Force For Motility Research Articles

Tension directs cancer cell migration over fiber alignment through energy minimization

Cell migration during many fundamental biological processes including metastasis requires cells to traverse tissue with heterogeneous mechanical cues that direct migration as well as determine force and energy requirements for motility. However, the influence of discrete structural and mechanical cues on migration remains challenging to determine as they are often coupled. Here, we decouple the pro-invasive cues of collagen fiber alignment and tension to study their individual impact on migration. When presented with both cues, cells preferentially travel in the axis of tension against fiber alignment. Computational and experimental data show applying tension perpendicular to alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Energy minimization directs migration trajectory, and tension can facilitate migration against fiber alignment. These findings provide a conceptual understanding of bioenergetics during migration through a fibrous matrix.

Motility driven glassy dynamics in confluent epithelial monolayers.

As wounds heal, embryos develop, cancer spreads, or asthma progresses, the cellular monolayer undergoes a glass transition between solid-like jammed and fluid-like flowing states. During some of these processes, the cells undergo an epithelial-to-mesenchymal transition (EMT): they acquire in-plane polarity and become motile. Thus, how motility drives the glassy dynamics in epithelial systems is critical for the EMT process. However, no analytical framework that is indispensable for deeper insights exists. Here, we develop such a theory inspired by a well-known glass theory. One crucial result of this work is that the confluency affects the effective persistence time-scale of active force, described by its rotational diffusivity, Deffr. Deffr differs from the bare rotational diffusivity, Dr, of the motile force due to cell shape dynamics, which acts to rectify the force dynamics: Deffr is equal to Dr when Dr is small and saturates when Dr is large. We test the theoretical prediction of Deffr and how it affects the relaxation dynamics in our simulations of the active Vertex model. This novel effect of Deffr is crucial to understanding the new and previously published simulation data of active glassy dynamics in epithelial monolayers.

ANGI-02. CELL MIGRATION SIMULATOR-BASED BIOMARKERS FOR GLIOBLASTOMA

Abstract Glioblastoma is the most aggressive malignant brain tumor with the poor survival due to its invasive nature driven by cell migration. There is still no effective treatment targeting glioma cell migration based on their transcriptomic information, due to the lack of understanding in the links to the fundamental mechanics of cell migration. In the previous studies, we developed a physics-based motor-clutch model, the cell migration simulator (CMS), to identify the basic mechanisms of cell migration in various cell types. In this study, we found that glioblastoma patient-derived (xenograft) (PD(X)) cell lines across mesenchymal (MES), proneural (PN), classical (CL) subtypes and two institutions (N = 13 patients) had optimal motility and traction force on stiffnesses around 9.3kPa, with otherwise heterogeneous and uncorrelated motility, traction, and F-actin flow. We applied the CMS as a consistent framework to describe the physical parameters of glioblastoma patient cell migration. Using single cell migration and force generation data on compliant 2D surfaces, we found all glioma PD(X) lines (11 patients) had balanced myosin/adhesion ratios resulting in effective migration. Mesenchymal (MES) lines had enhanced F-actin polymerization resulting in higher motility. Adherent PDX lines had increased myosin-adhesion amount resulting in higher traction force. The CMS also predicted the differential cell migration under cytoskeletal drugs between glioma subtypes. Finally, we found 18 genes in actin cytoskeleton and focal adhesion pathways significantly correlated with the glioma physical parameters, which were being used to estimate the physical parameters of a larger cohort (66 patients) and to strategize targeting their cell migration. Overall, we described a consistent physics-based framework for parameterizing individual glioblastoma patients, connecting to clinical transcriptomic data, and predicting drug effects, that can potentially be used to develop subtype and patient-specific anti-migratory therapeutic strategies.

CNK2 promotes cancer cell motility by mediating ARF6 activation downstream of AXL signalling

Cell motility is a critical feature of invasive tumour cells that is governed by complex signal transduction events. Particularly, the underlying mechanisms that bridge extracellular stimuli to the molecular machinery driving motility remain partially understood. Here, we show that the scaffold protein CNK2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to downstream activation of ARF6 GTPase. Mechanistically, AXL signalling induces PI3K-dependent recruitment of CNK2 to the plasma membrane. In turn, CNK2 stimulates ARF6 by associating with cytohesin ARF GEFs and with a novel adaptor protein called SAMD12. ARF6-GTP then controls motile forces by coordinating the respective activation and inhibition of RAC1 and RHOA GTPases. Significantly, genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model. Together, this work identifies CNK2 and its partner SAMD12 as key components of a novel pro-motility pathway in cancer cells, which could be targeted in metastasis.

