Systemic Clearance Research Articles

Inhibition of proteasome, but not lysosome, upregulates organic anion transporter 3 in vitro and in vivo

Organic anion transporter 3 (OAT3), an indispensable basolateral membrane transporter predominantly distributed in the kidney proximal tubules, mediated the systemic clearance of substrates including clinical drugs, nutrients, endogenous and exogenous metabolites, toxins, and critically sustains body homeostasis. Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3. Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms. The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. The upregulated OAT3 function by proteasome inhibition was attributed to the augment in maximum transport velocity and stability of membrane OAT3. Lastly, in vivo study using Sprague Dawley rats validated that proteasome inhibition using bortezomib induced enhancement of OAT3 ubiquitination and membrane expression in kidney. These data suggest that activity of proteasome but not lysosome could have an impact on the physiological function of OAT3, and proteasome displayed a promising target for OAT3 regulation in vitro and in vivo, and could be used in restoring OAT3 impairment under pathological conditions, avoiding OAT3-associated toxicity and diseases, ensuring drug efficacy and safety.

593. Population Pharmacokinetic Model Development for Epetraborole and Mycobacterium avium Complex (MAC) Lung Disease Patients Using Data from Phase 1 and 2 Studies

593. Population Pharmacokinetic Model Development for Epetraborole and <i>Mycobacterium avium</i> Complex (MAC) Lung Disease Patients Using Data from Phase 1 and 2 Studies

Positively charged and neutral drug-loaded liposomes as the aerosolized formulations for the efficiency assessment of tumor metastases to lungs

Tumor metastasis is the major contributor to mortality in cancer patients, and the metastases to the lungs have been aggressively concerned because neoplasms are more likely to metastasize in the lung than other organs. The development of effective pulmonary drug delivery is urgently needed to deal with this metastasis. Nebulization of liposomes receives increasing attention because it is one of the most promising methods for pulmonary drug deliveries. However, the study on the influence of liposome charges in the aerosolized formulation remains insufficient. Hence, the positively charged (CAB-DOX) and neutral (ACB-DOX) liposomes consisting of l-α-phosphatidylcholine, cholesterol, doxorubicin (DOX), and two types of azobenzene derivatives were prepared . ACB-DOX is stable throughout the compression-type nebulizer. CAB-DOX showed a lower inflammatory response to the lung tissue of normal mice. The inhalation of these formulations into the lung of mice with metastasis was conducted. On one hand, CAB-DOX suppressed the viability of tumor cells more than free doxorubicin (free-DOX), but ACB-DOX presented similar results to the free-DOX. On the other hand, the effective therapies in mice using CAB-DOX were demonstrated from bioluminescent signals, hematoxylin and eosin (H&E) staining, survival, and immunohistochemical (IHC) staining, and the biodistribution of DOX shows that CAB-DOX has a slower systemic absorption and clearance than free-DOX and ACB-DOX. It is suggested that the longer retention time in the lungs of inhaled CAB-DOX promotes the DOX exposure of pulmonary tumor cells, resulting in improved therapeutic effects.

Ambulance Path Clearance System With Health Monitoring Using Internet of Things

Countless people’s lives are jeopardized in India’s crowded metropolitan metropolises due to ambulances stuck in traffic. Most of the time, commuters are unable to move in these traffic jams to make way for approaching ambulances which has become one of the most pressing issues in India. The main reason in the back of that is growing population which ends up in multiplied number of cars, due to which emergency provider like ambulance gets affected. This paper proposes a solution to make such offerings effortlessly available to those in need. It includes a software application referred to as Ambulance service through which the user can book the ambulance with one faucet and track the ambulance by using Google map on his device, we also use microcontroller-based hardware module to provide an easy flow for ambulance to attain the preferred destination This is achieved by using imposing RF technology which might automatically control the visitors alerts within the course of ambulance accordingly minimizing the time required to reach the destination. Besides this, a portable health monitoring device will be placed in the ambulance which will measure patient’s body temperature, blood pressure which will be displayed in the hospital’s webpage.

