Passive Permeability Research Articles

Implications of Nanotechnology in Ameliorating Cancer

Despite medical advances, cancer is still one of the most dreaded disorders. Surgery, chemotherapies and radiotherapies have typically been the preferred form of treatment. However, their limited solubility and unstable nature hinder their widespread application. Drug resistance, hair loss, and the destruction of healthy cells are all frequent side effects. Nanotechnology provides new cancer treatment options in this regard and have emerged as an efficient platform in targeting chemotherapies to malignant cells and neoplasms particularly. It has also been known to improve the therapeutic efficacy of radiation based and other modern treatment modalities. All these factors tend to lower the risk of a patient suffering from cancer, thereby increasing its survival.
 Research on cancer therapy using nanotechnology extends beyond the development of existing drugs to create new ones that are only made possible by the special properties of nanomaterials. Despite being much smaller than cells, nanoparticles are large enough to hold a variety of microscopic substances. Additionally, ligands like small molecules, DNA or RNA strands, peptides, aptamers, or antibodies can be employed to functionalize the relatively large surface area of nanoparticles. These characteristics allow the targeted delivery of many drugs with theranostic action. Their physical characteristics such as energy absorption and re-radiation can also be employed to destroy malignant cells.
 By enabling therapeutic substances to be enclosed in nanoparticulate materials and delivered specifically to tumours via passive permeation and active internalisation mechanisms, nanomedicine technologies have paved the way for revolutionary targeted cancer therapies. One of the biggest barriers to traditional therapy, resistance, has been discovered to be reduced by employing nanotechnology for therapeutic purposes. Many cancer treatments using nanomedicines are already available on the market, and many more are in advanced stages of development and clinical testing. These treatments use a variety of nanosystems, including metallic nanoparticles, liposomes, quantum dots, carbon nanotubes, polymeric micelles, and nanospheres.

Mechanistic modeling-guided optimization of microneedle-based skin patch for rapid transdermal delivery of naloxone for opioid overdose treatment.

Naloxone, an FDA-approved opioid inhibitor, used to reverse opioid overdose complications has up till date faced challenges associated with its delivery. Limitations include the use of invasive delivery forms and the need for frequent redosing due to its short half-life. The goal of the current study was to design a transdermal rapidly dissolving polymeric microneedle (MN) patch with delivery and pharmacokinetic properties comparable to that seen with the commercially available NAL products, eliminating their delivery limitations. Patches of varying dimensions (500µm; 100 array,800µm; 100array, and 600µm; 225 array) were fabricated to evaluate the effect of increasing MN length, and density (no. of needles/unit area) on drug release. Drug dose in each of these patches was 17.89 ± 0.23mg, 17.2 ± 0.77mg, and 17.8 ± 1.01mg, respectively. Furthermore, the insertion efficiency of each of the MN patches was 94 ± 4.8%, 90.6 ± 1.69%, and 96 ± 1.29%, respectively. Compared to passive permeation, a reduced lag time of about 5-15min was observed with a significant drug flux of 15.09 ± 7.68g[Formula: see text]/cm2/h seen in the first 1h (p < 0.05) with the array of 100 needles (500µm long). Over 24h, a four and ten-fold increase in permeation was seen with the longer length and larger density MN patch, respectively, when compared to the 500µm (100 array) patch. Model simulations and analyses revealed the significance of needle base diameter and needle count in improving systemic pharmacokinetics of NAL.

Conformational Effects on the Passive Membrane Permeability of Synthetic Macrocycles.

Macrocyclic compounds (MCs) can have complex conformational properties that affect pharmacologically important behaviors such as membrane permeability. We measured the passive permeability of 3600 diverse nonpeptidic MCs and used machine learning to analyze the results. Incorporating selected properties based on the three-dimensional (3D) conformation gave models that predicted permeability with Q2 = 0.81. A biased spatial distribution of polar versus nonpolar regions was particularly important for good permeability, consistent with a mechanism in which the initial insertion of nonpolar portions of a MC helps facilitate the subsequent membrane entry of more polar parts. We also examined effects on permeability of 800 substructural elements by comparing matched molecular pairs. Some substitutions were invariably beneficial or invariably deleterious to permeability, while the influence of others was highly contextual. Overall, the work provides insights into how the permeability of MCs is influenced by their 3D conformational properties and suggests design hypotheses for achieving macrocycles with high membrane permeability.

