High Drug Concentrations Research Articles

Endovascular Infusion of Amphotericin B for the Treatment of Rhino-Orbito-Cerebral Mucormycosis: A Pilot Study Assessing Technical Feasibility and Safety

Abstract Objectives The aim of this study was to assess the technical feasibility and safety of intra-arterial infusion of amphotericin B for the management of rhino-orbito-cerebral mucormycosis in patients unable to receive full dose and schedule of intravenous amphotericin B and/or unsuitable for surgery. Materials and Methods Five consecutive patients underwent five sessions of intra-arterial infusion each via both external carotid arteries on alternate days. Liposomal amphotericin B (50 mg) was infused at each session. The baseline and follow-up investigations as well as local and systemic complications were charted. Results Procedure could be completed for all participants without any local complications. One patient had transient and another had progressive deterioration in renal parameters during the follow-up period of 30 days. Conclusions Authors conducted successfully a pilot study of multisession intra-arterial infusion of amphotericin B, with the premise that it can provide high concentration of drug at the desired site with reduced systemic complications. They recommend further larger randomized studies to evaluate its efficacy for the management of advanced rhino-oculo-cerebral mucormycosis.

Activity of N-Acetylcysteine Alone and in Combination with Colistin against Pseudomonas aeruginosa Biofilms and Transcriptomic Response to N-Acetylcysteine Exposure.

ABSTRACTChronic colonization by Pseudomonas aeruginosa is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating combination therapies, especially for drugs that can be administered by nebulization, which allows high drug concentrations to be reached at the site of infections while limiting systemic toxicity. Here, we investigated the potential antibiofilm activity of N-acetylcysteine (NAC) alone and in combination with colistin against a panel of P. aeruginosa strains (most of which are from CF patients) and the transcriptomic response of a P. aeruginosa CF strain to NAC exposure. NAC alone (8,000 mg/L) showed a limited and strain-dependent antibiofilm activity. Nonetheless, a relevant antibiofilm synergism of NAC-colistin combinations (NAC at 8,000 mg/L plus colistin at 2 to 32 mg/L) was observed with all strains. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. These findings suggested that NAC-colistin combinations might contribute to the management of biofilm-associated P. aeruginosa lung infections. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization.IMPORTANCE Pseudomonas aeruginosa biofilm-related chronic lung colonization contributes to cystic fibrosis (CF) disease progression. Colistin is often a last-resort antibiotic for the treatment of such P. aeruginosa infections, and it has been increasingly used in CF, especially by nebulization. N-acetylcysteine (NAC) is a mucolytic agent with antioxidant activity, commonly administered with antibiotics for the treatment of lower respiratory tract infections. Here, we show that NAC potentiated colistin activity against in vitro biofilms models of P. aeruginosa strains, with both drugs tested at the high concentrations achievable after nebulization. In addition, we report the first transcriptomic data on the P. aeruginosa response to NAC exposure.

Contact Lenses Loaded with Melatonin Analogs: A Promising Therapeutic Tool against Dry Eye Disease.

Melatonin analogs topically administered evoke a potent tear secretagogue effect in rabbits. This route of drug administration requires high drug concentration and frequent dosing due to its reduced ocular surface retention. Therefore, contact lenses (CLs) have emerged as an alternative drug-delivery system that prolongs drug retention in the cornea, improving its therapeutic performance. This study explores the in vitro ability of five commercially available hydrogel CLs to act as a delivery system for melatonin analogs and the in vivo secretagogue effect of melatonin analog-loaded CLs. We soaked CLs with melatonin or melatonin analog solutions (1 mM) for 12 h. Spectroscopic assays showed that IIK7-loaded CLs led to the inadequate delivery of this compound. Conventional hydrogel lenses loaded with agomelatine released more agomelatine than silicone ones (16–33% more). In contrast, the CLs of silicone materials are more effective as a delivery system of 5-MCA-NAT than CLs of conventional materials (24–29%). The adaptation of CLs loaded with agomelatine or 5-MCA-NAT in rabbits triggered a higher tear secretion than the corresponding eye drops (78% and 59% more, respectively). These data suggest that CLs preloaded with melatonin analogs could be an adequate strategy to combat aqueous tear deficient dry eye disease.

