Interstitial Fluid Concentrations Research Articles

Plasma and interstitial fluid antibiotic levels of subcutaneously implanted compounded florfenicol calcium sulfate beads in New Zealand White rabbits (Oryctolagus cuniculus).

To determine antibiotic levels in plasma and interstitial fluid (ISF) after SC placement of compounded florfenicol (FF) calcium sulfate beads (CSBs) in New Zealand White rabbits (Oryctolagus cuniculus). 6 juvenile female rabbits (n = 5 treatment and 1 control). An ultrafiltration probe and CSBs were placed SC in 6 rabbits (n = 5 for FF CSBs and 1 for control CSBs). Plasma (3, 6, 12, 24, and 48 hours and 7, 14, and 21 days) and ISF (daily for 21 days) samples were collected, and FF was measured by HPLC for pharmacokinetic analysis. Hematology, biochemistry, and histopathology were assessed. Means ± SD for the area under the curve, maximum concentration, time of maximum concentration, terminal half-life, and mean residence time to the last data point for plasma and ISF were 16.63 ± 8.16 and 17,902 ± 7,564 h·µg/mL, 0.79 ± 0.38 and 245 ± 223 µg/mL, 2.90 ± 0.3 and 59 ± 40 hours, 30.81 ± 16.9 and 27.3 ± 9.39 hours, 23.4 ± 10 and 73.7 ± 13 hours, respectively. Plasma FF was < 2 µg/mL at all time points. The ISF FF remained > 8 μg/mL for 109.98 to 231.58 hours. One rabbit death occurred during treatment, but the cause of death was undetermined. Local tissue inflammation was present, but no clinically significant systemic adverse effects were found on hematology, biochemistry, or histopathology in the remaining rabbits. Florfenicol CSBs maintained antibiotic concentrations in ISF at levels likely to be effective against bacteria sensitive to > 8 µg/mL for 5 to 10 days while maintaining low (< 2 µg/mL) plasma levels. Florfenicol CSBs may be effective for local antibiotic treatment in rabbit abscesses.

Hydrogel-Coated SERS Microneedles for Drug Monitoring in Dermal Interstitial Fluid.

In vivo drug monitoring is crucial for evaluating the effectiveness and safety of drug treatment. Blood sampling and analysis is the current gold standard but needs professional skills and cannot meet the requirements of point-of-care testing. Dermal interstitial fluid (ISF) showed great potential to replace blood for in vivo drug monitoring; however, the detection was challenging, and the drug distribution behavior in ISF was still unclear until now. In this study, we proposed surface-enhanced Raman spectroscopy (SERS) microneedles (MNs) for the painless and real-time analysis of drugs in ISF after intravenous injection. Using methylene blue (MB) and mitoxantrone (MTO) as model drugs, the innovative core-satellite structured Au@Ag SERS substrate, hydrogel coating over the MNs, rendered sensitive and quantitative drug detection in ISF of mice within 10 min. Based on this technique, the pharmacokinetics of the two drugs in ISF was investigated and compared with those in blood, where the drugs were analyzed via liquid chromatography-mass spectrometry. It was found that the MB concentration in ISF and blood was comparable, whereas the concentration of MTO in ISF was 2-3 orders of magnitude lower than in blood. This work proposed an efficient tool for ISF drug monitoring. More importantly, it experimentally proved that the penetration ratio of blood to ISF was drug-dependent, providing insightful information into the potential of ISF as a blood alternative for in vivo drug detection.

Quantification of Unencapsulated Drug in Target Tissues Demonstrates Pharmacological Properties and Therapeutic Effects of Liposomal Topotecan (FF-10850)

PurposeQuantifying unencapsulated drug concentrations in tissues is crucial for understanding the mechanisms underlying the efficacy and safety of liposomal drugs; however, the methodology for this has not been fully established. Herein, we aimed to investigate the enhanced therapeutic potential of a pegylated liposomal formulation of topotecan (FF-10850) by analyzing the concentrations of the unencapsulated drug in target tissues, to guide the improvement of its dosing regimen.MethodsWe developed a method for measuring unencapsulated topotecan concentrations in tumor and bone marrow interstitial fluid (BM-ISF) and applied this method to pharmacokinetic assessments. The ratios of the area under the concentration–time curves (AUCs) between tumor and BM-ISF were calculated for total and unencapsulated topotecan. DNA damage and antitumor effects of FF-10850 or non-liposomal topotecan (TPT) were evaluated in an ES-2 mice xenograft model.ResultsFF-10850 exhibited a much larger AUC ratio between tumor and BM-ISF for unencapsulated topotecan (2.96), but not for total topotecan (0.752), than TPT (0.833). FF-10850 promoted milder DNA damage in the bone marrow than TPT; however, FF-10850 and TPT elicited comparable DNA damage in the tumor. These findings highlight the greater tumor exposure to unencapsulated topotecan and lower bone marrow exposure to FF-10850 than TPT. The dosing regimen was successfully improved based on the kinetics of unencapsulated topotecan and DNA damage.ConclusionsTissue pharmacokinetics of unencapsulated topotecan elucidated the favorable pharmacological properties of FF-10850. Evaluation of tissue exposure to an unencapsulated drug with appropriate pharmacodynamic markers can be valuable in optimizing liposomal drugs and dosing regimens.