Exogenous laminin exhibits a unique vascular pattern in the brain via binding to dystroglycan and integrins

BackgroundUnlike other proteins that exhibit a diffusion pattern after intracerebral injection, laminin displays a vascular pattern. It remains unclear if this unique vascular pattern is caused by laminin-receptor interaction or laminin self-assembly.MethodsWe compared the distribution of various wild-type laminin isoforms in the brain after intracerebral injection. To determine what causes the unique vascular pattern of laminin in the brain, laminin mutants with impaired receptor-binding and/or self-assembly activities and function-blocking antibodies to laminin receptors were used. In addition, the dynamics of laminin distribution and elimination were examined at multiple time points after intracerebral injection.ResultsWe found that β2-containing laminins had higher affinity for the vessels compared to β1-containing laminins. In addition, laminin mutants lacking receptor-binding domains but not that lacking self-assembly capability showed substantially reduced vascular pattern. Consistent with this finding, dystroglycan (DAG1) function-blocking antibody significantly reduced the vascular pattern of wild-type laminin-111. Although failed to affect the vascular pattern when used alone, integrin-β1 function-blocking antibody further decreased the vascular pattern when combined with DAG1 antibody. EDTA, which impaired laminini-DAG1 interaction by chelating Ca2+, also attenuated the vascular pattern. Immunohistochemistry revealed that laminins were predominantly located in the perivascular space in capillaries and venules/veins but not arterioles/arteries. The time-course study showed that laminin mutants with impaired receptor-engaging activity were more efficiently eliminated from the brain compared to their wild-type counterparts. Concordantly, significantly higher levels of mutant laminins were detected in the cerebral-spinal fluid (CSF).ConclusionsThese findings suggest that intracerebrally injected laminins are enriched in the perivascular space in a receptor (DAG1/integrin)-dependent rather than self-assembly-dependent manner and eliminated from the brain mainly via the perivascular clearance system.

Human pharmacokinetics prediction with an in vitro–in vivo correction factor approach and in vitro drug-drug interaction profile of bictegravir, a potent integrase-strand transfer inhibitor component in approved biktarvy® for the treatment of HIV-1 infection

Bictegravir (BIC) is a potent small-molecule integrase strand-transfer inhibitor (INSTI) and a component of Biktarvy®, a single-tablet combination regimen that is currently approved for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. The in vitro properties, pharmacokinetics (PK), and drug-drug interaction (DDI) profile of BIC were characterised in vitro and in vivo. BIC is a weakly acidic, ionisable, lipophilic, highly plasma protein-bound BCS class 2 molecule, which makes it difficult to predict human PK using standard methods. Its systemic plasma clearance is low, and the volume of distribution is approximately the volume of extracellular water in nonclinical species. BIC metabolism is predominantly mediated by cytochrome P450 enzyme (CYP) 3A and UDP-glucuronosyltransferase 1A1. BIC shows a low potential to perpetrate clinically meaningful DDIs via known drug metabolising enzymes or transporters. The human PK of BIC was predicted using a combination of bioavailability and volume of distribution scaled from nonclinical species and a modified in vitro-in vivo correlation (IVIVC) correction for clearance. Phase 1 studies in healthy subjects largely bore out the prediction and supported the methods used. The approach presented herein could be useful for other drug molecules where standard projections are not sufficiently accurate.

Ex vivo quantitative amyloid and perivascular space measurements in white and gray matter on immunohistological microscopy in AD, MCI, and cognitively intact individuals

AbstractBackgroundAmyloid (Aβ) clearance is disturbed in late‐onset Alzheimer’s disease (AD), and a local relationship between Aβ clearance and gray (GM) and white matter (WM) perivascular spaces (PVS) is presumed. Most in vivo primate work has focused on MRI‐visible PVS in WM, described as pathological and enlarged, while nearly all in vivo mechanistic work in animals interrogates “normal” GM PVS. This disparity describes a critical knowledge gap in the morphology, location and relevance of PVS to Aβ clearance.Method: Ex vivo samples from four locations in 22 NIA‐Layton Oregon Alzheimer’s Disease Research Center participants (mean age at death=92y). Pre mortem diagnoses were cognitively intact (“C”, N=9), mild cognitive impairment (“MCI”, N=5), and AD (N=8, confirmed post mortem). Samples were sliced, stained and digitized (3.52µm/pixel). GM masks and morphometrics of vessels&gt;6200µm2 in WM (n=1403) and GM (n=237) were derived from trichrome stain (Fig. 2H,C, and D). Aβ coverage (% positive 4G8 pixels in GM pixels within 1.76mm of the nearest GM pixel to the vessel center‐of‐mass) and normalized PVS size were used as dependent variables (Fig. 2D&amp;F).ResultThere were significant main effects of group for Aβ burden in GM nearest to WM vessels (C&lt;MCI&lt;AD). Main group effects for Aβ around GM vessels were ordered differently (C&gt;AD&gt;MCI) (Fig. 3D). There were significant main effects of group for PVS size in GM (AD&gt;MCI and C) but not WM (Fig. 3C). There were no vessel‐by‐vessel associations between PVSnorm and nearby cortical amyloid within group. (Fig. 3E)ConclusionIn MCI, relatively smaller PVS and high cortical Aβ near GM vessels may reflect a functional waste clearance system in those for whom Aβ burden is not yet advanced. In AD, large GM PVS and an absence of a relationship with cortical Aβ may describe a clearance system overwhelmed by Aβ deposition. Absence of group‐wise differences of relative PVS size in WM vessels, along with no vessel‐by‐vessel association of PVS size with nearby GM Aβ suggests that WM PVS size may not be relevant to Aβ clearance at this scale in individuals of advanced age.