Analysis of EMT induction in a non-invasive breast cancer cell line by mesenchymal stem cell supernatant: Study of 2D and 3D microfluidic based aggregate formation and migration ability, and cytoskeleton remodeling.

The process of Epithelial-to-mesenchymal transition (EMT) as a phenotypic invasive shift and the factors affecting it, are under extensive research. Application of supernatants of human adipose-derived mesenchymal stem cells (hADMSCs) on non-invasive cancer cells is a well known method of in vitro induction of EMT like process. While previous researches have focused on the effects of hADMSCs supernatant on the biochemical signaling pathways of the cells through expression of different proteins and genes, we investigated pro-carcinogic alterations of physico-mechanical cues in terms of changes in cell motility and aggregated formation in 3D microenvironments, and cytoskeletal actin-myosin content and fiber arrangement. MCF-7 cancer cells were treated by the supernatant from 48hour-starved hADMSCs, and their vimentin/E-cadherin expressions were evaluated. The invasive potential of treated and non-treated cells was measured and compared through aggregate formation and migration capability. Furthermore, alterations in cell and nucleus morphologies were studied, and F-actin and myosin-II alterations in terms of content and arrangement were investigated. Results indicated that application of hADMSCs supernatant enhanced vimentin expression as the biomarker of EMT, and induced pro-carcinogenic effects on non-invasive cancer cells through increased invasive potential by higher cell motility and reduced aggregate formation, rearrangement of actin structure and generation of more stress fibers, together with increased myosin II that lead to enhanced cell motility and traction force. Our results indicated that in vitro induction of EMT through mesenchymal supernatant influenced biophysical features of cancer cells through cytoskeletal remodeling that emphasizes the interconnection of chemical and physical signaling pathways during cancer progress and invasion. Results give a better insight to EMT as a biological process and the synergy between biochemical and biophysical parameters that contribute to this process, and eventually assist in improving cancer treatment strategies.

Collective vortical motion and vorticity reversals of self-propelled particles on circularly patterned substrates.

The collective behavior of self-propelled particles (SPPs) under the combined effects of a circularly patterned substrate and circular confinement is investigated through coarse-grained molecular dynamics simulations of polarized and disjoint ring polymers. The study is performed over a wide range of values of the SPPs packing fraction ϕ[over ¯], motility force F_{D}, and area fraction of the patterned region. At low packing fractions, the SPPs are excluded from the system's center and exhibit a vortical motion that is dominated by the substrate at intermediate values of F_{D}. This exclusion zone is due to the coupling between the driving force and torque induced by the substrate, which induces an outward spiral motion of the SPPs. For high values of F_{D}, the SPPs exclusion from the center is dominated by the confining boundary. At high values of ϕ[over ¯], the substrate pattern leads to reversals in the vorticity, which become quasiperiodic with increasing ϕ[over ¯]. We also found that the substrate pattern is able to separate SPPs based on their motilities.

Mechanisms of pathogenicity in the hypertrophic cardiomyopathy-associated TPM1 variant S215L.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder often caused by mutations to sarcomeric genes. Many different HCM-associated TPM1 mutations have been identified but they vary in their degrees of severity, prevalence, and rate of disease progression. The pathogenicity of many TPM1 variants detected in the clinical population remains unknown. Our objective was to employ a computational modeling pipeline to assess pathogenicity of one such variant of unknown significance, TPM1 S215L, and validate predictions using experimental methods. Molecular dynamic simulations of tropomyosin on actin suggest that the S215L significantly destabilizes the blocked regulatory state while increasing flexibility of the tropomyosin chain. These changes were quantitatively represented in a Markov model of thin-filament activation to infer the impacts of S215L on myofilament function. Simulations of in vitro motility and isometric twitch force predicted that the mutation would increase Ca2+ sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher Ca2+ sensitivity compared with wild type. Three-dimensional genetically engineered heart tissues expressing TPM1 S215L exhibited hypercontractility, upregulation of hypertrophic gene markers, and diastolic dysfunction. These data form a mechanistic description of TPM1 S215L pathogenicity that starts with disruption of the mechanical and regulatory properties of tropomyosin, leading thereafter to hypercontractility and finally induction of a hypertrophic phenotype. These simulations and experiments support the classification of S215L as a pathogenic mutation and support the hypothesis that an inability to adequately inhibit actomyosin interactions is the mechanism whereby thin-filament mutations cause HCM.