Macromolecules in polysorbate 80 for injection: an important cause of anaphylactoid reactions

Polysorbate 80 for injection (TW80) is a common excipient used for injection whose macromolecular impurities, including those that cause anaphylactoid reactions, are frequently ignored. The main aim of this study was to prove that the macromolecular impurities in the excipient are an important cause of anaphylactoid reactions. Component A (containing macromolecules > 100 kDa), Component B (containing macromolecules from 10 to 100 kDa), and Component C (containing substances < 10 kDa) were prepaired from the original TW80 using ultrafilters. The original TW80 contained numerous substances with molecular weights > 10kD. The original TW80 and Components A and B caused strong anaphylactoid reactions in both guinea pigs and rabbits by intravenous administration. Moreover, the original TW80 and Components A and B even caused strong passive cutaneous anaphylactoid (PCA) reactions and pulmonary capillary permeability. The PCA reaction and increased permeability were partly prevented by cromolyn sodium. Additionally, the original TW80 and Components A and B caused vasodilation and severe hemolysis in vitro. The anaphylactoid reactions were associated with histamine release but not with mast cell degranulation. Nevertheless, Component C almost caused no anaphylactoid reactions or hemolysis and was weaker in the few reactions that ocurred. Taken together, these results suggest that macromolecular substances are one of the main risk factors responsible for anaphylactoid reactions and hemolysis caused by TW80.

Systematic Exploration of Passive Permeability in Tetrapeptides with Hydrogen-Bond-Accepting Amino Acid Side Chains.

We synthesized and experimentally tested the passive permeability of more than thirty tetrapeptides mimicking the N-terminus of the pro-apoptotic protein Smac (Second mitochondria-derived activator of caspases). Each peptide bore one or two unnatural Hydrogen Bond Acceptor-bearing Amino Acid (HBA-AA) residues, such that intramolecular hydrogen bonding with proximal backbone amide N-H donors is feasible. Passive permeability of the synthetic peptides was determined using the parallel artificial membrane permeability assay (PAMPA). Experimental permeability values were found to span three orders of magnitude, providing useful empirical guidance for the design of more permeable Smac mimetics specifically, and peptidic ligands generally.

Property-Driven Development of Passively Permeable Macrocyclic Scaffolds Using Heterocycles.

Passive membrane permeability is a fundamental challenge in the development of bioactive macrocycles. To achieve this objective, chemists have resorted to various strategies, the most common of which is deployment of N-methylated amino acids and/or D-amino acids. Here we investigate the effect of heterocyclic grafts on the passive membrane permeability of macrocycles and report the structural consequences of iterative amino acid replacement by azole rings. Through stepwise substitution of amino acid residues for heterocycles, we show that lipophilicity and PAMPA permeability of a macrocycle can be vastly improved. Overall, changes in permeability do not scale linearly as more heterocycles are incorporated, underscoring the subtleties of conformation-property relationships in this class of molecule. NMR analysis and molecular dynamics simulations provide insights into the structural consequences of the added heterocycles and these frameworks can now be applied as macrocyclic scaffolds for drug discovery.

Property‐Driven Development of Passively Permeable Macrocyclic Scaffolds Using Heterocycles**

AbstractPassive membrane permeability is a fundamental challenge in the development of bioactive macrocycles. To achieve this objective, chemists have resorted to various strategies, the most common of which is deployment of N‐methylated amino acids and/or D‐amino acids. Here we investigate the effect of heterocyclic grafts on the passive membrane permeability of macrocycles and report the structural consequences of iterative amino acid replacement by azole rings. Through stepwise substitution of amino acid residues for heterocycles, we show that lipophilicity and PAMPA permeability of a macrocycle can be vastly improved. Overall, changes in permeability do not scale linearly as more heterocycles are incorporated, underscoring the subtleties of conformation‐property relationships in this class of molecule. NMR analysis and molecular dynamics simulations provide insights into the structural consequences of the added heterocycles and these frameworks can now be applied as macrocyclic scaffolds for drug discovery.

Transporters in Drug Development: International Transporter Consortium Update on Emerging Transporters of Clinical Importance.

During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers.