Parenchymal CSF Fraction and DTI‐ALPS in Brain Aging

AbstractBackgroundThe perivascular space (PVS) functions as part of a brain clearance system that drains interstitial fluid and solutes including Aβ and tau from brain parenchyma and is associated with the development of AD. Recent developed imaging tools of cerebral parenchymal CSF fraction (CSFF)1 and diffusion tensor imaging along perivascular spaces index (DTI‐ALPS)2 provide an insight of the perivascular CSF flow and early PVS pathology in the cerebral cortex. We applied CSFF and DTI‐ALPS in an elderly cognitive normal cohort to investigate its value in brain aging.MethodA total of 27 cognitive normal subjects in this study are within age 57 and 82 years (mean=67.7, std=6.4). We conducted MR FASTT2 scan for CSFF, T1W, T2W for PVS burden, and DTI for DTI_ALPS index. CSFF was fitted using three compartment model with 6 TEs = 0, 7.5, 17.5, 67.5, 147.5, 307.5 ms. PVS burden was calculated by using imaging processing based PVS segmentation on enhance PVS image (T2W/T1W). DTI_ALPS was calculated by mean(D𝑥_𝑝𝑟𝑜𝑗,D𝑥_𝑎𝑠𝑠𝑜𝑐)/mean (D𝑦_𝑝𝑟𝑜𝑗, D𝑧_𝑎𝑠𝑠𝑜𝑐), where Dx, Dy and Dz are the diffusivity along x‐, y‐ and x‐axis, and the projection, association regions. Multi‐variate linear model Age∼1+DTI_ALPS+CSFF was compared with Age∼1+DTI_ALPS+PVS_burden.ResultFigure 1 (a) and (b) show the PVS mask on T2W and CSFF map. Figure 2 shows the scattered plot of linear model between age and PVS burden+DTI_ALPS, which is not significant (p=0.06, R2=0.14). Figure 3 is the plot of the model between age and CSFF+DTI_ALPS, which is significant (p&lt;0.01, R2=0.34).ConclusionThe preliminary results showed CSFF and DTI‐ALPS can better predict brain aging. The conventional PVS burden only measures the MRI visible PVS in brain white matter while CSFF can assess micro‐scale PVS along small arteriole and capillary in the cerebral cortex, which is closely related to glymphatic system and may reflect the PVS function alert at early stage. CSFF and DTI‐ALPS could be a potential marker for detecting early stage of dysfunction of glymphatic clearance. Reference: 1. Liangdong Zhou, BioRvix:2021.02.01.429067, 2021. 2. Toshiaki Taoka, Jpn.J.Radiol. 2022;40:147–58.3.

