upon drug discontinuation has been characterized with an indirect response model.

A Soft Mechanical Phenotype of SH-SY5Y Neuroblastoma and Primary Human Neurons Is Resilient to Oligomeric Aß(1-42) Injury.

Aggregated amyloid beta (Aß) is widely reported to cause neuronal dystrophy and toxicity through multiple pathways: oxidative stress, disrupting calcium homeostasis, and cytoskeletal dysregulation. The neuro-cytoskeleton is a dynamic structure that reorganizes to maintain cell homeostasis in response to varying soluble and physical cues presented from the extracellular matrix (ECM). Due this relationship between cell health and the ECM, we hypothesize that amyloid toxicity may be directly influenced by physical changes to the ECM (stiffness and dimensionality) through mechanosensitive pathways, and while previous studies demonstrated that Aß can distort focal adhesion signaling with pathological consequences, these studies do not address the physical contribution from a physiologically relevant matrix. To test our hypothesis that physical cues can adjust Aß toxicity, SH-SY5Y human neuroblastoma and primary human cortical neurons were plated on soft and stiff, 2D polyacrylamide matrices or suspended in 3D collagen gels. Each cell culture was exposed to escalating concentrations of oligomeric or fibrillated Aß(1-42) with MTS viability and lactate dehydrogenase toxicity assessed. Actin restructuring was further monitored in live cells by atomic force microscopy nanoindentation, and our results demonstrate that increasing either matrix stiffness or exposure to oligomeric Aß promotes F-actin polymerization and cell stiffening, while mature Aß fibrils yielded no apparent cell stiffening and minor toxicity. Moreover, the rounded, softer mechanical phenotype displayed by cells plated onto a compliant matrix also demonstrated a resilience to oligomeric Aß as noted by a significant recovery of viability when compared to same-dosed cells plated on traditional tissue culture plastic. This recovery was reproduced pharmacologically through inhibiting actin polymerization with cytochalasin D prior to Aß exposure. These studies indicate that the cell-ECM interface can modify amyloid toxicity in neurons and the matrix-mediated pathways that promote this protection may offer unique targets in amyloid pathologies like Alzheimer's disease.

Reduced Extracellular Matrix Stiffness Prompts SH-SY5Y Cell Softening and Actin Turnover To Selectively Increase Aß(1-42) Endocytosis.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by the extracellular deposition of dense amyloid beta plaques. Emerging evidence suggests that the production of these plaques is initiated by the intracellular uptake and lysosomal preconcentration of the amyloid-beta (Aß) peptide. All previous endocytosis studies assess Aß uptake with cells plated on traditional tissue culture plastic; however, brain tissue is distinctly soft with a low-kPa stiffness. Use of an ultrastiff plastic/glass substrate prompts a mechanosensitive response (increased cell spreading, cell stiffness, and membrane tension) that potentially distorts a cell's endocytic behavior from that observed in vivo or in a more physiologically relevant mechanical environment. Our studies demonstrate substrate stiffness significantly modifies the behavior of undifferentiated SH-SY5Y neuroblastoma, where cells plated on soft (∼1 kPa) substrates display a rounded morphology, decreased actin polymerization, reduced adhesion (decreased ß1 integrin expression), and reduced cell stiffness compared to cells plated on tissue culture plastic. Moreover, these neuroblastoma on softer substrates display a preferential increase in the uptake of the Aß(1-42) compared to Aß(1-40), while both isoforms display a clear stiffness-dependent increase of uptake relative to cells plated on plastic. Considering the brain is a soft tissue that continues to soften with age, this mechanosensitive endocytosis of Aß has significant implications for understanding age-related neurodegeneration and the mechanism behind Aß uptake and fibril production. Overall, identifying these physical factors that contribute to the pathology of AD may offer novel avenues of therapeutic intervention.

Mechanosensitive Endocytosis of High-Stiffness, Submicron Microgels in Macrophage and Hepatocarcinoma Cell Lines.

The mechanical properties of submicron particles offer a unique design space for advanced drug-delivery particle engineering. However, the recognition of this potential is limited by a poor consensus about both the specificity and sensitivity of mechanosensitive endocytosis over a broad particle stiffness range. In this report, our model series of polystyrene-co-poly(N-isopropylacrylamide) (pS-co-NIPAM) microgels have been prepared with a nominally constant monomer composition (50 mol % styrene and 50 mol % NIPAM) with varied bis-acrylamide cross-linking densities to introduce a tuned spectrum of particle mechanics without significant variation in particle size and surface charge. While previous mechanosensitive studies use particles with moduli ranging from 15 kPa to 20 MPa, the pS-co-NIPAM particles have Young's moduli (E) ranging from 300 to 700 MPa, which is drastically stiffer than these previous studies as well as pure pNIPAM. Despite this elevated stiffness, particle uptake in RAW264.7 murine macrophages displays a clear stiffness dependence, with a significant increase in particle uptake for our softest microgels after a 4 h incubation. Preferential uptake of the softest microgel, pS-co-NIPAM-1 (E = 310 kPa), was similarly observed with nonphagocytic HepG2 hepatoma cells; however, the uptake kinetics were distinct relative to that observed for RAW264.7 cells. Pharmacological inhibitors, used to probe for specific routes of particle internalization, identify actin- and microtubule-dependent pathways in RAW264.7 cells as sensitive particle mechanics. For our pS-co-NIPAM particles at nominally 300-400 nm in size, this microtubule-dependent pathway was interpreted as a phagocytic route. For our high-stiffness microgel series, this study provides evidence of cell-specific, mechanosensitive endocytosis in a distinctly new stiffness regime that will further broaden the functional landscape of mechanics as a design space for particle engineering.

Core and surface microgel mechanics are differentially sensitive to alternative crosslinking concentrations.

Microgel mechanics are central to the swelling of stimuli-responsive materials and furthermore have recently emerged as a novel design space for tuning the uptake of nanotherapeutics. Despite this importance, the techniques available to assess mechanics, at the sub-micron scale, remain limited. In this report, all mechanical moduli for a series of air-dried, polystyrene-co-poly(N-isopropylacrylamide) (pS-co-NIPAM) microgels of varying composition in monomer and crosslinker (N,N'-methylene-bisacrylamide (BIS)) mol% have been determined using Brillouin light scattering (BLS) and AFM nanoindentation. These techniques sample the material through distinct means and provide complementary nanomechanical data. An initial demonstration of this combined approach is used to evaluate size-dependent nanomechanics in pS particles of varying diameter. For the pS-co-NIPAM series, our BLS results demonstrate an increase in Young's (E) and shear moduli with increasing NIPAM and/or BIS mol%, while the Poisson's ratio decreased. The same rank order in E was observed from AFM and the two techniques correlate well. However, at low BIS crosslinking, an inverted particle structure persists and small increases in BIS yield a higher increase in E from AFM relative to BLS, consistent with a higher density at the particle surface. At higher BIS incorporation, the microgel reverts to a typical, dense-core structure and further increasing BIS yields changes to core-particle mechanics reflected in BLS. Lastly, at 75 mol% NIPAM, the microgels displayed a broad volume phase transition and increased crosslinking resulted in a minor, yet unexpected, increase in swelling ratio. This complementary approach offers new insight into nanomechanics critical for microgel design and application.