Enabling oral novel Taxanes-based Chemotherapy with Lipophilic prodrug Self-nanoemulsifying drug delivery system.

Larotaxel (LTX) and SB-T-1214 (SBT), two new synthetic experimental toxoids, have shown broad-spectrum antitumor activity, especially against tumors that are resistant to other drugs. However, their poor solubility, membrane permeability, and first-pass effect limits their use in oral administration. We designed and synthesized two long-chain triglyceride-mimic prodrugs of LTX (LTXSSTG) and SBT (SBTSSTG), which are bridged by disulfide bonds and efficiently incorporated them into Self-nanoemulsifying drug delivery system (SNEDDS). These prodrugs can bypass hepatic metabolism by entering the blood through intestinal lymphatic transport, following a similar oral absorption pathway to dietary lipids. It was found that LTXSSTG and SBTSSTG significantly improved oral bioavailability (about 4.5-fold for LTX and 3.4-fold for SBT) compared to their solution forms. Moreover, with LTXSSTG and SBTSSTG incorporating reduction stimulus-responsive spacer were much more effective in suppressing tumor growth in vivo with eliminated adverse effects than solution form. To sum up, this strategy provides a new avenue to enhance oral delivery of new toxoids.

Simplified method to quantify valve back-leak uncovers severe mesenteric lymphatic valve dysfunction in mice deficient in connexins 43 and 37.

KEY POINTS: Lymphatic valve defects are one of the major causes of lymph transport dysfunction; however, there are no accessible methods for quantitatively assessing valve function. This report describes a novel technique for quantifying lymphatic valve back-leak. Postnatal endothelial-specific deletion of connexin 43 (Cx43) in connexin 37 null (Cx37-/- ) mice results in rapid regression of valve leaflets and severe valve dysfunction. This method can also be used for assessing the function of venous and lymphatic valves from various species, including humans. ABSTRACT: The lymphatic system relies on robust, spontaneous contractions of collecting lymphatic vessels and one-way secondary lymphatic valves to efficiently move lymph forward. Secondary valves prevent reflux and allow for the generation of propulsive pressure during each contraction cycle. Lymphatic valve defects are one of the major causes of lymph transport dysfunction. Genetic mutations in multiple genes have been associated with the development of primary lymphoedema in humans; and many of the same mutations in mice result in valve defects that subsequently lead to chylous ascites or chylothorax. At present the only experimental technique for the quantitative assessment of lymphatic valve function utilizes the servo-null micropressure system, which is highly accurate and precise, but relatively inaccessible and difficult to use. We developed a novel, simplified alternative method for quantifying valve function and determining the degree of pressure back-leak through an intact valve in pressurized, single-valve segments of isolated lymphatic vessels. With this diameter-based method, the competence of each lymphatic valve is challenged over a physiological range of pressures (e.g. 0.5-10cmH2 O) and pressure back-leak is extrapolated from calibrated, pressure-driven changes in diameter upstream from the valve. Using mesenteric lymphatic vessels from C57BL/6J, Ub-CreERT2 ;Rasa1fx/fx , Foxc2Cre/+ , Lyve1-Cre;Cx43fx/fx , and Prox1-CreERT2 ;Cx43fx/fx ;Cx37-/- mice, we tested our method on lymphatic valves displaying a wide range of dysfunction, from fully competent to completely incompetent. Our results were validated by simultaneous direct measurement of pressure back-leak using a servo-null micropressure system. Our diameter-based technique can be used to quantify valve function in isolated lymphatic valves from a variety of species. This method also revealed that haplodeficiency in Foxc2 (Foxc2Cre/+ ) is not sufficient to cause significant valve dysfunction; however, postnatal endothelial-specific deletion of Cx43 in Cx37-/- mice results in rapid regression of valve leaflets and severe valve dysfunction.

The relationship between lymphangion chain length and maximum pressure generation established through in vivo imaging and computational modeling.