Interplay of cell motility and self-secreted extracellular polymeric substance induced depletion effects on spatial patterning in a growing microbial colony.

Reproducing bacteria self-organize to develop patterned biofilms in various conditions. Various factors contribute to the shaping of a multicellular bacterial organization. Here we investigate how motility force and self-secreted extracellular polymeric substances (EPS) influence bacterial cell aggregation, leading to phase-separated colonies using a particle-based/individual-based model. Our findings highlight the critical role of the interplay between motility force and depletion effects in regulating phase separation within a growing colony under far-from-equilibrium conditions. We observe that increased motility force hinders depletion-induced cell aggregation and phase segregation, necessitating a higher depletion effect for highly motile bacteria to undergo phase separation within a growing biofilm. We present a phase diagram illustrating the systematic variation of motility force and repulsive mechanical force, shedding light on the combined contributions of these two factors: self-propulsive motion and aggregation due to the depletion effect, resulting in the presence of small to large bacterial aggregates. Furthermore, our study reveals the dynamic nature of clustering, marked by changes in cluster size over time. Additionally, our findings suggest that differential dispersion among the components can lead to the localization of EPS at the periphery of a growing colony. Our study enhances the understanding of the collective dynamics of motile bacterial cells within a growing colony, particularly in the presence of a self-secreted polymer-driven depletion effect.

Motor Dysfunction of Gastric Antral Smooth Muscle in Diabetic Rats: Contribution of ATP-Dependent Potassium Channels.

The goal of the current research was to further elucidate the role of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels in the motility and contractility force of gastric smooth muscle of diabetic rats. Male Wistar rats (190-230 g) were grouped into control and streptozotocin (STZ)-induced diabetes (55 mg/kg) rats. Thirty days later, gastric muscle contractility was measured using a myograph and a force transducer of antral segments immersed in a tissue bath. Gastric emptying response was measured through feeding of standard pellet. Furthermore, the expression of KATP channel subunits in antral smooth muscle was determined by western blot technique. The amplitude of KCl-evoked twitch contractions of diabetic antral strips was about 25% more than control (P < 0.05). Application of minoxidil, a KATP channel opener, dose dependently decreased the force of twitch contractions in both normal and diabetic antral strips. Application of 10 μM glibenclamide, a KATP channel blocker, did not antagonize the minoxidil-induced relaxation of antral strips. Diabetic gastric emptying was faster than normal, although not significant. Despite the relaxant effect of minoxidil on gastric emptying rate in normal rats (P < 0.05), this effect was not observed in diabetic rats. Also, glibenclamide increased gastric emptying and antagonized minoxidil-induced relaxation in normal rats (P < 0.05). Furthermore, the expression of KATP Kir6.1 and SUR2B subunits was substantially reduced in antral smooth muscle in diabetic condition (P < 0.01). These results propose that KATP channels may contribute to the development of gastric motility disorders in diabetes.

Borrelia burgdorferi modulates the physical forces and immunity signaling in endothelial cells

SummaryBorrelia burgdorferi (Bb), a vector-borne bacterial pathogen and the causative agent of Lyme disease, can spread to distant tissues in the human host by traveling in and through monolayers of endothelial cells (ECs) lining the vasculature. To examine whether Bb alters the physical forces of ECs to promote its dissemination, we exposed ECs to Bb and observed a sharp and transient increase in EC traction and intercellular forces, followed by a prolonged decrease in EC motility and physical forces. All variables returned to baseline at 24 h after exposure. RNA sequencing analysis revealed an upregulation of innate immune signaling pathways during early but not late Bb exposure. Exposure of ECs to heat-inactivated Bb recapitulated only the early weakening of EC mechanotransduction. The differential responses to live versus heat-inactivated Bb indicate a tight interplay between innate immune signaling and physical forces in host ECs and suggest their active modulation by Bb.

Twin-Engine Janus Supramolecular Nanomotors with Counterbalanced Motion.