Linezolid brain penetration in neurointensive care patients.

Linezolid exposure in critically ill patients is associated with high inter-individual variability, potentially resulting in subtherapeutic antibiotic exposure. Linezolid exhibits good penetration into the CSF, but its penetration into cerebral interstitial fluid (ISF) is unknown. To determine linezolid penetration into CSF and cerebral ISF of neurointensive care patients. Five neurocritical care patients received 600 mg of linezolid IV twice daily for treatment of extracerebral infections. At steady state, blood and CSF samples were collected from arterial and ventricular catheters, and microdialysate was obtained from a cerebral intraparenchymal probe. The median fAUC0-24 was 57.6 (24.9-365) mg·h/L in plasma, 64.1 (43.5-306.1) mg·h/L in CSF, and 27.0 (10.7-217.6) mg·h/L in cerebral ISF. The median penetration ratio (fAUCbrain_or_CSF/fAUCplasma) was 0.5 (0.25-0.81) for cerebral ISF and 0.92 (0.79-1) for CSF. Cerebral ISF concentrations correlated well with plasma (R = 0.93, P < 0.001) and CSF levels (R = 0.93, P < 0.001).The median fAUC0-24/MIC ratio was ≥100 in plasma and CSF for MICs of ≤0.5 mg/L, and in cerebral ISF for MICs of ≤0.25 mg/L. The median fT>MIC was ≥80% of the dosing interval in CSF for MICs of ≤0.5 mg/L, and in plasma and cerebral ISF for MICs of ≤0.25 mg/L. Linezolid demonstrates a high degree of cerebral penetration, and brain concentrations correlate well with plasma and CSF levels. However, substantial variability in plasma levels, and thus cerebral concentrations, may result in subtherapeutic tissue concentrations in critically ill patients with standard dosing, necessitating therapeutic drug monitoring.

Demonstrating the Analytical Potential of a Wearable Microneedle-Based Device for Intradermal CO2 Detection.

Monitoring of carbon dioxide (CO2) body levels is crucial under several clinical conditions (e.g., human intensive care and acid-base disorders). To date, painful and risky arterial blood punctures have been performed to obtain discrete CO2 measurements needed in clinical setups. Although noninvasive alternatives have been proposed to assess CO2, these are currently limited to benchtop devices, requiring trained personnel, being tedious, and providing punctual information, among other disadvantages. To the best of our knowledge, the literature and market lack a wearable device for real-time, on-body monitoring of CO2. Accordingly, we have developed a microneedle (MN)-based sensor array, labeled as CO2-MN, comprising a combination of potentiometric pH- and carbonate (CO32-)-selective electrodes together with the reference electrode. The CO2-MN is built on an epidermal patch that allows it to reach the stratum corneum of the skin, measuring pH and CO32- concentrations directly into the interstitial fluid (ISF). The levels for the pH-CO32- tandem are then used to estimate the PCO2 in the ISF. Assessing the response of each individual MN, we found adequate response time (t95 < 5s), sensitivity (50.4 and -24.6 mV dec-1 for pH and CO32-, respectively), and stability (1.6 mV h-1 for pH and 2.1 mV h-1 for CO32-). We validated the intradermal measurements of CO2 at the ex vivo level, using pieces of rat skin, and then, with in vivo assays in anesthetized rats, showing the suitability of the CO2-MN wearable device for on-body measurements. A good correlation between ISF and blood CO2 concentrations was observed, demonstrating the high potential of the developed MN sensing technology as an alternative to blood-based analysis in the near future. Moreover, these results open new horizons in the noninvasive, real-time monitoring of CO2 as well as other clinically relevant gases.

Intratesticular Testosterone and Its Precursors among Azoospermic Men: A Pilot Study.