Abstract 2080: LAG3-targeted IL15/IL15Rα-Fc (LAG3 x IL15) fusion proteins for preferential TIL expansion

Abstract The IL2Rβ/γc binding human cytokines IL2 and IL15 aid in the activation, proliferation, and survival of T and NK cells, and their therapeutic potential has been well established in animal models and human trials. However, therapeutic approaches utilizing these cytokines have suffered from low tolerability, fast clearance, and limited therapeutic window due to extensive activity on peripheral lymphocytes. Conversely, higher drug concentration and prolonged exposure are desirable to allow lymphocyte activation and proliferation at the tumor site, but this can be difficult to achieve due to dose-limiting toxicities associated with this axis. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, which is presented in trans to lymphocytes expressing IL2Rβ/γc. We hypothesized that we could selectively target tumor-reactive TILs by combining a reduced potency IL15/IL15Rα heterocomplex with an immune checkpoint(CP)-targeting arm to bias binding and activation to CP-positive TILs, potentially improving therapeutic index. Lymphocyte activation gene 3 (LAG3) was chosen as the CP targeting-arm due to its frequent co-expression with PD1, bias to CD8+ T cells, ability to easily combine with anti-PD1 agents, and recent promising results with anti-LAG3 agents in the clinic. First, potency-reduced IL15/IL15Rα were created by engineering amino acid substitutions in IL15 - at the IL2Rβ/γc interface - that reduced in vitro potency by at least 1000-fold. We then designed LAG3 x IL15 fusion proteins containing single-chain IL15/IL15Rα and LAG3-targeting arms attached to a heterodimeric-Fc region, relying on targeting avidity to recover potency on LAG3+ cells. In vitro proliferation of lymphocytes in human PBMCs, stimulated with sub-optimal concentrations of anti-CD3 to induce LAG3 expression, was monitored by CFSE dilution or by counting Ki67+ cells after incubation with LAG3 x IL15 for 4 days. In vivo activity was evaluated using humanized mouse models by measuring the extent of human leukocyte expansion. Lead LAG3 x IL15 were evaluated for pharmacodynamic activity, pharmacokinetics, and tolerability in non-human primates. LAG3 x IL15 showed >500-fold selectivity compared to a non-targeted IL15 in an in vitro proliferation assay of lymphocytes stimulated for induced LAG3 expression. In vitro potency was greatest on effector memory CD8 T cells. LAG3 x IL15 were 3-fold more potent on CD8 T cells compared to CD4 T cells and had very weak activity on NK cells, consistent with minimal LAG3 expression on this population. In mouse models, treatment with LAG3 x IL15 promoted significantly increased numbers of T cells. Moreover, LAG3 x IL15 combined productively with anti-PD1 to promote additional T cell expansion. These results demonstrate that LAG3 x IL15 show a promising profile of selective IL15 delivery to TIL with minimal peripheral activity. Citation Format: Matthew J. Bernett, Suzanne Schubbert, Michael Hackett, Lukasz J. Ochyl, Lizett E. Scott, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Connie Ardila, Umesh S. Muchhal, Norman J. Barlow, Rena Bahjat, John R. Desjarlais. LAG3-targeted IL15/IL15Rα-Fc (LAG3 x IL15) fusion proteins for preferential TIL expansion [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 2080.