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. In the present study, we proposed and validated that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrated increases in pumping pressure along the length of the tail. Computational simulations based on a microstructurally motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was used to provide insights into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lymphangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, whereas the frequency of contraction has no effect. In vivo administration of nitric oxide attenuated lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest that the reduction in contractile strength of smooth muscle cells in the presence of nitric oxide can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus, combining modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity noninvasively in vivo while also providing insights into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient. NEW & NOTEWORTHY Here, we report the first minimally invasive in vivo measurements of the relationship between lymphangion chain length and lymphatic pumping pressure. We also provide the first in vivo validation of lumped parameter models of lymphangion chains previously developed through data obtained from isolated vessel testing.

The Lymphatic System in Disease Processes and Cancer Progression.

Advances in our understanding of the structure and function of the lymphatic system have made it possible to identify its role in a variety of disease processes. Because it is involved not only in fluid homeostasis but also in immune cell trafficking, the lymphatic system can mediate and ultimately alter immune responses. Our rapidly increasing knowledge of the molecular control of the lymphatic system will inevitably lead to new and effective therapies for patients with lymphatic dysfunction. In this review, we discuss the molecular and physiological control of lymphatic vessel function and explore how the lymphatic system contributes to many disease processes, including cancer and lymphedema.

Numerical investigation on flow-induced wall shear stress variation of metastatic cancer cells in lymphatics with elastic valves.

Hematogenous metastasis occurs when cancer cells detach from the extracellular matrix in the primary tumor into the bloodstream or lymphatic system. Elucidating the response of metastatic tumor cells in suspension to the flow conditions in lymphatics with valves from a mechanical/fluidic perspective is necessary. A physiologically relevant computational model of a lymphatic vessel with valves was constructed using fully coupled fluid-cell-vessel interactions to investigate the effects of lymphatic vessel contractility, valve properties, and cell size and stiffness on the variations in magnitude and gradient of the flow-induced wall shear stress (WSS) experienced by suspended tumor cells. Results indicated that the maximum WSSmax increased with the increments in cell diameter, vessel contraction amplitude, and valve stiffness. The decrease in vessel contraction period and valve aspect ratio also increased the maximum WSSmax. The influence of the properties of the valve on the WSS was more significant among the factors mentioned above. The maximum WSSmax acting on the cancer cell when the cell reversed the direction of its motion in the valve region increased by 0.5-1.4 times that before the cell entered the valve region. The maximum change in WSS was in the range of 0.004-0.028 Pa/µm depending on the factors studied. They slightly exceeded the values associated with breast cancer cell apoptosis. The results of this study provide biofluid mechanics-based support for mechanobiological research on the metastasis of metastatic cancer cells in suspension within the lymphatics.

Parameter sensitivity analysis of a lumped-parameter model of a chain of lymphangions in series.

Any disruption of the lymphatic system due to trauma or injury can lead to edema. There is no effective cure for lymphedema, partly because predictive knowledge of lymphatic system reactions to interventions is lacking. A well-developed model of the system could greatly improve our understanding of its function. Lymphangions, defined as the vessel segment between two valves, are the individual pumping units. Based on our previous lumped-parameter model of a chain of lymphangions, this study aimed to identify the parameters that affect the system output the most using a sensitivity analysis. The system was highly sensitive to minimum valve resistance, such that variations in this parameter caused an order-of-magnitude change in time-average flow rate for certain values of imposed pressure difference. Average flow rate doubled when contraction frequency was increased within its physiological range. Optimum lymphangion length was found to be some 13-14.5 diameters. A peak of time-average flow rate occurred when transmural pressure was such that the pressure-diameter loop for active contractions was centered near maximum passive vessel compliance. Increasing the number of lymphangions in the chain improved the pumping in the presence of larger adverse pressure differences. For a given pressure difference, the optimal number of lymphangions increased with the total vessel length. These results indicate that further experiments to estimate valve resistance more accurately are necessary. The existence of an optimal value of transmural pressure may provide additional guidelines for increasing pumping in areas affected by edema.