Optoacoustic imaging of the glymphatic system

Background: The glymphatic system is a brain waste clearance system mediated via cerebrospinal fluid (CSF) flow,1 with implications for influence on neurodegenerative diseases.2 Most preclinical glymphatic studies employ fluorescence imaging, which provides higher specificity, but a smaller field-of-view (FOV), or magnetic resonance imaging (MRI), which provides brain-wide FOV, but lower specificity. Functional optoacoustic neuro-tomography3 (FONT) offers a larger FOV compared to classical optical methods, and higher specificity compared to MRI. However, FONT has not yet been applied to probe the glymphatic system. In this study, we used fluorescence and optoacoustic imaging of a near-infrared dye, Janelia Fluor 669 (JF669), to track CSF and multimodal CSF-hemodynamic flows in mice.&#x0D; Methods: After observing strong fluorescence and optoacoustic signatures of JF669 in phantom experiments, we performed a series of in vivo experiments in isoflurane-anesthetized C57BL/6 mice (n=3 fluorescence and n=4 FONT experiments, respectively) (Figure 1A). The lumbar injection was applied to deliver JF669 at a rate of 2 µL/min for 30 minutes. A polyethylene tube was placed intrathecally at the lumbar region (L4-L5). The scalp was removed. Fluorescence or FONT images were obtained every 5 minutes after injection.&#x0D; Results: Fluorescence imaging and FONT probe CSF flow Images (Figure 1B) and time traces (Figure 1C) revealed time-dependent anatomical routes of paravascular influx, including the transport along the olfactory artery (OFA), superior cerebellar artery (SCA), and bilateral middle cerebral artery (MCA). For FONT imaging, since the OFA showed strong fluorescence (Figure 1B), we positioned the ultrasound transducer array at the anterior of the mouse brain with a FOV of 5x5 mm2 (Figure 1A). Standard filtered back-projection reconstruction was applied. Besides the OFA route of the paravascular influx, the dynamic images (Figure 1D) and time-traces (Figure 1E) also revealed time-dependent anatomical routes of CSF-interstitial fluid (ISF) exchange in the olfactory bulb (OFB) and paravascular efflux in the superior sagittal sinus (SSS) and the bilateral inferior cerebral vein (ICV). Next, we studied the aquaporin-4 (AQP4) dependence of glymphatic flow by subcutaneously injecting AQP4 inhibitor TGN020, in addition to the prior procedures. Under fluorescence imaging and FONT, we observed that TGN020 significantly decreased and spatially restricted the spread of JF669 in the brain. FONT spectral unmixing separates CSF and blood We swept the OPO through 680 nm to 750 nm, with 10 nm steps at 10 Hz in the phantom and in each animal. The multispectral reconstructions were unmixed using the known absorption spectra of hemoglobin and the JF669 OA spectrum obtained from the phantom.4 This enabled the separation of blood and JF669 signal (Figure 1D).&#x0D; Conclusions: We characterized anatomical routes of paravascular influx (OFA), CSF-ISF exchange (OFB) and paravascular efflux using FONT, while fluorescence imaging only enabled visualization of the paravascular influx. We thus demonstrated the feasibility and advantages of FONT for probing the glymphatic system. These findings present great opportunities for monitoring and understanding the glymphatic system during development, aging, disease, pharmacological interventions and genetic modifications.

Probing cerebrospinal fluid mobility for human brain clearance imaging MRI: water transport across the blood-cerebrospinal fluid barrier and mobility of cerebrospinal fluid in perivascular spaces

Background. The growing interest in the brain clearance system in the last decade has led to great insights into how waste clearance via perivascular spaces acts like a lymphatic-like system. However, most of these observations have been done in rodent studies, often with invasive techniques. When aiming to understand the human brain clearance system, the main technology has so far relied on intrathecal injections1 and cerebrospinal fluid (CSF) flow in larger structures like the fourth ventricle2 or the aqueduct. The availability of non-invasive imaging technology would be an important driver to probe human brain clearance in health and disease.&#x0D; Methods. When looking at the current knowledge on brain clearance, it is clear that CSF and interstitial fluid (ISF) are the main solvents that propel waste out of the brain. The insight that CSF and ISF mainly consist of water makes magnetic resonance imaging (MRI) an attractive modality, since many possibilities exist to measure cerebral water dynamics, such as transitions between compartments, as well as water flow/diffusion in sub-compartments. MRI does provide excellent opportunities to image CSF/ISF, due to the long T2 of these compared to background tissue. By using long echo-time imaging, MRI sequences can be tuned towards CSF and ISF. This approach is applied both to arterial spin labeling (ASL) MRI to measure water transport across the blood-CSF barrier, as well as to high spatial resolution imaging at 7 tesla MRI to measure CSF mobility in perivascular spaces.&#x0D; Results. By using ASL that magnetically labels inflowing blood, we could prove that water exchange into CSF is not only taking place in the choroid plexus, but also in the subarachnoid space.3 We refer to the reference for a complete description of the method and results.3 The second technique also exploits long echo times to isolate CSF-signal, but combines this with high spatial resolution readouts and motion-sensitizing gradients to allow measurement of the CSF-mobility in the perivascular spaces of penetrating arteries (Figure 1) and e.g. the subarachnoid space around the MCA. Retrospective triggering allows studying how the cardiac and respiratory cycle influence the CSF-mobility, i.e. the driving forces of propulsion and mixing processes within the perivascular spaces (PVS). Preliminary results show approximately equal contributions from the cardiac and respiratory cycles in smaller PVS.4 Conclusions The exchange of water between the vascular and CSF compartments does not exclusively happen in the choroid plexus, but also in the subarachnoid arteries along the cortex. CSF mobility is influenced both by cardiac and respiratory cycles in approximately equal contributions in the PVS of penetrating arteries.