The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their significance in the testicular microenvironment and their correlation with testicular sperm retrieval outcomes. We analyzed 37 TIF samples, including 5 from men with obstructive azoospermia (OA) and 32 from men with non-obstructive azoospermia (NOA). Liquid chromatography with tandem mass spectrometry quantified testosterone and precursor levels. Comparative assessments of the outcomes of testicular sperm retrieval were performed between the OA and NOA groups as well as among men with NOA. Men with NOA who had not undergone hormone treatment exhibited significantly higher intratesticular concentrations of testosterone (median 1,528.1 vs. 207.5 ng/mL), androstenedione (median 10.6 vs. 1.9 ng/mL), and 17-OH progesterone (median 13.0 vs. 1.8 ng/mL) than men diagnosed with OA. Notably, in the subgroup of patients with NOA subjected to medical treatment, men with successful sperm retrieval had significantly reduced levels of androstenedione (median androstenedione 5.7 vs. 18.5 ng/mL, p=0.004). Upon a more detailed analysis of these men who underwent hormone manipulation treatment, the testosterone/androstenedione ratio (indicative of HSD17B3 enzyme activity) was markedly increased in men with successful sperm retrieval (median: 365.8 vs. 165.0, p=0.008) compared with individuals with NOA who had unsuccessful sperm recovery. Furthermore, within the subset of men with NOA who did not undergo medical treatment before microdissection testicular sperm extraction but achieved successful sperm retrieval, the ratio of 17-OH progesterone/progesterone (indicative of CYP17A1 activity) was substantially higher. The study suggests distinct testosterone biosynthesis pathways in men with compromised spermatogenesis and those with normal spermatogenesis. Among NOA men with successful retrieval after hormone optimization therapy, there was decreased androstenedione and increased HSD17B3 enzyme activity. These findings have diagnostic and therapeutic implications for the future.

Comparative Study of Dermal Pharmacokinetics Between Topical Drugs Using Open Flow Microperfusion in a Pig Model

PurposeAccurate methods to determine dermal pharmacokinetics are important to increase the rate of clinical success in topical drug development. We investigated in an in vivo pig model whether the unbound drug concentration in the interstitial fluid as determined by dermal open flow microperfusion (dOFM) is a more reliable measure of dermal exposure compared to dermal biopsies for seven prescription or investigational drugs. In addition, we verified standard dOFM measurement using a recirculation approach and compared dosing frequencies (QD versus BID) and dose strengths (high versus low drug concentrations).MethodsDomestic pigs were topically administered seven different drugs twice daily in two studies. On day 7, drug exposures in the dermis were assessed in two ways: (1) dOFM provided the total and unbound drug concentrations in dermal interstitial fluid, and (2) clean punch biopsies after heat separation provided the total concentrations in the upper and lower dermis.ResultsdOFM showed sufficient intra-study precision to distinguish interstitial fluid concentrations between different drugs, dose frequencies and dose strengths, and had good reproducibility between studies. Biopsy concentrations showed much higher and more variable values. Standard dOFM measurements were consistent with values obtained with the recirculation approach.ConclusionsdOFM pig model is a robust and reproducible method to directly determine topical drug concentration in dermal interstitial fluid. Dermal biopsies were a less reliable measure of dermal exposure due to possible contributions from drug bound to tissue and drug associated with skin appendages.

(Invited) Continuous Molecular Monitoring in Interstitial Fluid Via Wearable Microneedle Aptamer-Based Sensors

The ability to continuously monitor the concentration of specific molecules in the body is a long-sought goal of biomedical research. For this purpose, interstitial fluid (ISF) has been proposed as the ideal target biofluid because its composition can rapidly equilibrate with that of systemic blood, allowing the assessment of molecular concentrations that reflect full-body physiology. To tap into this measurement compartment, it is possible to use microneedle sensor arrays for continuous molecular monitoring. In this presentation, I will discuss our efforts to develop microneedle-supported electrochemical, aptamer-based (E-AB) sensors. This platform allows affinity-based monitoring of target concentrations in ISF, and takes advantage of the versatility of aptamers to selectively bind a large variety of molecular targets. I will discuss our current state of platform development, including fabrication challenges, in-vivo deployment in rodent models, and variability of pharmacokineitc profiles as a function of microneedle penetration depth in the skin.