Piperine Derivatives Enhance Fusion and Axonal Transport of Mitochondria by Activating Mitofusins

Piperine (1-piperoylpiperidine) is the major pungent component of black pepper (Piper nigrum) and exhibits a spectrum of pharmacological activities. The molecular bases for many of piperine’s biological effects are incompletely defined. We noted that the chemical structure of piperine generally conforms to a pharmacophore model for small bioactive molecules that activate mitofusin (MFN)-mediated mitochondrial fusion. Piperine, but not its isomer chavicine, stimulated mitochondrial fusion in MFN-deficient cells with EC50 of ~8 nM. We synthesized piperine analogs having structural features predicted to optimize mitofusin activation and defined structure-activity relationships (SAR) in live-cell mitochondrial elongation assays. When optimal spacing was maintained between amide and aromatic groups the derivatives were potent mitofusin activators. Compared to the prototype phenylhexanamide mitofusin activator, 2, novel molecules containing the piperidine structure of piperine exhibited markedly enhanced passive membrane permeability with no loss of fusogenic potency. Lead compounds 5 and 8 enhanced mitochondrial motility in cultured murine Charcot-Marie-Tooth disease type 2A (CMT2A) neurons, but only 8 improved mitochondrial transport in sciatic nerve axons of CMT2A mice. Piperine analogs represent a new chemical class of mitofusin activators with potential pharmaceutical advantages.

A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation.

To mimic the levels of spatiotemporal control that exist in nature, tools for chemically induced dimerization (CID) are employed to manipulate protein-protein interactions. Although linker composition is known to influence speed and efficiency of heterobifunctional compounds, modeling or in vitro experiments are often insufficient to predict optimal linker structure. This can be attributed to the complexity of ternary complex formation and the overlapping factors that impact the effective concentration of probe within the cell, such as efflux and passive permeability. Herein, we synthesize a library of modular chemical tools with varying linker structures and perform quantitative microscopy in live cells to visualize dimerization in real-time. We use our optimized probe to demonstrate our ability to recruit a protein of interest (POI) to the mitochondria, cell membrane, and nucleus. Finally, we induce and monitor local and global phase separation. We highlight the importance of quantitative approaches to linker optimization for dynamic systems and introduce new, synthetically accessible tools for the rapid control of protein localization.

Abstract 3461: Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy

Abstract Background: Incidence of patients having brain metastasis of breast cancer has increased due to enhanced treatment for peripheral disease. Breast cancer brain metastasis is often fatal and occurs in 15-20% of patients diagnosed with stage IV breast cancer. Traditional chemotherapy delivery to the brain is limited by the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Low intensity focused ultrasound (LIFU) is a unique technique recently investigated to non-invasively disrupt these barriers and aid neuro-modulation. Herein, we demonstrate the use of a clinical LIFU device to modulate blood-tumor permeability and deliver combined chemotherapy in a novel preclinical model of brain metastases of breast cancer. Methods: Brain colonizing MDA-MB-231Br cells transfected with luciferase were injected into athymic Nu/Nu female mice by intracardiac injection into the left ventricle (per mouse 175,000 cells). Brain metastasis was allowed to develop, and tumor burden was monitored by bioluminescent imaging IVIS Spectrum CT (Perkin Elmer). Mice were randomized into sham or single treatment or combined treatment groups on Day 21 (Doxil nanoparticles 5.6 mg/kg or Paclitaxel 10mg/kg). Mice receiving LIFU were placed on a specially designed animal restraint with the skull partially immersed in degassed water. T1 weighted MRI was used to identify and localize sonication spots within the cortex, and mice were sonicated using 0.4uL/kg Definity© at 0.3 cavitation dose for 60sec. Treatment with LIFU was continued once weekly for three consecutive weeks. Survival and tumor burden was recorded until mice developed neurological symptoms. LIFU induced blood-brain barrier breakdown in healthy mice was also analyzed by in-situ brain perfusion with 14CSucrose, 3HGlucose, and 3HIvermectin to monitor BBB integrity and transporter function. Results: Mice receiving LIFU+paclitaxel+Doxil-NP showed significantly lower tumor burden (peak area 635.9) and higher median survival of 60 days as compared to vehicle (29d) and LIFU+vehicle (32d) groups. Analysis of BBB permeability in healthy mice demonstrated significantly higher passive permeability marker accumulation than non-sonicated contralateral hemisphere. Conclusion: Timing the combined chemotherapy with LIFU induced windows of maximal BBB opening resulted in slower tumor progression and increased survival in a preclinical model of breast cancer brain metastasis. Future directions aim to combine these results and evaluate underlying mechanisms of enhanced permeability post-LIFU. Citation Format: Tasneem A. Arsiwala, Kathryn E. Blethen, Samuel A. Sprowls, Ross A. Fladeland, Brooke Keilkowski, Morgan Glass, Trenton Pritt, Peng Wang, Ali Rezai, Paul R. Lockman. Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3461.