Factors determining the oral absorption and systemic disposition of zeaxanthin in rats: in vitro, in situ, and in vivo evaluations

Context Zeaxanthin is a yellow‑coloured dietary carotenoid widely recognized as an essential component of the macula. It exerts blue light filtering and antioxidant activities, offering eye health and vision benefits. Objective This study explores the oral absorption and systemic disposition of zeaxanthin from biopharmaceutical and pharmacokinetic perspectives. Materials and methods In vivo intravenous (5 and 10 mg/kg) and intraportal (5 mg/kg) pharmacokinetic studies were performed to determine intrinsic tissue‑blood partition coefficient, elimination pathway, and hepatic clearance, of zeaxanthin in rats. Moreover, in vitro physicochemical property test, in situ closed loop study, in vivo oral pharmacokinetic study (20 and 100 mg/kg), and in vivo lymphatic absorption study (100 mg/kg) were conducted to investigate the gut absorption properties of zeaxanthin and assess the effects of several lipids on the lymphatic absorption of zeaxanthin in rats. Results Zeaxanthin exhibited poor solubility (≤144 ng/mL) and stability (6.0–76.9% of the initial amount remained at 24 h) in simulated gut luminal fluids. Gut absorption of zeaxanthin occurred primarily in the duodenum, but the major fraction (≥84.7%) of the dose remained unabsorbed across the entire gut tract. Considerable fractions of intravenous zeaxanthin accumulated in the liver, lung, and spleen (21.3, 11.7, and 2.0%, respectively). It was found that the liver is the major eliminating organ of zeaxanthin, accounting for 53.5–90.1% of the total clearance process (hepatic extraction ratio of 0.623). Discussion and conclusions To our knowledge, this is the first systematic study to report factors that determine the oral bioavailability and systemic clearance of zeaxanthin.

Blinatumomab Alternating With Low-Intensity Chemotherapy (CT) Treatment for Older Adults With Newly Diagnosed Philadelphia (Ph)-Negative B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is Well Tolerated and Efficacious: Safety Run-In Results for the Phase 3 Randomized Controlled Golden Gate Study

Introduction: Outcomes have improved for younger adults with BCP-ALL through better understanding of the biological mechanisms of BCP-ALL and development of intensive CT regimens (overall survival [OS], ≥60%). However, outcomes in patients (pts) >55 y remain poor (5 y OS, <10-40%) as toxicities limit use of intensive CT regimens. Novel treatment strategies incorporating immunotherapy to reduce CT-related morbidity and mortality may improve outcomes in this older pt population. Blinatumomab, a CD3/CD19-directed bispecific T-cell engager (BiTE®) molecule, directs T cells to lyse CD19+ B-lineage cells and has demonstrated efficacy in pediatric and adult pts with relapsed or refractory CD19+ BCP-ALL or minimal residual disease (MRD) ≥0.1%. We report results from the single-arm safety run-in for the randomized controlled phase 3 Golden Gate Study in older adults with newly diagnosed Ph-negative BCP-ALL (NCT04994717). The safety run-in evaluated blinatumomab alternating with low-intensity CT and a shorter blinatumomab dose step (4 days) than presently included in the blinatumomab label. Methods: Pts aged ≥55 y (or 40 to <55 y with severe, pre-defined comorbidities) with newly diagnosed Ph-negative BCP-ALL, Eastern Cooperative Oncology Group performance status ≤2 and adequate organ function were included. Clearance of central nervous system (CNS) leukemia with intrathecal (IT) CT was required before enrollment. Pts in the safety run-in (Figure) were to receive 7 cycles (C) of blinatumomab (2 in induction, 2 in consolidation and 3 in maintenance) alternating with CT, including IT CT. Rituximab (8 doses) was given if CD20 expression was ≥20%. Permitted pre-phase CT consisted of cyclophosphamide, vincristine, dexamethasone and/or hydroxyurea; IT CT was requested per local standard of care. Primary objective of the safety run-in was to evaluate the safety and tolerability of blinatumomab alternating with low-intensity CT. Secondary objectives were to evaluate efficacy endpoints (complete remission [CR] within 14 weeks of starting induction C1; MRD response, defined as <10-4 by quantitative PCR [central lab] within 14 weeks of starting induction C1; relapse-free survival) and pharmacokinetics (PK) of blinatumomab. Results: Ten pts enrolled in the safety run-in between November 2021 and 28 June 2022. Median age was 69 y (range, 57-77); 4 (40%) pts were ≥70 y (Table). Nine (90%) pts had bone marrow blasts ≥50% at diagnosis; 4 (40%) pts were CD20+ and received rituximab. All 10 pts completed induction C1. Seven pts remain on treatment (induction C2, n=5 [ongoing, n=2; completed, n=3]; consolidation C1 completed, n=1; consolidation C2 ongoing, n=1) and 3 pts discontinued treatment (2 for planned allogeneic stem cell transplant in post C2 induction CR and 1 due to isolated extramedullary relapse during induction C2). From the data cut off (28 June 2022), the most common all-grade treatment emergent adverse events (TEAEs) were cytokine release syndrome (CRS; n=5), decreased neutrophils (n=5) and decreased white blood cell count (WBC; n=5). All pts had grade ≥3 TEAEs; grade ≥3 TEAEs in ≥20% of pts were neutropenia (n=6), decreased WBC (n=5), anemia (n=3), thrombocytopenia (n=3) and bacteremia (n=2). All grade 4 TEAEs were hematologic. No deaths occurred. One pt had a serious adverse event (AE; grade ≥3, bilirubin increased). Grade ≥3 CNS TEAEs occurred in 1 pt (vasovagal and peripheral neuropathy unrelated to blinatumomab); no grade ≥3 CNS or CRS AEs related to blinatumomab occurred. Four pts had COVID-19 (grade 2, n=3; grade 3, n=1); blinatumomab was interrupted in 4/4 pts until COVID-19 negative and treatment then resumed without unusual AEs. After induction C1, all 10 pts achieved CR and 9/10 pts achieved a MRD response <10-4 per central lab. After induction C2, 5 pts had MRD results available; all 5 pts had a MRD response <10-4. Blinatumomab PK results will be presented. Conclusions: Blinatumomab alternating with low-intensity CT had an acceptable safety profile and was well tolerated. No deaths occurred. The regimen was efficacious in this older adult population as all 10 pts achieved CR and 90% had a MRD response <10-4 after induction C1. These data support the planned regimen for the randomized controlled phase 3 study of blinatumomab alternating with low-intensity CT versus standard of care CT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intratumoral nanobody-IL-2 fusions that bind the tumor extracellular matrix suppress solid tumor growth in mice.