Reduced graphene oxide-functionalized polymer microneedle for continuous and wide-range monitoring of lactate in interstitial fluid

Despite substantial developments in minimally invasive lactate monitoring microneedle electrodes, most such electrode developments have focused on either sensitivity or invasiveness while ignoring a wide range of detection, which is the most important factor in measuring the normal range of lactate in interstitial fluid (ISF). Herein, we present a polymer-based planar microneedle electrode fabrication using microelectromechanical and femtosecond laser technology for the continuous monitoring of lactate in ISF. The microneedle is functionalized with two-dimensional reduced graphene oxide (rGO) and electrochemically synthesized platinum nanoparticles (PtNPs). A particular quantity of Nafion (1.25 wt%) is applied on top of the lactate enzyme to create a diffusion-controlled membrane. Due to the combined effects of the planar structure of the microneedle, rGO, and membrane, the biosensor exhibited excellent linearity up to 10 mM lactate with a limit of detection of 2.04 μM, high sensitivity of 43.96 μA mM−1cm−2, a reaction time of 8 s and outstanding stability, selectivity, and repeatability. The feasibility of the microneedle is evaluated by using it to measure lactate concentrations in artificial ISF and human serum. The results demonstrate that the microneedle described here has great potential for use in real-time lactate monitoring for use in sports medicine and treatment.

Microneedles coated with composites of phenylboronic acid-containing polymer and carbon nanotubes for glucose measurements in interstitial fluids

A microneedle (MN) sensor coated with a sensing composite material was proposed for measuring glucose concentrations in interstitial fluid (ISF). The sensing composite material was prepared by blending a polymer containing glucose-responsive phenylboronic acid (PBA) moieties (i.e., polystyrene-block-poly(acrylic acid-co-acrylamidophenylboronic acid)) with conductive carbon nanotubes (CNTs). The polymer exhibited reversible swelling behavior in response to glucose concentrations, which influenced the distribution of the embedded CNTs, resulting in sensitive variations in electrical percolation, even when coated onto a confined surface of the MN in the sensor. We varied the ratio of PBA moieties and the loading amount of CNTs in the sensing composite material of the MN sensor and tested them in vitro using an ISF-mimicking gel with physiological glucose concentrations to determine the optimal sensitivity conditions. When tested in animal models with varying blood glucose concentrations, the MN sensor coated with the selected sensing material exhibited a strong correlation between the measured electrical currents and blood glucose concentrations, showing accuracy comparable to that of a glucometer in clinical use.

Population pharmacokinetic modeling of the influence of chronic and acute biofilm-forming Pseudomonas aeruginosa lung infection on ciprofloxacin free pulmonary and epithelial lining fluid concentrations

We previously reported that ciprofloxacin (CIP) free lung interstitial concentrations are decreased by biofilm-forming Pseudomonas aeruginosa pulmonary chronic (14 d) infection. To get a better understanding on the influence of infection on CIP lung distribution, in the present study free lung interstitial fluid and epithelial lining fluid (ELF) concentrations were determined by microdialysis in biofilm-forming P. aeruginosa acutely (2 d) and chronically infected (14 d) Wistar rats following CIP 20 mg/kg i.v. bolus dosing. A popPK model was developed, using NONMEM® (version 7.4.3) with FOCE+I, with plasma data described as a three-compartment model with first-order elimination. For lung data inclusion, the model was expanded to four compartments and ELF concentrations were described as a fraction of lung levels estimated as a distribution factor (ƒD). Acute infection had a minor impact on plasma and lung CIP distribution and both infection stages did not alter ELF drug penetration. Probability of target attainment of ƒAUC0–24/MIC ≥ 90 using 20 mg q8h, equivalent to 400 mg q8h in humans, showed that CIP free concentrations in plasma are adequate to successfully treat lung infections. However, lung and ELF free interstitial concentrations might be insufficient to result in efficacious treatment of biofilm-forming P. aeruginosa chronic infection. However, lung and ELF free interstitial concentrations might be insufficient to result in efficacious treatment of biofilm-forming P. aeruginosa chronic infection.