Abstract 3346: BBT-207, a novel, 4th generation, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with broad-spectrum activity to both treatment-emergent and drug-naïve mutants for the treatment of NSCLC

Abstract Background: Activating mutations in EGFR, e.g., L858R (L) and Del19 (D), are observed in 14.1-32.3% of NSCLC worldwide and defines a druggable entity. Single agent EGFR TKI treatment was established in clinical trials. However, a treatment-emergent mutation of T790M (T) was identified as a major source of drug resistance. Irreversible 2nd and 3rd generation EGFR TKIs were shown effective. Mutant-specific inhibition by 3rd generation EGFR TKIs reduced adverse events such as skin toxicities. The emergence of a missense mutation at C797 (C), to which irreversible TKIs covalently bind, limited their efficacy. Patients harboring treatment-emergent C mutations are left with few therapeutic options. Despite anecdotal reports of efficacy from 1st generation TKIs, these complex molecular variants involving C797 are an increasing clinical concern. To prevent the emergence of drug-resistant subclones, a broad-spectrum EGFR TKI against both treatment-emergent and drug-naïve mutants is required. Such a 4th generation EGFR TKI must also enable monotherapy, allowing precise dose adjustment in case of toxicity. Method: Derived from BBT-176 which is under clinical evaluation (NCT04820023), we synthesized BBT-207 and other related molecules. Using Promega ADP-Glo™ kinase assay, we tested compounds against wild-type (WT) and mutated EGFR proteins. Ba/F3 cells harboring WT and mutated EGFR genes were created and cell viability was measured by CellTiter-Glo® and SpectraMax iD3®. In vivo anti-tumor activity was evaluated in Ba/F3 cell-derived xenograft models and an Osimertinib-resistant patient-derived xenograft model. Single dose pharmacokinetic (PK) study was performed in animals. Results: BBT-207 showed IC50 values < 10 nM against EGFR triple mutants (DTC and LTC) and comparable IC50 values against double mutants (DC and LC), while Osimertinib was ineffective in both groups. BBT-207 was also as active as Gefitinib to single mutants (D and L). Double mutants containing T790M (DT and LT) were inhibited effectively by BBT-207 and Osimertinib at similar concentrations, while not by Gefitinib. In in vivo models to which Osimertinib was inactive (LC and DTC), significant tumor growth inhibition was observed from BBT-207 alone, which was enhanced by the addition of Osimertinib. PK studies in mouse, rat, dog and monkey were consistent with high exposure and sustained drug concentrations above the IC50 values, with evidence of penetration into the central nervous system. Conclusion: BBT-207 is a reversible, mutant-specific, broad-spectrum TKI, active to clinically observed mutations of EGFR and is expected to be compatible with monotherapy of QD schedule in humans. BBT-207 is well-positioned to augment the treatment of EGFR mutated NSCLC, either for acquired drug resistance or in earlier line. Citation Format: Taiguang Jimmy Jin, Sang-Uk Kang, Chulwon Kim, Jehrod Brenneman, Mihee Song, Pavel Printsev, Bo-Bae Seo, Yong-Hee Lee, Sang-Yoon Lee. BBT-207, a novel, 4th generation, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with broad-spectrum activity to both treatment-emergent and drug-naïve mutants for the treatment of NSCLC [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 3346.

Statistical optimization of process conditions for pyrvinium pamoate elimination using UV/zeolite‐based nanostructures/H 2 O 2

Statistical optimization of process conditions for pyrvinium pamoate elimination using UV/zeolite‐based nanostructures/H ₂ O ₂

WPI Hydrogels with a Prolonged Drug-Release Profile for Antimicrobial Therapy.

Infectious sequelae caused by surgery are a significant problem in modern medicine due to their reduction of therapeutic effectiveness and the patients’ quality of life.Recently, new methods of local antimicrobial prophylaxis of postoperative sequelae have been actively developed. They allow high local concentrations of drugs to be achieved, increasing the antibiotic therapy’s effectiveness while reducing its side effects. We have developed and characterized antimicrobial hydrogels based on an inexpensive and biocompatible natural substance from the dairy industry—whey protein isolate—as matrices for drug delivery. The release of cefazolin from the pores of hydrogel structures directly depends on the amount of the loaded drug and occurs in a prolonged manner for three days. Simultaneously with the antibiotic release, hydrogel swelling and partial degradation occurs. The WPI hydrogels absorb solvent, doubling in size in three days and retaining cefazolin throughout the duration of the experiment. The antimicrobial activity of cefazolin-loaded WPI hydrogels against Staphylococcus aureus growth is prolonged in comparison to that of the free cefazolin. The overall cytotoxic effect of cefazolin-containing WPI hydrogels is lower than that of free antibiotics. Thus, our work shows that antimicrobial WPI hydrogels are suitable candidates for local antibiotic therapy of infectious surgical sequelae.