Lack of an Effect of Polysorbate 80 on Intestinal Drug Permeability in Humans.

Despite no broad, direct evidence in humans, there is a potential concern that surfactants alter active or passive drug intestinal permeation to modulate oral drug absorption. The purpose of this study was to investigate the impact of the surfactant polysorbate 80 on active and passive intestinal drug absorption in humans. The human (n = 12) pharmacokinetics (PK) of three probe substrates of intestinal absorption, valacyclovir, chenodeoxycholic acid (CDCA), and enalaprilat, were assessed. Endogenous bile acid levels were assessed as a secondary measure of transporter and microbiota impact. Polysorbate 80 did not inhibit peptide transporter 1 (PepT1)- or apical sodium bile acid transporter (ASBT)-mediated PK of valacyclovir and CDCA, respectively. Polysorbate 80 did not increase enalaprilat absorption. Modest increases in unconjugated secondary bile acid Cmax ratios suggest a potential alteration of the in vivo intestinal microbiota by polysorbate 80. Polysorbate 80 did not alter intestinal membrane fluidity or cause intestinal membrane disruption. This finding supports regulatory relief of excipient restrictions for Biopharmaceutics Classification System-based biowaivers.

In silico-in vitro estimation of lipophilicity and permeability association for succinimide derivatives using chromatographic anisotropic systems and parallel artificial membrane permeability assay.

Passive permeability is one of the key features that determine absorbability and one of the most studied properties in the early phases of drug development. Newly synthesized succinimide derivatives from two different series (1-aryl-3-methylsuccinimides and 1-aryl-3-ethyl-3-methylsuccinimides) with high biological potential have been subjected to estimation of their passive permeability and their association with (a) experimentally obtained anisotropic lipophilicity, (b) in silico-calculated lipophilicity and (c) in silico-predicted permeability and absorbability. Non-cellular-based parallel artificial membrane permeability assay was applied for quantifying their passive permeation, expressed as logPapp . Passive permeation was governed by the lipophilicity of the analysed compounds, and anisotropic lipophilicity was related with statistically significant passive transcellular diffusion (r2 = 0.614, P < 0.001). Moreover, experimentally determined passive permeability, logPapp , was statistically significantly associated with both in silico-predicted absorption constant, ka (r2 = 0.7886, P < 0.001), and human intestinal absorption (HIA) in percentage (r2 = 0.484, P < 0.001), respectively. However, there was no statistically significant relationship between experimentally obtained permeability on non-cellular-based model and in silico-predicted Caco-2 permeability based on the predictions conducted on two different software. Based on the obtained results, anisotropic systems are promising surrogates for determining lipophilicity, except for compounds with acidic functional groups that are completely ionized under (pH = 7.4).

Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells.

BackgroundTriple-negative breast cancer (TNBC) responds poorly to the available drugs; thus, the mortality rate associated with TNBC remains high. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells.Methods7HF uptake by MDA-MB-468 cells was assessed using spectrophotometry. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. Then, the effect of 7HF treatment (6, 12, and 24 µM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. Polymerization of α- and β-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs.ResultsWe observed that 7HF was able to enter the MDA-MB-468 cells. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. 7HF reduced polymerization of α-, β-tubulin in dose-dependent manner. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. According to molecular docking analysis, 7HF preferred to bind to β-tubulin over α-tubulin. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF.ConclusionsThis is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. The findings will be useful for developing 7HF-based treatment for patients with TNBC.

Grid inhomogeneous solvation theory for cross-solvation in rigid solvents.

Grid Inhomogeneous Solvation Theory (GIST) has proven useful to calculate localized thermodynamic properties of water around a solute. Numerous studies have leveraged this information to enhance structure-based binding predictions. We have recently extended GIST toward chloroform as a solvent to allow the prediction of passive membrane permeability. Here, we further generalize the GIST algorithm toward all solvents that can be modeled as rigid molecules. This restriction is inherent to the method and is already present in the inhomogeneous solvation theory. Here, we show that our approach can be applied to various solvent molecules by comparing the results of GIST simulations with thermodynamic integration (TI) calculations and experimental results. Additionally, we analyze and compare a matrix consisting of 100 entries of ten different solvent molecules solvated within each other. We find that the GIST results are highly correlated with TI calculations as well as experiments. For some solvents, we find Pearson correlations of up to 0.99 to the true entropy, while others are affected by the first-order approximation more strongly. The enthalpy-entropy splitting provided by GIST allows us to extend a recently published approach, which estimates higher order entropies by a linear scaling of the first-order entropy, to solvents other than water. Furthermore, we investigate the convergence of GIST in different solvents. We conclude that our extension to GIST reliably calculates localized thermodynamic properties for different solvents and thereby significantly extends the applicability of this widely used method.

Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer

IntroChronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate. MethodsFirst, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [11C]AZD1283 with radioactive precursor [11C]NH4CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [11C]AZD1283 via dynamic PET imaging of naïve wild-type mice, P2Y12R knockout mouse, and a rhesus macaque. ResultsWe determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [11C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/μmol (average of n = 3). Of note, we found [11C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV)). ConclusionDespite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [11C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [11C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development.

Mechanistic Modeling of Central Nervous System Pharmacokinetics and Target Engagement of HER2 Tyrosine Kinase Inhibitors to Inform Treatment of Breast Cancer Brain Metastases.

This study evaluated the central nervous system (CNS) pharmacokinetics and target engagement of lapatinib, neratinib, and tucatinib in patients with cancer, using a physiologically based pharmacokinetic (PBPK) modeling approach. Drug-specific parameters for in vitro metabolism, binding to plasma proteins and brain tissues, transcellular passive permeability, and interactions with efflux transporters were determined. Whole-body PBPK models integrated with a 4-compartment permeability-limited brain model was developed and verified for predicting plasma and CNS pharmacokinetics. Target engagement ratio (TER), defined as the ratio of the average steady-state unbound drug brain concentration (Css,ave,br) to in vitro IC50 for HER2 inhibition, was used as a predictor of intracranial efficacy. PBPK models predicted that following 1 cycle of standard dosing, tucatinib and lapatinib achieved similar Css,ave,br (14.5 vs. 16.8 nmol/L), while neratinib Css,ave,br (0.68 nmol/L) was 20-fold lower. Tucatinib and neratinib were equally potent for HER2 inhibition (IC50, 6.9 vs. 5.6 nmol/L), while lapatinib was less potent (IC50, 109 nmol/L). The model-predicted population mean TER in the human normal brain was 2.1 for tucatinib, but < 0.20 for lapatinib and neratinib. The PBPK modeling suggests that tucatinib induces sufficient HER2 inhibition (TER > 2.0) in not only brain metastases with a disrupted blood-brain barrier (BBB), but also micrometastases where the BBB largely remains intact. These findings, in line with available clinical pharmacokinetics and efficacy data, support the therapeutic value of tucatinib for treatment of brain metastases and warrant further clinical investigation for the prevention of brain metastases in patients with HER2-positive breast cancer.

A physiologically based pharmacokinetic model to predict pegylated liposomal doxorubicin disposition in rats and human.

The use of nanoparticles (NPs) can support an enhancement of drug distribution, resulting in increased drug penetration into key tissues. Experimental in vitro data can be integrated into computational approaches to simulate NP absorption, distribution, metabolism and elimination (ADME) processes and provide quantitative pharmacokinetic predictions. The aim of this study is to develop a novel mechanistic and physiologically based pharmacokinetic (m-PBPK) model to predict the biodistribution of NPs focusing on Doxil. The main processes underpinning NPs ADME were represented considering molecular and cellular mechanisms such as stability in biological fluids, passive permeability and uptake activity by macrophages. A whole-body m-PBPK rat and human models were designed in Simbiology v. 9.6.0 (MATLAB R2019a). The m-PBPK models were successfully qualified across doxorubicin and Doxil® in both rat and human since all PK parameters AUC0-inf, Cmax, t1/2, Vd and Cl were within twofold, with an AUC0-inf absolute average-fold error (AAFE) value of 1.23 and 1.16 and 1.76 and 1.05 for Doxorubicin and Doxil® in rat and human, respectively. The time to maximum concentration in tissues for doxorubicin in both rat and human models was before 30min of administration, while for Doxil®, the tmax was after 24h of administration. The organs that accumulate most NP are the spleen, liver and lungs, in both models. The m-PBPK represents a predictive platform for the integration of in vitro and formulation parameters in a physiological context to quantitatively predict the NP biodistribution. Schematic diagram of the whole-body m-PBPK models developed for Doxil® in rat and human physiology.