Supramolecular nanomotors were created with two types of propelling forces that were able to counterbalance each other. The particles were based on bowl-shaped polymer vesicles, or stomatocytes, assembled from the amphiphilic block copolymer poly(ethylene glycol)-block-polystyrene. The first method of propulsion was installed by loading the nanocavity of the stomatocytes with the enzyme catalase, which enabled the decomposition of hydrogen peroxide into water and oxygen, leading to a chemically induced motion. The second method of propulsion was attained by applying a hemispherical gold coating on the stomatocytes, on the opposite side of the opening, making the particles susceptible to near-infrared laser light. By exposing these Janus-type twin engine nanomotors to both hydrogen peroxide (H2O2) and near-infrared light, two competing driving forces were synchronously generated, resulting in a counterbalanced, “seesaw effect” motion. By precisely manipulating the incident laser power and concentration of H2O2, the supramolecular nanomotors could be halted in a standby mode. Furthermore, the fact that these Janus stomatocytes were equipped with opposing motile forces also provided a proof of the direction of motion of the enzyme-activated stomatocytes. Finally, the modulation of the “seesaw effect”, by tuning the net outcome of the two coexisting driving forces, was used to attain switchable control of the motile behavior of the twin-engine nanomotors. Supramolecular nanomotors that can be steered by two orthogonal propulsion mechanisms hold considerable potential for being used in complex tasks, including active transportation and environmental remediation.

Sperm Ion Transporters and Channels in Human Asthenozoospermia: Genetic Etiology, Lessons from Animal Models, and Clinical Perspectives

In mammals, sperm fertilization potential relies on efficient progression within the female genital tract to reach and fertilize the oocyte. This fundamental property is supported by the flagellum, an evolutionarily conserved organelle that provides the mechanical force for sperm propulsion and motility. Importantly several functional maturation events that occur during the journey of the sperm cells through the genital tracts are necessary for the activation of flagellar beating and the acquisition of fertilization potential. Ion transporters and channels located at the surface of the sperm cells have been demonstrated to be involved in these processes, in particular, through the activation of downstream signaling pathways and the promotion of novel biochemical and electrophysiological properties in the sperm cells. We performed a systematic literature review to describe the currently known genetic alterations in humans that affect sperm ion transporters and channels and result in asthenozoospermia, a pathophysiological condition defined by reduced or absent sperm motility and observed in nearly 80% of infertile men. We also present the physiological relevance and functional mechanisms of additional ion channels identified in the mouse. Finally, considering the state-of-the art, we discuss future perspectives in terms of therapeutics of asthenozoospermia and male contraception.

Collective motion of cells modeled as ring polymers.

In this article, we use a coarse-grained model of disjoint semi-flexible ring polymers to investigate computationally the spatiotemporal collective behavior of cell colonies. A ring polymer in this model is self-propelled by a motility force along the cell's polarity, which depends on its historical kinetics. Despite the repulsive interaction between the cells, a collective behavior sets in as a result of cells pushing against each other. This cooperative motion emerges as the amplitude of the motility force is increased and/or their areal density is increased. The degree of collectivity, characterized by the average cluster size, the velocity field order parameter, and the polarity field nematic order parameter, is found to increase with increasing the amplitude of the motility force and area coverage of the cells. Furthermore, the degree of alignment exhibited by the cell velocity field within a cluster is found to be stronger than that exhibited by the cell polarity. Comparison between the collective behavior of elongated cells and that of circular cells, at the same area coverage and motility force, shows that elongated cells exhibit a stronger collective behavior than circular cells, in agreement with earlier studies of self-propelled anisotropic particles. An investigation of two-cell collisions shows that while two clustered cells move in tandem, their polarities are misaligned. As such the cells push against each other while moving coherently.

Cluster size distribution of cells disseminating from a primary tumor.