Enhancing Oral Performance of Paclitaxel Lipid-Mimic Prodrug via Modulating Type of Fatty Acids.

Owing to the serious clinical side effects of intravenous Taxol, an oral chemotherapeutic strategy is expected to be promising for paclitaxel (PTX) delivery. However, its poor solubility and permeability, high first-pass metabolism, and gastrointestinal toxicity need to be overcome. A triglyceride (TG)-like prodrug strategy facilitates oral drug delivery by bypassing liver metabolism. However, the effect of fatty acids (FAs) in sn-1,3 on the oral absorption of prodrugs remains unclear. Herein, a series of TG-mimetic prodrugs of PTX is explored with different carbon chain lengths and degrees of unsaturation of FAs at the sn-1,3 position in an attempt to enhance oral antitumor effect and to guide the design of TG-like prodrugs. Interestingly, the different FA lengths exhibit great influence on in vitro intestinal digestion behavior, lymph transport efficiency, and up to fourfold differences in plasma pharmacokinetics. The prodrug with long-chain FAs shows a more effective antitumor effect, whereas the degree of unsaturation has a negligible impact. The findings illustrate how FAs structures affect the oral delivery efficiency of TG-like PTX prodrugs and thus provide a theoretical basis for their rational design.

An Enhanced 3D Model of Intravascular Lymphatic Valves to Assess Leaflet Apposition and Transvalvular Differences in Wall Distensibility.

Lymphatic valves operate in a fluid-dynamically viscous environment that has little in common with that of cardiac valves, and accordingly have a different, axially lengthened, shape. A previously developed 3D fluid/structure interaction model of a lymphatic valve was extended to allow the simulation of stages of valve closure after the leaflets come together. This required that the numerical leaflet be prevented from passing into space occupied by the similar other leaflet. The resulting large deflections of the leaflet and lesser deflections of the rest of the valve were mapped as functions of the transvalvular pressure. In a second new development, the model was reconstructed to allow the vessel wall to have different material properties on either side of where the valve leaflet inserts into the wall. As part of this, a new pre-processing scheme was devised which allows easier construction of models with modified valve dimensions, and techniques for successfully interfacing the CAD software to the FE software are described. A two-fold change in wall properties either side of the leaflet made relatively little difference to valve operation apart from affecting the degree of sinus distension during valve closure. However, the numerically permitted strains were modest (<14%), and did not allow examination of the large-scale highly nonlinear elastic properties exhibited by real lymphatic vessels. A small series of murine popliteal, mesenteric, and inguinal-axillary lymphatic vessel segments containing a valve were experimentally investigated ex vivo. The pressure-diameter curves measured just upstream and just downstream of the valve were parameterised by computing the difference in tubular distensibility at three values of transmural pressure. In the popliteal and mesenteric segments, it was found that the distensibility was usually greater just downstream, i.e., in the sinus region, than upstream, at low and intermediate transmural pressure. However, there was wide variation in the extent of difference, and possible reasons for this are discussed.

Modelling the coupling of the M-clock and C-clock in lymphatic muscle cells.

Chronic dysfunction of the lymphatic vascular system results in fluid accumulation between cells: lymphoedema. The condition is commonly acquired secondary to diseases such as cancer or the associated therapies. The primary driving force for fluid return through the lymphatic vasculature is provided by contractions of the muscularized lymphatic collecting vessels, driven by electrochemical oscillations. However, there is an incomplete understanding of the molecular and bioelectric mechanisms involved in lymphatic muscle cell excitation, hampering the development and use of pharmacological therapies. Modelling in silico has contributed greatly to understanding the contributions of specific ion channels to the cardiac action potential, but modelling of these processes in lymphatic muscle remains limited. Here, we propose a model of oscillations in the membrane voltage (M-clock) and intracellular calcium concentrations (C-clock) of lymphatic muscle cells. We modify a model by Imtiaz and colleagues to enable the M-clock to drive the C-clock oscillations. This approach differs from typical models of calcium oscillators in lymphatic and related cell types, but is required to fit recent experimental data. We include an additional voltage dependence in the gating variable control for the L-type calcium channel, enabling the M-clock to oscillate independently of the C-clock. We use phase-plane analysis to show that these M-clock oscillations are qualitatively similar to those of a generalised FitzHugh-Nagumo model. We also provide phase plane analysis to understand the interaction of the M-clock and C-clock oscillations. The model and methods have the potential to help determine mechanisms and find targets for pharmacological treatment of lymphoedema.