Confining cytokine exposure to the tumors would greatly enhance cancer immunotherapy safety and efficacy. Immunocytokines, cytokines fused to tumor-targeting antibodies, have been developed with this intention, but without significant clinical success to date. A critical limitation is uptake by receptor-expressing cells in the blood, that decreases the dose at the tumor and engenders toxicity. Small-format immunocytokines, constructed with antibody fragments, are hypothesized to improve tumor specificity due to rapid systemic clearance. However, effective design criteria for small-format immunocytokines need further examination. Here, we engineer small interleukin-2 (IL-2) immunocytokines fused to nanobodies with nanomolar to picomolar affinities for the tumor-specific EIIIB domain of fibronectin (also known as EDB). Upon intravenous delivery into immunocompetent mice, such immunocytokines led to similar tumor growth delay as size-matched untargeted IL-2. Intratumoral (i.t.) delivery imparted improved survival dependent on affinity to EIIIB. I.t. administration offers a promising avenue to deliver small-format immunocytokines, given effective affinity for the tumor microenvironment.

Diffusion Tensor Imaging Analysis Along the Perivascular Space Index in Primary Parkinson’s Disease Patients With and Without Freezing of Gait

Parkinson’s disease (PD) is a common neurodegeneration disease associated with the abnormal deposition and spread of misfolded proteins (α-synuclein and Tau protein), which progressively damages the glymphatic system. This research intended to investigate the activity of the glymphatic system in PD individuals with freezing of gait (PD-FOG) and PD patients without it (PD-nFOG), as well as their relationship to the clinical neural scale. Diffusion tensor imaging (DTI) was performed in 28 PD-FOG individuals, 31 PD-nFOG individuals, and 34 healthy controls (HC). The DTI analysis along the perivascular space (DTI-ALPS) index was computed after post-processing of DTI images, representing brain glymphatic functions. The DTI-ALPS index was assessed for the association with the clinical variables. Compared to the HC group, the DTI-ALPS index of both PD-FOG and PD-nFOG patients was significantly decreased; however, no notable difference was found between the PD-FOG and PD-nFOG group. In addition, the DTI-ALPS index of PD-nFOG patients were positively correlated with disease duration, Unified Parkinson’s Disease Rating-III Right (UPDRS-III R), UPDRS-III TOTAL, UPDRS-IV. Taken together, these findings highlighted the weakening of function of the glymphatic system in PD individuals, which is associated with motor symptoms and treatment complications. We speculate that treatment aimed at enhancing the flow and clearance of the glymphatic system may alleviate clinical symptoms of PD.