The first stage of the metastatic cascade often involves motile cells emerging from a primary tumor either as single cells or as clusters. These cells enter the circulation, transit to other parts of the body and finally are responsible for growth of secondary tumors in distant organs. The mode of dissemination is believed to depend on the EMT nature (epithelial, hybrid or mesenchymal) of the cells. Here, we calculate the cluster size distribution of these migrating cells, using a mechanistic computational model, in presence of different degree of EMT-ness of the cells; EMT is treated as given rise to changes in their active motile forces (μ) and cell-medium surface tension (Γ). We find that, for (μ > μmin, Γ > 1), when the cells are hybrid in nature, the mean cluster size, [Formula: see text], where μmin increases with increase in Γ. For Γ ≤ 0, [Formula: see text], the cells behave as completely mesenchymal. In presence of spectrum of hybrid states with different degree of EMT-ness (motility) in primary tumor, the cells which are relatively more mesenchymal (higher μ) in nature, form larger clusters, whereas the smaller clusters are relatively more epithelial (lower μ). Moreover, the heterogeneity in μ is comparatively higher for smaller clusters with respect to that for larger clusters. We also observe that more extended cell shapes promote the formation of smaller clusters. Overall, this study establishes a framework which connects the nature and size of migrating clusters disseminating from a primary tumor with the phenotypic composition of the tumor, and can lead to the better understanding of metastasis.

Mechanistic underpinning of cell aspect ratio-dependent emergent collective motions in swarming bacteria.

Self-propelled bacteria can exhibit a large variety of non-equilibrium self-organized phenomena. Swarming is one such fascinating dynamical scenario where a number of motile individuals group into dynamical clusters and move in synchronized flows and vortices. While precedent investigations into rod-like particles confirm that an increased aspect-ratio promotes alignment and order, recent experimental studies in bacteria Bacillus subtilis show a non-monotonic dependence of the cell-aspect ratio on their swarming motion. Here, by computer simulations of an agent-based model of self-propelled, mechanically interacting, rod-shaped bacteria under overdamped conditions, we explore the collective dynamics of a bacterial swarm subjected to a variety of cell-aspect ratios. When modeled with an identical self-propulsion speed across a diverse range of cell aspect ratios, simulations demonstrate that both shorter and longer bacteria exhibit slow dynamics whereas the fastest speed is obtained at an intermediate aspect ratio. Our investigation highlights that the origin of this observed non-monotonic trend of bacterial speed and vorticity with the cell-aspect ratio is rooted in the cell-size dependence of motility force. The swarming features remain robust for a wide range of surface density of the cells, whereas asymmetry in friction attributes a distinct effect. Our analysis identifies that at an intermediate aspect ratio, an optimum cell size and motility force promote alignment, which reinforces the mechanical interactions among neighboring cells leading to the overall fastest motion. Mechanistic underpinning of the collective motions reveals that it is a joint venture of the short-range repulsive and the size-dependent motility forces, which determines the characteristics of swarming.

Surgical management of ileocolic intussusception in a German shepherd dog

Intussusception occurs when excessive peristaltic motility forces one segment of the intestine into another slightly larger part of the intestine. The incidence of intussusception is more in German shepherd dogs. A six-month old German shepherd dog weighing 17 kg was presented with a history of anorexia, vomiting, diarrhoea, and lethargy for the past 7 days. Clinical examination revealed a congested conjunctival mucous membrane, bounding pulse and tachycardia. Abdominal palpation evinced pain and a bunch of coiled sausage shaped mass was identified during deep palpation. Abdominal ultrasonography revealed bulls eye appearance in the intestine confirming the occurrence of intussusception. Pre-operatively, the animal was hydrated and stabilized with intravenous fluids. Prophylactic antibiotic therapy using ceftriaxone was given at a dose rate of 25 mg per kg bodyweight and pre-emptive analgesia with meloxicam at a dose rate of 0.2 mg per kg bodyweight was also given. Emergency laparotomy was performed under general anaesthesia maintained with isoflurane. A 20 cm long necrosed irreducible intestinal segment was identified. The necrosed loop was resected after ligating the mesenteric vessels. The cut ends of the intestine being uneven, was cut at right angles and the smaller segment was further cut at an oblique angle to correct the disparity in the lumen size. End to end anastomosis was performed using polydioxanone suture in a simple continues pattern. The abdominal cavity was lavaged with normal saline. Laparotomy wound was closed in a routine pattern. Post operatively, the dog was treated using fluids, antibiotics, and analgesics for seven days. The owner was advised to give easily digestible liquid diet in small quantities from fourth day onwards and gradually shift to normal food. The skin sutures were removed on 10th post-operative day and the animal made an uneventful recovery. Keywords: Ultrasonography, Intestinal telescoping, Resection, Anastomosis, Surgical management, Deworming