Modelling secondary lymphatic valves with a flexible vessel wall: how geometry and material properties combine to provide function.

A three-dimensional finite-element fluid/structure interaction model of an intravascular lymphatic valve was constructed, and its properties were investigated under both favourable and adverse pressure differences, simulating valve opening and valve closure, respectively. The shear modulus of the neo-Hookean material of both vascular wall and valve leaflet was varied, as was the degree of valve opening at rest. Also investigated was how the valve characteristics were affected by prior application of pressure inflating the whole valve. The characteristics were parameterised by the volume flow rate through the valve, the hydraulic resistance to flow, and the maximum sinus radius and inter-leaflet-tip gap on the plane of symmetry bisecting the leaflet, all as functions of the applied pressure difference. Maximum sinus radius on the leaflet-bisection plane increased with increasing pressure applied to either end of the valve segment, but also reflected the non-circular deformation of the sinus cross section caused by the leaflet, such that it passed through a minimum at small favourable pressure differences. When the wall was stiff, the inter-leaflet gap increased sigmoidally during valve opening; when it was as flexible as the leaflet, the gap increased more linearly. Less pressure difference was required both to open and to close the valve when either the wall or the leaflet material was more flexible. The degree of bias of the valve characteristics to the open position increased with the inter-leaflet gap in the resting position and with valve inflation pressure. The characteristics of the simulated valve were compared with those specified in an existing lumped-parameter model of one or more collecting lymphangions and used to estimate a revised value for the constant in that model which controls the rate of valve opening/closure with variation in applied pressure difference. The effects of the revised value on the lymph pumping efficacy predicted by the lumped-parameter model were evaluated.

Efficient Intestinal Digestion and On Site Tumor-Bioactivation are the Two Important Determinants for Chylomicron-Mediated Lymph-Targeting Triglyceride-Mimetic Docetaxel Oral Prodrugs.

The oral absorption of chemotherapeutical drugs is restricted by poor solubility and permeability, high first-pass metabolism, and gastrointestinal toxicity. Intestinal lymphatic transport of lipophilic prodrugs is a promising strategy to improve the oral delivery efficiency of anticancer drugs via entrapment into a lipid formulation and to avoid first-pass metabolism. However, several basic principles have still not been clarified, such as intestinal digestibility and stability and on-site tumor bioactivation. Herein, triglyceride-mimetic prodrugs of docetaxel (DTX) are designed by conjugating them to the sn-2 position of triglyceride (TG) through different linkage bonds. The role of intestinal digestion in oral absorption of TG-like prodrugs is then investigated by introducing significant steric-hindrance α-substituents into the prodrugs. It is surprisingly found that poor intestinal digestion leads to an unsatisfactory bioavailability but efficient intestinal digestion of TG-like prodrugs with a less steric-hindrance linkage (DTX-S-S-TG) facilitating oral absorption. Moreover, it is found that the TG-like reduction-sensitive prodrug (DTX-S-S-TG) has good stability during intestinal transport and blood circulation, and on-demand release of docetaxel at the tumor site, leading to a significantly improved antitumor efficiency with negligible gastrointestinal toxicity. In summary, the chylomicron-mediated lymph-targeting triglyceride-mimetic oral prodrug approach provides a good foundation for the development of oral chemotherapeutical formulations.