Optimization and Validation of Glycerol-induced Acute Renal Failure Model in Rats in Predicting the Pharmacokinetics in Patients with Acute Renal Failure

The study standardized the glycerol induced (intra-peritoneal and intra-muscular) acute renal failure (ARF) models in Sprague-Dawley rats and validated its potential to predict the change in pharmacokinetics of the selected probe drugs in renal impairment. We assessed the induction of ARF by measuring the serum creatinine and blood-urea-nitrogen (BUN) levels at different time points (baseline, 6 h, 12 h, 24 h, 48 h and 72 h). Glycerol administrations via both intra-peritoneal and intra-muscular routes caused a significant increase in serum creatinine and BUN levels in rats at 6 h; however, the levels decreased within 24 h with intra-peritoneal administration. In contrast, with intra-muscular administration, a sustained increase in serum creatinine and BUN levels was observed up to 72 h post glycerol treatment demonstrating a sustained ARF like condition in rats that was suitable to evaluate the pharmacokinetics of drugs. Therefore, intra-muscular glycerol administration model was used to evaluate the pharmacokinetics of selected probe drugs including metformin, digoxin, atorvastatin and linezolid. The pharmacokinetics of metformin and digoxin were significantly altered in ARF versus control rats with increase in plasma exposure, prolonged elimination half-life and decreased systemic clearance. However, the pharmacokinetics of atorvastatin and linezolid did not show significant differences between ARF and control rats. These results demonstrated the utility of the ARF model in rats in predicting pharmacokinetic changes in patients with renal impairment. Such models may prove useful in early discovery work to screen and prioritize compounds requiring dose adjustment in patients with renal impairment.

Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte’s ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes.In this work we aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm.These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.

Mouse Pharmacokinetics and In Vitro Metabolism of SH-11037 and SH-11008, Synthetic Homoisoflavonoids for Retinal Neovascularization.

Cremastranone is a member of the homoisoflavanone family with anti-angiogenic activity in the eyes. SH-11037, a potent and selective synthetic homoisoflavonoid derived from cremastranone, was studied here for pharmacokinetics and metabolism characterization with a special focus on esterase-mediated hydrolysis. SH-11037 was shown to be converted rapidly and nearly completely to SH-11008 following an intravenous dose in mice. SH-11008 showed a high systemic clearance well exceeding the hepatic blood flow in mice. Neither SH-11037 nor SH-11008 were detected in plasma following oral administration of SH-11037 and SH-11008 in mice. Carboxylesterase was shown to be responsible for the rapid and quantitative hydrolysis of SH-11037 to SH-11008 in mouse plasma; the hydrolytic bioconversion was much slower in dog and human plasma, with butyrylcholinesterase and paraoxonase 1 likely being responsible. In vitro metabolism studies with liver S9 fractions suggested that SH-11008 was likely to have a high hepatic metabolic clearance with a predicted hepatic extraction ratio close to 1 in both mouse and human. In conclusion, SH-11037 and SH-11008 both appear to possess pharmacokinetic profiles suboptimal as a systemic agent. SH-11008 is suggested to possess a low potential for systemic toxicity suitable as a topical ocular therapeutic agent.

Perivascular spaces as a marker of disease severity and neurodegeneration in patients with behavioral variant frontotemporal dementia.

Behavioural Variant Frontotemporal Dementia (bvFTD) is a rapidly progressing neurodegenerative proteinopathy. Perivascular spaces (PVS) form a part of the brain's glymphatic clearance system. When enlarged due to poor glymphatic clearance of toxic proteins, PVS become larger and more conspicuous on MRI. Therefore, enlarged PVS may be a useful biomarker of disease severity and progression in neurodegenerative proteinopathies such as bvFTD. This study aimed to determine the utility of PVS as a biomarker of disease progression in patients with bvFTD. Serial baseline and week 52 MRIs acquired from ten patients with bvFTD prospectively recruited and followed in a Phase 1b open label trial of sodium selenate for bvFTD were used in this study. An automated algorithm quantified PVS on MRI, which was visually inspected and validated by a member of the study team. The number and volume of PVS were extracted and mixed models used to assess the relationship between PVS burden and other measures of disease (cognition, carer burden scale, protein biomarkers). Additional exploratory analysis investigated PVS burden in patients who appeared to not progress over the 12 months of selenate treatment (i.e., "non-progressors"). Overall, PVS cluster number (ß = -3.27, CI [-7.80 - 1.27], p = 0.267) and PVS volume (ß = -36.8, CI [-84.9 - 11.3], p = 0.171) did not change over the paired MRI scans 12 months apart. There was association between cognition total composite scores and the PVS burden (PVS cluster ß = -0.802e-3, CI [9.45e - 3 - -6.60e - 3, p ≤ 0.001; PVS volume ß = -1.30e - 3, CI [-1.55e - 3 - -1.05e - 3], p ≤ 0.001), as well as between the change in the cognition total composite score and the change in PVS volume (ß = 4.36e - 3, CI [1.33e - 3 - 7.40e - 3], p = 0.046) over the trial period. There was a significant association between CSF t-tau and the number of PVS clusters (ß = 2.845, CI [0.630 - 5.06], p = 0.036). Additionally, there was a significant relationship between the change in CSF t-tau and the change in the number of PVS (ß = 1.54, CI [0.918 - 2.16], p < 0.001) and PVS volume (ß = 13.8, CI [6.37 - 21.1], p = 0.003) over the trial period. An association was found between the change in NfL and the change in PVS volume (ß = 1.40, CI [0.272 - 2.52], p = 0.045) over time. Within the "non-progressor" group (n = 7), there was a significant relationship between the change in the CSF total-tau (t-tau) levels and the change in the PVS burden (PVS cluster (ß = 1.46, CI [0.577 - 2.34], p = 0.014; PVS volume ß = 14.6, CI [3.86 - 25.4], p = 0.032) over the trial period. Additionally, there was evidence of a significant relationship between the change in NfL levels and the change in the PVS burden over time (PVS cluster ß = 0.296, CI [0.229 - 0.361], p ≤ 0.001; PVS volume ß = 3.67, CI [2.42 - 4.92], p = 0.002). Analysis of serial MRI scans 12 months apart in patients with bvFTD demonstrated a relationship between PVS burden and disease severity as measured by the total cognitive composite score and CSF t-tau. Further studies are needed to confirm PVS as a robust marker of neurodegeneration in proteinopathies.