#3827 BICARBONATE PROFILING IN HEMODIALYSIS: MATHEMATICAL MODELING OF DATA FROM A CLINICAL TRIAL

Abstract Background and Aims During hemodialysis (HD), bicarbonate and other bases are added to the dialysis fluid to correct chronic acidosis that may develop in kidney-impaired patients, increasing plasma bicarbonate concentration to physiological levels. Previous studies have shown that both exceedingly high and low post-dialysis bicarbonate concentrations are associated with adverse clinical outcomes; however, no clear guidelines for the prescription of bicarbonate concentration in the dialysis fluid are available. Data from a recent controlled clinical trial, carried out at the Department of Nephrology, Dialysis and Internal Medicine, Medical University of Warsaw (Poland), were used to identify transport parameters of a mathematical model of acid-base homeostasis in HD patients, as the first step in the implementation of a tool to optimize bicarbonate profiling during HD. Method The study was carried out in 20 chronic patients, undergoing thrice weekly 4-hr HD. The patients underwent three treatment regimens: A) constant dialysis fluid bicarbonate concentration (HCO3,D) equal to 35 mEq/L; B) HCO3,D decreasing from 35 to 30 mEq/L after 2 hrs; C) HCO3,D increasing from 30 to 35 mEq/L after 2 hrs. The patients were divided in two cohorts: for the first (n = 11) treatment schedule was: treatment A during the first and third week, B during the second week and C during the fourth week. For the second cohort (n = 9) treatment C was carried out during the second week and treatment B during the fourth. Data were collected only during the second and third sessions of the week, and acid-base composition was determined using a blood gas analyzer. The mathematical model describes the transport of bicarbonate and dissolved CO2 from dialysis fluid to the patient and can predict changes in the acid-base equilibrium of arterial and venous plasma and erythrocytes, interstitial fluid, and tissue cells, using mass transport equations. The dialysances for bicarbonate (KHCO3) and dissolved CO2 (KCO2) were estimated via least square minimization. Results No significant difference was found comparing patients in the two cohorts, or comparing sessions of the same week. Pre-dialysis plasma bicarbonate, pH, or CO2 partial pressure (PCO2) were significantly lower for treatment B compared to treatment A. Average plasma bicarbonate concentrations for the different treatments are shown in Figure 1 for data and model simulations. Table 1 reports the average values of the estimated parameters for week. No statistically significant difference was found between sessions, cohorts, or treatments. The model fitted bicarbonate data with an average error of 2.5 ± 1.4%, and PCO2 with an error of 4.5 ± 3.6%. Simulations of mixed-venous blood, interstitial fluid, and tissue cells concentrations showed a similar response to different treatments as in arterial blood. Conclusion The model accurately describes plasma bicarbonate data with high accuracy regardless of the treatment conditions. The parameters estimated showed considerable variability between treatments (less so between the sessions of the same week), but without statistical significance. The model can be used to simulate the effect of dialysate bicarbonate profiling on acid-base equilibrium in HD patients.

Should Airway Interstitial Fluid Be Used to Evaluate the Pharmacokinetics of Macrolide Antibiotics for Dose Regimen Determination in Respiratory Infection?

Macrolide antibiotics are important drugs to combat infections. The pharmacokinetics (PK) of these drugs are essential for the determination of their optimal dose regimens, which affect antimicrobial pharmacodynamics and treatment success. For most drugs, the measurement of their concentrations in plasma/serum is the surrogate for drug concentrations in target tissues for therapy. However, for macrolides, simple reliance on total or free drug concentrations in serum/plasma might be misleading. The macrolide antibiotic concentrations of serum/plasma, interstitial fluid (ISF), and target tissue itself usually yield very different PK results. In fact, the PK of a macrolide antibiotic based on serum/plasma concentrations alone is not an ideal predictor for the in vivo efficacy against respiratory pathogens. Instead, the PK based on drug concentrations at the site of infection or ISF provide much more clinically relevant information than serum/plasma concentrations. This review aims to summarize and compare/discuss the use of drug concentrations of serum/plasma, airway ISF, and tissues for computing the PK of macrolides. A better understanding of the PK of macrolide antibiotics based on airway ISF concentrations will help optimize the antibacterial dose regimen as well as minimizing toxicity and the emergence of drug resistance in clinical practice.

Characterization of Viloxazine Effects on Cortical Serotonin Neurotransmission at Doses Relevant for ADHD Treatment