Solubility and Permeability Enhancement of Oleanolic Acid by Solid Dispersion in Poloxamers and γ-CD.

Oleanolic acid (OA) is a pentacyclic triterpenoid widely found in the Oleaceae family, and it represents 3.5% of the dry weight of olive leaves. OA has many pharmacological activities, such as hepatoprotection, anti-inflammatory, anti-oxidant, anti-diabetic, anti-tumor, and anti-microbic activities. Its therapeutic application is limited by its poor water solubility, bioavailability, and permeability. In this study, solid dispersions (SDs) were developed to overcome these OA limitations. Solubility studies were conducted to evaluate different hydrophilic polymers, drug-to-polymer ratios, and preparation methods. Poloxamer 188, Poloxamer 407, and γ-CD exhibited the highest increases in terms of OA solubility, regardless of the method of preparation. Binary systems were characterized using differential scanning calorimetry (DSC), X-ray diffraction (XRPD), and Fourier transform infrared spectroscopy (FTIR). In addition, pure compounds and SDs were analyzed using scanning electron microscopy (SEM) in order to observe both the morphology and the particle surface. In vitro dissolution studies were performed for P407, P188, and γ-CD SDs. Preparation using the solvent evaporation method (SEM) produced the highest increase in the dissolution profiles of all three polymers with respect to the OA solution. Finally, the effect of SDs on OA permeability was evaluated with an in vitro parallel artificial membrane permeability assay (PAMPA). The formulation improved passive permeation across the simulated barrier due to OA increased solubility. The dissolution and PAMPA results indicate that the amorphization of OA by SD preparation could be a useful method to enhance its oral absorption, and it is also applicable on an industrial scale.

PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION

You have accessJournal of UrologyCME1 May 2022PD36-08 BLOOD TESTIS BARRIER-MIMETIC DROPLET INTERFACE BILAYERS (DIBS) FOR PASSIVE PERMEABILITY PREDICTION Jaime Korner, Meghan Robinson, Katherine Elvira, Ryan Flannigan, and Stephanie Willerth Jaime KornerJaime Korner More articles by this author , Meghan RobinsonMeghan Robinson More articles by this author , Katherine ElviraKatherine Elvira More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Stephanie WillerthStephanie Willerth More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The blood testis barrier (BTB) serves as a “gatekeeper” for the apical compartment of the seminiferous epithelium where spermatogenesis takes place. Tight junctions between adjacent Sertoli cells create the BTB, regulate transport of molecules, and protect the luminal compartment from toxins and pathogens. Investigating the impact of toxins and drugs, and the regulation of human spermatogenesis requires realistic in vitro models. The purpose of this study was to develop an artificial cellular membrane to mimic the BTB using droplet interface bilayers (DIBs) derived from human Sertoli cell lipid extracts. METHODS: Human Sertoli cells were collected from a dissociated human testicular biopsy using magnetic activated sorting for Follicle-Stimulating Hormone Receptor (FSHR). Sertoli cells were expanded in vitro and lipids were extracted using a Lipid Extraction Kit (Cell Biolabs). Permeability experiments were conducted using a poly(dimethylsiloxane) (PDMS) microfluidic device which allows for the controlled formation of pairs of differing droplet compartments connected by a phospholipid bilayer. A solution of Sertoli lipids in squalene (10 mg/mL) was used as the outer phase. Donor droplets contained fluorophore (100 µM in buffer) and acceptor droplets contained only buffer (both pH 7.4). The DIB-connected droplet pairs were formed at 50 °C and the temperature was decreased to 37 °C for observation. RESULTS: These results represent the first DIBs to be made using human lipid extracts and the first use of DIBs to model permeability of the BTB. We found that the BTB-mimetic DIBs were impermeable to both fluorophores tested: fluorescein and calcein. Calcein is normally membrane-impermeable and serves as a control experiment to confirm the presence of an intact phospholipid bilayer. Fluorescein has been observed to permeate DIBs made using single, synthetic lipids and intestine-mimetic DIBs but not most blood-brain barrier-mimetic DIBs. CONCLUSIONS: DIBs can be made from human Sertoli cell lipid extracts and show great potential to more accurately model BTB permeability. Source of Funding: Collaborative Healthy Grant © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e635 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jaime Korner More articles by this author Meghan Robinson More articles by this author Katherine Elvira More articles by this author Ryan Flannigan More articles by this author Stephanie Willerth More articles by this author Expand All Advertisement PDF DownloadLoading ...