Swimming force and behavior of optically trapped micro-organisms

We demonstrate how optical tweezers combined with a three-dimensional force detection system and high-speed camera are used to study the swimming force and behavior of trapped micro-organisms. By utilizing position sensitive detection, we measure the motility force of trapped particles, regardless of orientation. This has the advantage of not requiring complex beam shaping or microfluidic controls for aligning trapped particles in a particular orientation, leading to unambiguous measurements of the propulsive force at any time. Correlating the direct force measurements with position data from a high-speed camera enables us to determine changes in the particle’s behavior. We demonstrate our technique by measuring the swimming force and observing distinctions between swimming and tumbling modes of the Escherichia coli (E. coli) strain MC4100. Our method shows promise for application in future studies of trappable but otherwise arbitrary-shaped biological swimmers and other active matter.

Instability and fingering of interfaces in growing tissue

Interfaces in tissues are ubiquitous, both between tissue and environment as well as between populations of different cell types. The propagation of an interface can be driven mechanically. Computer simulations of growing tissues are employed to study the stability of the interface between two tissues on a substrate. From a mechanical perspective, the dynamics and stability of this system is controlled mainly by four parameters of the respective tissues: (i) the homeostatic stress (ii) cell motility (iii) tissue viscosity and (iv) substrate friction. For propagation driven by a difference in homeostatic stress, the interface is stable for tissues which differ in their substrate friction even for very large differences of homeostatic stress; however, it becomes unstable above a critical stress difference when the tissue with the larger homeostatic stress has a higher viscosity. A small difference in directed bulk motility between the two tissues suffices to result in propagation with a stable interface, even for otherwise identical tissues. Larger differences in motility force, however, result in a finite-wavelength instability of the interface. Interestingly, the instability is apparently bound by nonlinear effects and the amplitude of the interface undulations only grows to a finite value in time.

Stability phase diagram of active Brownian particles

Phase separation in a low-density gas-like phase and a high-density liquid-like one is a common trait of biological and synthetic self-propelling particles' systems. The competition between motility and stochastic forces is assumed to fix the boundary between the homogeneous and the phase-separated phase. Here we demonstrate that motility does also promote the homogeneous phase allowing particles to resolve their collisions. This new understanding allows quantitatively predicting the spinodal-line of hard self-propelling Brownian particles, the prototypical model exhibiting a motility induced phase separation. Furthermore, we demonstrate that frictional forces control the physical process by which motility promotes the homogeneous phase. Hence, friction emerges as an experimentally variable parameter to control the motility induced phase diagram.

Biorelevant In Vitro Release Testing and In Vivo Study of Extended-Release Niacin Hydrophilic Matrix Tablets

Niacin (nicotinic acid, NA) is administered orally as an antihyperlipidemic agent in extended-release (ER) tablets in high doses. Due to rapid absorption and extensive metabolism (non-linear pharmacokinetics), the drug plasma levels are highly variable, which may correlate with side effects. Interestingly, this erratic drug delivery behavior of niacin ER products cannot be clarified by compendial in vitro release testing. The standard dissolution tests do not allow to mimic the selected GI tract characteristics in order to estimate the robustness of formulation under the variability of the physiological conditions. These are characterized by the pH value, impact of motility forces and composition, as well as volume of GI liquids. Our paper demonstrates a comparison of a newly developed ER HPMC niacin formulation with an originator product. The research aimed to design a robust matrix tablet of comparable biopharmaceutical behavior, safety and efficacy. The extensive in vitro investigation, including dynamic studies in flow-through cell apparatus and stress test device, forms the basis for the evaluation of nicotinic acid plasma concentrations in vivo. The occurrence of erratic, multiple NA plasma peaks after the administration of both extended-release products is a result of its local input excess over the metabolic threshold (at the level corresponding to maximum 2% of the administered dose, i.e., 20 mg of drug) due to the mechanical stresses of physiological intensity. We demonstrate how this behavior is similar for both marketed and test products. In this context, we describe how a robust ER matrix and well-designed formulation does not guarantee the test product’s bioequivalence to the comparator one out of reasons unrelated to technology and biopharmaceutical properties, but because of the active compound’s intrinsic pharmacokinetic characteristics, i.e., highly variable, extensive metabolism of nicotinic acid.