Probing the effect of morphology on lymphatic valve dynamic function.

The lymphatic system is vital to the circulatory and immune systems, performing a range of important functions such as transport of interstitial fluid, fatty acid, and immune cells. Lymphatic vessels are composed of contractile walls and lymphatic valves, allowing them to pump lymph against adverse pressure gradients and to prevent backflow. Despite the importance of the lymphatic system, the contribution of mechanical and geometric changes of lymphatic valves and vessels in pathologies of lymphatic dysfunction, such as lymphedema, is not well understood. We develop a fully coupled fluid-solid, three-dimensional computational model to interrogate the various parameters thought to influence valve behavior and the consequences of these changes to overall lymphatic function. A lattice Boltzmann model is used to simulate the lymph, while a lattice spring model is used to model the mechanics of lymphatic valves. Lymphatic valve functions such as enabling lymph flow and preventing backflow under varied lymphatic valve geometries and mechanical properties are investigated to provide an understanding of the function of lymphatic vessels and valves. The simulations indicate that lymphatic valve function is optimized when valves are of low aspect ratio and bending stiffness, so long as these parameters are maintained at high enough values to allow for proper valve closing. This suggests that valve stiffening could have a profound effect on overall lymphatic pumping performance. Furthermore, dynamic valve simulations showed that this model captures the delayed response of lymphatic valves to dynamic flow conditions, which is an essential feature of valve operation. Thus, our model enhances our understanding of how lymphatic pathologies, specifically those exhibiting abnormal valve morphologies such as has been suggested to occur in cases of primary lymphedema, can lead to lymphatic dysfunctions.

Roe-derived phospholipid administration enhances lymphatic docosahexaenoic acid-containing phospholipid absorption in unanesthetized rats.

Plasma n-3 fatty acids are important as the supplying pool of n-3 fatty acids to various tissues including the brain, although the relationship between dietary n-3 fatty acids and their molecular species in the plasma are not fully clarified. We investigated the intestinal absorption of docosahexaenoic acid (DHA) derived from fish roe phospholipid (Roe-PL) and compared it with fish oil triacylglycerol and free DHA using unanesthetized lymph-cannulated rats. The DHA absorption from intraduodenally administered three samples were not significantly different, whereas Roe-PL administration resulted in a significantly higher level of DHA in the phospholipid fraction than the other two samples administrations. DHA in Roe-PL at the sn-2 position was less hydrolyzed by pancreatin than by purified phospholipase A2 in vitro and simultaneous administration of free DHA and lysophosphatidylcholine did not produce the same results as the Roe-PL administration. Our results indicate that dietary DHA-containing phospholipid is effective to increase the systemic DHA incorporated into phospholipids via intestinal absorption and biosynthesis.

Quantification of the passive and active biaxial mechanical behaviour and microstructural organization of rat thoracic ducts.

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague-Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH2O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1-48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural 'fibre families' oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development.

Incorporating measured valve properties into a numerical model of a lymphatic vessel.

An existing lumped-parameter model of multiple lymphangions (lymphatic vascular segments) in series is adapted for the incorporation of recent physiological measurements of lymphatic vascular properties. The new data show very marked nonlinearity of the passive pressure-diameter relation during distension, relative to comparable blood vessels, and complex valve behaviour. Since lymph is transported as a result of either the active contraction or the passive squeezing of vascular segments situated between two one-way valves, the performance of these valves is of primary importance. The valves display hysteresis (the opening and closing pressure drop thresholds differ), a bias to staying open (both state changes occur when the trans-valve pressure drop is adverse) and pressure-drop threshold dependence on transmural pressure. These properties, in combination with the strong nonlinearity that valve operation represents, have in turn caused intriguing numerical problems in the model, and we describe numerical stratagems by which we have overcome the problems. The principal problem is also generalised into a relatively simple mathematical example, for which solution detail is provided using two different solvers.