INSP-17. Augmented Drug Delivery for Pediatric Diffuse Midline Glioma using Convection Enhanced Delivery

Convection enhanced delivery (CED), an alternative to systemic chemotherapy, spans nearly two decades of clinical experience. The application of CED in pediatric neuro-oncology has been mainly used in children with DMG. This exploratory period is highlighted by widely variable features including procedural technique, disease extent, device interface, infusion parameters, and therapeutic agent. This presentation is meant to critically assess if CED for DMG is a logical therapeutic strategy. Most convincing to the perpetuation of this platform is the reproducible demonstration of augmented drug delivery. Post-treatment ratios of intralesional to systemic drug concentrations consistently exceed 1000. CED in the brain stem satisfies a desirable goal of CNS drug delivery: achieving high target tissue drug concentrations while avoiding systemic exposure. Surgical targeting of the brain stem and cannula deployment are solidly established as safe and accurate. Catheter insertion complications such as clinically relevant hemorrhage, CNS infection, and neurological injury are exceptional. Likewise, targeting error is on a millimeter scale with infrequent need for catheter repositioning. The use of intrinsic MRI signals, direct drug labeling, and surrogate tracers have all been used for measuring distribution of the infusion. The experience using a radio-labelled theragnostic agent has provided detailed distribution and dosimetry information. Based on estimates of tumor and hence target volumes, a rationale goal is to reach volumes of distribution in the range of 15-25 cm3. Optimizing this goal has focused on surgical targeting, infusion duration, and multiple treatment plans. Computational predictive modeling is being employed as a preoperative adjunct for maximizing distribution. Experience in DMG thus far has been limited to feasibility and safety studies. There is a paucity of Phase 2 studies and hence outcome has not yet been rigorously tested. Anecdotal increase in survival and long-term survival has been reproducibly reported. Of particular interest are the occasional cases of children exhibiting late out-of-treatment field and even extra-CNS recurrence. Available data is convincing that CED should serve be further explored in future clinical trials designs for children with DMG. Important features of any Phase 2 study design however should include monitoring of drug distribution, measures of tumor coverage, early response monitoring techniques, and possibly combination with whole CNS axis treatment.

Immune response during regional chemotherapy.

e14537 Background: Regional chemotherapy is known to yield high drug concentrations in the treated tumor area while keeping systemic drug concentrations low. Regional Chemotherapy can be performed as an intra arterial infusion or isolated perfusion. Known perfusion techniques are isolated limb perfusion, hypoxic pelvic perfusion, hypoxic abdominal perfusion, upper abdominal perfusion, and isolated thoracic perfusion, Only little is known on the effect of chemotherapy on immune cells, especially not if chemotherapy is applied regionally. Since more and more immuno oncologic treatment possibilities arise, that often are combined with chemotherapy, knowledge on the effects on immune cells is crucial. Since systemic chemotherapy often decreases the number of immune cells in general, the more targeted approach of regional chemotherapy is investigated in this study. Methods: In this work, we investigate subpopulations of T-lymphocytes, Natural Killer cells and B-lymphocytes during regional chemotherapy and report on their changes after cycles of intra-arterial chemotherapy infusion followed by chemofiltration. Surface phenotyping of cells from EDTA blood was performed with combinations of monoclonal antibodies (anti- CD3,CD16, CD56, CD45, CD4, CD19, CD8, CD28, HLA-DR, CD39, CD25, CD127, CD45RA, CD45RO) for FACS analysis. 58 Patients with advanced breast cancer, ovarian cancer, head and neck cancer, lung cancer, or bladder cancer received regional chemotherapy as an intra arterial infusion therapy or an isolated perfusion therapy. Results: Contrary to the mild immune cell depletion during the first two cycles a more activated immune status is seen after treatment cycle three. Non-significant increase or stable numbers are observed for T-Lymphocytes and its subsets of CD4+ T-helper cells, CD8+ cytotoxic T-cells, and terminal memory T-cells (CD8+/CD28-/CD57+). Significant increase of cell numbers is observed for B-lymphocytes, regulative CD8+ T-cells (CD8+/CD28-/CD57-), activated HLA-DR+ T cells, and Natural Killer cells while regulative CD4+ T cells are significantly decreased. Conclusions: The data show that regional chemotherapy has controversial effects on the immune cell subpopulations. As every chemotherapy, it does affect immune cells, however to a limited extend, but importantly has an activating potential. Immune activation prevalently takes place after the third cycle of therapy which might be a hint for auto-vaccination caused by phases of repeated tumor necrosis after locally high drug concentrations. Finding the optimal dosage of chemotherapeutics may be crucial to balance direct tumor response, immune toxicity and immune activation.