AbstractIntroductionMost ADHD treatments are thought to be effective due to augmentation of dopamine (DA) and norepinephrine (NE). Our prior preclinical studies found that the ADHD treatment, viloxazine, may augment serotonin (5-HT) in addition to NE and DA; however, it was unclear if these effects occurred at clinically relevant concentrations. To further understand these potential 5-HT effects, we conducted a series of experiments with two objectives: 1) Can we confirm and better elucidate the previously observed serotonergic effects of viloxazine and determine if they occur at clinically relevant concentrations? 2) Are these effects observed in species with close physiology to humans?MethodsObjective 1: The affinity of viloxazine for human isoforms of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors was assessed via cell-based binding assays. Viloxazine agonism of 5-HT2C and antagonism at 5-HT7 was elucidated with IP1, Ca2+, β-arrestin, internalization, and cAMP assays in cells expressing human receptor isoforms. A microdialysis study was conducted in rats to determine the relationship between viloxazine concentrations in the interstitial fluid (ISF) and changes in NE, DA, 5-HT, and their metabolite concentrations in the prefrontal cortex (PFC). Objective 2: A PET imaging study using a 5-HT2A/2C radioligand agonist, [11C]CIMBI-36, is being conducted in non-human primates (NHPs) to evaluate if viloxazine binds these receptors and/or increases 5-HT release.Animal research was approved by animal care and use committees. Animals were cared for according to international standards.ResultsObjective 1: Cell-based assays to measure viloxazine affinity for NET, 5-HT2B, 5-HT2C, and 5-HT7 found Ki values of 0.14, 0.65, 0.84, 1.90 μM respectively. These values were lower than therapeutically relevant rat ISF concentrations (3.5 ± 1.6 μM) approximating pediatric ADHD patients unbound plasma concentrations (2.1-3.3 μM), indicating receptor recruitment. Binding affinity and functional activity assays found viloxazine had negligible activity for 5-HT2A and SERT at therapeutic concentrations. Viloxazine 5-HT2C agonism activated Gq-protein signaling (EC50=1.6 μM, Ca2+ assay), but not β-arrestin or internalization pathways (EC50 values &gt;150 μM). Viloxazine 5-HT7 antagonism decreased Gs-protein signaling (IC50 =6.7 μM). The microdialysis study found that at therapeutically relevant ISF concentrations, 5-HT levels were significantly increased over baseline; no changes were seen in the 5-HIAA metabolite, indicating 5-HT increase is not due to 5-HT reuptake inhibition. Objective 2: PET imaging studies are ongoing.ConclusionsTo date, our experiments to further elucidate the potential 5-HT effects of viloxazine have shown that the previously observed effects of viloxazine on 5-HT receptors and its augmentation of 5-HT in rat PFC occur at clinically relevant concentrations. Further exploration is needed to ascertain if these effects occur in NHPs and are relevant to ADHD.FundingSupernus Pharmaceuticals, Inc.

Viloxazine Increases Cortical Serotonin Without Inhibiting Serotonin Reuptake at Doses Used to Treat ADHD

AbstractBackgroundMost FDA-approved ADHD treatments increase norepinephrine (NE) and dopamine (DA); however, our prior preclinical studies of the non-stimulant ADHD treatment viloxazine ER (Qelbree®) demonstrated that viloxazine also increases serotonin (5-HT). A prior microdialysis study showed increases in NE, DA, and 5-HT in the rat prefrontal cortex (PFC); however, the 50 mg/kg dose resulted in supratherapeutic plasma concentrations. Viloxazine is a moderate affinity selective NE reuptake inhibitor, structurally different than traditional SSRI antidepressants. Viloxazine has negligible activity at the serotonin reuptake transporter (SERT), suggesting viloxazine has a different mechanism of 5-HT PFC elevation than SSRIs. The current microdialysis study was undertaken to further characterize if viloxazine affects 5-HT and its 5-HIAA metabolite at therapeutically relevant plasma concentrations. Results are compared to similar microdialysis studies of SSRIs.MethodsRats were implanted with I-shaped microdialysis probes connected to a microperfusion pump, delivering artificial cerebrospinal fluid, in the PFC. After a 2-hour baseline period, viloxazine (1, 3, 10, or 30 mg/kg) was administered (ip). Dialysate samples were collected from the interstitial fluid (ISF) of the PFC before and after dosing. LC-MS/MS was used to determine the dialysate concentrations of viloxazine and viloxazine-induced changes in NE, 5-HT, and their respective metabolites, DHPG and 5-HIAA. Viloxazine plasma concentrations were also measured.Animal research was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.ResultsViloxazine administration resulted in significant dose-dependent increases in ISF NE levels and corresponding decreases in DHPG (NE metabolite) at all doses tested, reflecting viloxazine’s activity as a NET inhibitor. Viloxazine treatment also resulted in a dose-dependent elevation of ISF 5-HT levels in the PFC. Of the doses tested, 30 mg/kg was found to be clinically relevant as it induced ISF concentrations approximating unbound plasma concentrations in pediatric ADHD patients. At this dose, 5-HT levels were significantly increased over baseline and higher than vehicle levels. Coincident changes in 5-HIAA concentrations were not observed, reaffirming viloxazine’s lack of activity as a SERT inhibitor.ConclusionViloxazine induced dose-dependent increases in NE and 5-HT in the PFC, a critical target region for ADHD therapies. At clinically relevant viloxazine plasma concentrations, 5-HT was increased in the PFC. Unlike SSRIs, which correspondingly decrease the 5-HT metabolite in the PFC (indicating serotonin reuptake inhibition), viloxazine did not affect 5-HIAA levels. Thus, viloxazine increases cortical 5-HT levels by a different mechanism than SSRIs. Whether 5-HT effects aid in viloxazine therapeutic efficacy in ADHD is yet unknown.FundingSupernus Pharmaceuticals, Inc.