Chitosan/xanthan gum-based (Hydroxypropyl methylcellulose-co-2-Acrylamido-2-methylpropane sulfonic acid) interpenetrating hydrogels for controlled release of amorphous solid dispersion of bioactive constituents of Pueraria lobatae

Pueraria lobatae (Willd) Ohwi is a traditional Chinese medicine used to treat alcohol intoxication, diabetes, cerebrovascular and cardiovascular diseases. Some of its active components include the flavonoids puerarin, daidzin, daidzein, and genistin. The therapeutic efficacy of these agents is hampered by their poor pharmacokinetic profiles (rapid systemic clearance, low oral bioavailability, short half-life) and physicochemical properties (such as poor aqueous solubility and stability). In the current study, chitosan/xanthan gum-based (hydroxypropyl methylcellulose-co-2-acrylamido-2-methylpropane sulfonic acid) hydrogels for the controlled release of Pueraria lobata-solid dispersion (SD) were successfully prepared and characterized. A total of 61 compounds were identified in the Pueraria lobatae-SD using UHPLC-Q-TOF-MS analysis. Hydrogel structure was confirmed by FTIR, XRD, TGA, DSC, and SEM showed a porous structure. Correlations between hydrogels structural properties was also investigated. The hydrogels showed higher swelling after 48 h at pH 1.2 (21.15 %) than pH 7.4 (15.91 %). In vitro drug release study demonstrated that drug release was maximum at pH 1.2 (63 %) compared to pH 7.4 (49 %) after 48 h. The gel fraction of the synthesized hydrogel was increased with the increase in the polymer and crosslinker concentrations. Furthermore, in vitro studies demonstrated that the developed hydrogels possess good antioxidant and antimicrobial properties.

Use of gold nanoparticle-silibinin conjugates: A novel approach against lung cancer cells.

Lung cancer presents one of the most challenging carcinomas with meager 5-year survival rates (less than 20%), high metastasis and high recurrence due to chemo- and radio- resistance. An alternative or complementation to existing prognosis modalities is the use of phytochemicals such as silibinin, which targets essential cytokines, angiogenic factors and transcription factors for a profound anti-tumor effect. However, the problems of low solubility in an aqueous physiological environment, poor penetration, high metabolism and rapid systemic clearance limit the therapeutic use of silibinin. Conjugation of gold nanoparticles (GNPs) with silibinin may overcome the above challenges along with distinct advantages of biocompatibility, optical properties for monitoring and causation of cytotoxicity in cancer cells. The current study thus aims to develop silibinin conjugated gold nanoparticles (Sb-GNPs) with pH responsive release in the cancer microenvironment, optimizing several parameters for its higher activity and further evaluate the nanoplatform for their efficacy in inducing cell death in vitro against A549 lung cancer cells. GNPs was synthesized using trisodium citrate dihydrate as the reducing agent and further used for the conjugation of silibinin. The synthesized GNPs were found to be monodispersed and spherical in shape. The silibinin was successfully conjugated with gold nanoparticles and long-term stability of GNPs and Sb-GNPs nanoconjugates in suspension phase was confirmed by FTIR and DLS. Anticancer properties of Sb-GNPs were confirmed by different assay using MTT, Trypan blue dye exclusion assay and cell cycle analysis assay. After conjugation of silibinin with GNPs, the efficacy of silibinin increased 4–5 times in killing the cancer cells. This is the first report on using silibinin gold nanoconjugate system for lung cancer therapy with promising future applications.