CHANGES OF VASCULAR REACTIVITY TO ADENOSINE A1 AND A2A RECEPTOR AGONISTS AFTER HYPERBARIC OXYGENATION

Objective: Hyperbaric oxygenation (HBO) affects vascular reactivitiy, which plays an important role in the pathophysiology of hypertension and other cardiovascular diseases. New data suggest important effects of HBO on oxidative stress and alteration of production of vasoactive metabolites in blood vessels; however, the role of adenosine A1 and A2a receptors is still underinvestigated.This study aimed to determine the functional role of adenosine A1 and A2a receptors in changes of vascular reactivity in isolated middle cerebral arteries (MCA) of Spague-Dawley rats, after acute and intermittent HBO. Design and method: Male and female healthy Sprague-Dawley rats aged 8–10 weeks were divided into 3 groups: CTRL (control non HBO), A-HBO (single animals exposed to HBO for 2 hours) and 4D-HBO (animals exposed to HBO at a pressure od 2 bars for 2 hours daily for 4 days, with vascular experiments conducted on the fifth day). The rats were anesthetized with ketamine-chloride (3 ml/kg) and midazolam (0,5 ml/kg) and decapitated. The MCA were isolated, cannulated and pressurized for 60’at 80 mmHg to assess basal diameter using pressure myograph Danish Myo Technology. A1 adenosin receptor selective agonist CCPA or A2a adenosin receptor agonist CGS in concentrations from 10–5 to 10–10 M were added to the vessel chamber, and blood vessel diameter was measured after incubation of the drug for 15 minutes. Statistical analyses were performed with Two-way ANOVA tests; p < 0.05 was considered significant. Results: The vasoconstriction in response to A1 agonist administration was similar between the CTRL and 4D-HBO group, while it was significantly reduced in A-HBO compared to the CTRL and 4D-HBO groups. The vasodilation in response to A2a stimulation was significantly reduced in rats exposed to A-HBO and I-HBO compared to CTRL at higher drug concentrations (10–7 to 10–5 M). Conclusions: Acute exposure of rats to HBO leads to decrease in the vasoconstrictive response to adenosine A1 receptor stimulation, while acute and intermittent exposure of rats to HBO is associated with a decrease in the vasodilatory response of MCA to adenosine A2a receptor activation, suggesting that HBO alters sensitivity to or expression of receptors.

A Comparison of High Drug Concentrations in Impaired Driving and Postmortem Casework in Harris County, TX.