Dietary nitrate supplementation increases nitrate and nitrite concentrations in human skin interstitial fluid

Acute dietary nitrate (NO3−) supplementation can increase [NO3−], but not nitrite ([NO2−]), in human skeletal muscle, though its effect on [NO3−] and [NO2−] in skin remains unknown. In an independent group design, 11 young adults ingested 140 mL of NO3−-rich beetroot juice (BR; 9.6 mmol NO3−), and 6 young adults ingested 140 mL of a NO3−-depleted placebo (PL). Skin dialysate, acquired through intradermal microdialysis, and venous blood samples were collected at baseline and every hour post-ingestion up to 4 h to assess dialysate and plasma [NO3−] and [NO2−]. The relative recovery rate of NO3− and NO2− through the microdialysis probe (73.1% and 62.8%), determined in a separate experiment, was used to estimate skin interstitial [NO3−] and [NO2−]. Baseline [NO3−] was lower, whereas baseline [NO2−] was higher in the skin interstitial fluid relative to plasma (both P < 0.001). Acute BR ingestion increased [NO3−] and [NO2−] in the skin interstitial fluid and plasma (all P < 0.001), with the magnitude being smaller in the skin interstitial fluid (e.g., 183 ± 54 vs. 491 ± 62 μM for Δ[NO3−] from baseline and 155 ± 190 vs. 217 ± 204 nM for Δ[NO2−] from baseline at 3 h post BR ingestion, both P ≤ 0.037). However, due to the aforementioned baseline differences, skin interstitial fluid [NO2−] post BR ingestion was higher, whereas [NO3−] was lower relative to plasma (all P < 0.001). These findings extend our understanding of NO3− and NO2− distribution at rest and indicate that acute BR supplementation increases [NO3−] and [NO2−] in human skin interstitial fluid.

Adaptation to the intracellular environment of primary human macrophages influences drug susceptibility of Mycobacterium tuberculosis

As a facultative intracellular pathogen, M. tuberculosis (Mtb) is highly adapted to evading antibacterial mechanisms in phagocytic cells. Both the macrophage and pathogen experience transcriptional and metabolic changes from the onset of phagocytosis. To account for this interaction in the assessment of intracellular drug susceptibility, we allowed a 3-day preadaptation phase post-macrophage infection prior to drug treatment. We found that intracellular Mtb in human monocyte-derived macrophages (MDM) presents dramatic alterations in susceptibility to isoniazid, sutezolid, rifampicin and rifapentine when compared to axenic culture. Infected MDM gradually accumulate lipid bodies, adopting a characteristic appearance reminiscent of foamy macrophages in granulomas. Furthermore, TB granulomas in vivo develop hypoxic cores with decreasing oxygen tension gradients across their radii. Accordingly, we evaluated the effects of hypoxia on preadapted intracellular Mtb in our MDM model. We observed that hypoxia induced greater lipid body formation and no additional shifts in drug tolerance, suggesting that the adaptation of intracellular Mtb to baseline host cell conditions under normoxia dominates changes to intracellular drug susceptibility. Using unbound plasma concentrations in patients as surrogates for free drug concentrations in lung interstitial fluid, we estimate that intramacrophage Mtb in granulomas are exposed to bacteriostatic concentrations of most study drugs.

Predictive Performance of Physiologically Based Pharmacokinetic Modelling of Beta-Lactam Antibiotic Concentrations in Adipose, Bone, and Muscle Tissues.

Physiologically based pharmacokinetic (PBPK) models consist of compartments representing different tissues. As most models are only verified based on plasma concentrations, it is unclear how reliable associated tissue profiles are. This study aimed to assess the accuracy of PBPK-predicted beta-lactam antibiotic concentrations in different tissues and assess the impact of using effect site concentrations for evaluation of target attainment. Adipose, bone, and muscle concentrations of five beta-lactams (piperacillin, cefazolin, cefuroxime, ceftazidime, and meropenem) in healthy adults were collected from literature and compared with PBPK predictions. Model performance was evaluated with average fold errors (AFEs) and absolute AFEs (AAFEs) between predicted and observed concentrations. In total, 26 studies were included, 14 of which reported total tissue concentrations and 12 unbound interstitial fluid (uISF) concentrations. Concurrent plasma concentrations, used as baseline verification of the models, were fairly accurate (AFE: 1.14, AAFE: 1.50). Predicted total tissue concentrations were less accurate (AFE: 0.68, AAFE: 1.89). A slight trend for underprediction was observed but none of the studies had AFE or AAFE values outside threefold. Similarly, predictions of microdialysis-derived uISF concentrations were less accurate than plasma concentration predictions (AFE: 1.52, AAFE: 2.32). uISF concentrations tended to be overpredicted and two studies had AFEs and AAFEs outside threefold. Pharmacodynamic simulations in our case showed only a limited impact of using uISF concentrations instead of unbound plasma concentrations on target attainment rates. The results of this study illustrate the limitations of current PBPK models to predict tissue concentrations and the associated need for more accurate models. SIGNIFICANCE STATEMENT: Clinical inaccessibility of local effect site concentrations precipitates a need for predictive methods for the estimation of tissue concentrations. This is the first study in which the accuracy of PBPK-predicted tissue concentrations of beta-lactam antibiotics in humans were assessed. Predicted tissue concentrations were found to be less accurate than concurrent predicted plasma concentrations. When using PBPK models to predict tissue concentrations, this potential relative loss of accuracy should be acknowledged when clinical tissue concentrations are unavailable to verify predictions.

Brain pharmacokinetics of mono- and bispecific amyloid-β antibodies in wild-type and Alzheimer’s disease mice measured by high cut-off microdialysis

BackgroundTreatment with amyloid-β (Aβ) targeting antibodies is a promising approach to remove Aβ brain pathology in Alzheimer's disease (AD) and possibly even slow down or stop progression of the disease. One of the main challenges of brain immunotherapy is the restricted delivery of antibodies to the brain. However, bispecific antibodies that utilize the transferrin receptor (TfR) as a shuttle for transport across the blood–brain barrier (BBB) can access the brain better than traditional monospecific antibodies. Previous studies have shown that bispecific Aβ targeting antibodies have higher brain distribution, and can remove Aβ pathology more efficiently than monospecific antibodies. Yet, there is only limited information available on brain pharmacokinetics, especially regarding differences between mono- and bispecific antibodies.MethodsThe aim of the study was to compare brain pharmacokinetics of Aβ-targeting monospecific mAb3D6 and its bispecific version mAb3D6-scFv8D3 that also targets TfR. High cut-off microdialysis was used to measure intravenously injected radiolabelled mAb3D6 and mAb3D6-scFv8D3 antibodies in the interstitial fluid (ISF) of hippocampus in wild-type mice and the AppNL−G−F mouse model of AD. Distribution of the antibodies in the brain and the peripheral tissue was examined by ex vivo autoradiography and biodistribution studies.ResultsBrain concentrations of the bispecific antibody were elevated compared to the monospecific antibody in the hippocampal ISF measured by microdialysis and in the brain tissue at 4–6 h after an intravenous injection. The concentration of the bispecific antibody was approximately twofold higher in the ISF dialysate compared to the concentration of monospecific antibody and eightfold higher in brain tissue 6 h post-injection. The ISF dialysate concentrations for both antibodies were similar in both wild-type and AppNL−G−F mice 24 h post-injection, although the total brain tissue concentration of the bispecific antibody was higher than that of the monospecific antibody at this time point. Some accumulation of radioactivity around the probe area was observed especially for the monospecific antibody indicating that the probe compromised the BBB to some extent at the probe insertion site.ConclusionThe BBB-penetrating bispecific antibody displayed higher ISF concentrations than the monospecific antibody. The concentration difference between the two antibodies was even larger in the whole brain than in the ISF. Further, the bispecific antibody, but not the monospecific antibody, displayed higher total brain concentrations than ISF concentrations, indicating association to brain tissue.

Comparing the Rise in Glucose Concentration in Blood, Aqueous and Interstitial Fluid During a Glucose Tolerance Test.

The purpose of the study was to determine if aqueous glucose levels rise in a comparable time frame to interstitial fluid and could therefore be suitable for a continuous glucose monitoring (CGM) site. An intravenous glucose tolerance test was performed on five New Zealand white rabbits. Aqueous humor from the posterior and anterior chamber of the eye and venous blood were sampled for glucose concentration measurement. Glucose concentrations in the interstitial fluids were monitored using a CGM system. A compartment model was created to map the glucose response curves in each compartment. The delay in rising glucose concentrations between blood and interstitial fluid and aqueous humor in the posterior chamber and anterior chamber of the eye were analyzed. The results showed a statistically similar time lag and rate of change in glucose concentrations between blood and interstitial fluids or aqueous humor in either the posterior or anterior chamber. The results of this study add further support to the aqueous humor being used as an alternative CGM site. The study provides the basis for developing an intraocular continuous glucose sensor that can overcome limitations of current CGM systems.