Drug-impaired driving is a growing public safety issue. Addressing impairment due to drugs other than ethanol can be challenging for forensic toxicologists as many factors need to be considered including the type of drug(s), drug-drug interaction, the dose(s) and the individual's physiological condition and drug use history. Interpretation of blood drug test results is additionally difficult as drug concentrations in impaired driving cases may overlap levels typically viewed as toxic. This study compares blood concentrations of drugs in impaired driving cases to those in postmortem cases in Houston, TX, from 2014 to 2020. Blood drug concentrations from driving while intoxicated (DWI) or driving under the influence of drugs (DUID) cases submitted to Houston Forensic Science Center (HFSC) and Harris County Institute of Forensic Sciences (HCIFS) were compared to postmortem blood test results from HCIFS. Eight DWI/DUID cases had drugs that exceeded impaired driving concentrations reported in the literature. These drugs included fentanyl (220 ng/mL), oxycodone (680 ng/mL), hydrocodone (310 and 490 ng/mL), clonazepam (330 ng/mL), methamphetamine (3,500 and 7,100 ng/mL) and tetrahydrocannabinol (THC) (160 ng/mL). For oxycodone and hydrocodone, the presented DWI/DUID cases exceeded 91% and 96% of postmortem concentrations, respectively. The 7,100 ng/mL methamphetamine DWI/DUID result was greater than 98% of postmortem cases. The presented DWI/DUID concentrations were higher than all but one postmortem case for clonazepam and higher than all postmortem cases for THC. This study demonstrates that extremely high drug concentrations in DWI/DUID casework blur the line between therapeutic/recreational and toxic concentrations.

In situ photo‐crosslinked hydrogels prepared from acrylated 4‐arm‐poly(ethylene glycol)‐poly(ε‐caprolactone) block copolymers for local cancer therapy

AbstractAmphiphilic block copolymers were synthesized by ring‐opening polymerization of ε‐caprolactone (ε‐CL) using 4‐arm poly(ethylene glycol) (4aPEG) as macroinitiator. 4aPEG and 4aPEG‐PCL copolymers were end functionalized by introducing acrylic groups, and characterized using NMR, GPC, FT‐IR and DSC analyses. Photo‐crosslinked hydrogels were prepared by irradiation of acrylated polymer solutions under visible light (405 nm), using lithium phenyl‐2,4,6‐trimethylbenzoy‐lphosphinate (LAP) as a photoinitiator. In situ gelation was achieved by subcutaneous injection of LAP containing copolymer solution into the abdomen of mice, followed by exposure to visible light. The hydrogels exhibit highly porous structure, high swelling up to 3300%, and excellent biocompatibility. The swelling ratio decreases with increase of poly(ɛ‐caprolactone) (PCL) block length and polymer concentration of hydrogels. Doxorubicin hydrochloride (DOX·HCl) was loaded in hydrogels by soaking dried gel in a DOX solution. The drug loading efficiency is dependent on the swelling performance, reaching a maximum of 94.3%. In vitro drug release studies showed a burst release for the first 6 h, followed by a slower release up to 82% at 28 days. The release rate decreased with increase of hydrophobic block length or polymer concentration of hydrogels. Higher anti‐cancer activity on A549 lung cancer cells was obtained for hydrogels with higher drug concentration, and shorter PCL block length. Therefore, 4aPEG‐PCL hydrogels with outstanding biocompatibility, in situ gelation and prolonged drug release could be very promising for long‐term local cancer therapy.

Time-course changes in DNA damage of corneal epithelial cells in rabbits following ocular instillation with genotoxic compounds

BackgroundIn eye-drop drug development, the additional genotoxicity tests in some cases might be necessary to assess genotoxicity in the ocular surface since the ocular surface is exposed directly to high drug concentrations. Recently, an in vivo comet assay using corneal epithelial cells in rabbits following single ocular instillation was developed as an assay to evaluate genotoxicity in ocular tissues. In this study, we investigated the time-course changes in DNA damage after ocular instillation of genotoxic compounds to evaluate the optimal sampling timing for in vivo comet assay of the ocular surface tissue. Ethidium bromide (EtBr), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4-NQO) were administered to the eyes of the rabbits. Corneas were collected at 0.5, 2, 4, 6, and 24 h after administration, and the comet assay was performed. In addition, the in vitro comet assay was performed to assess the time-course changes in DNA damage induced by short-time exposure to the genotoxic compounds.ResultsThe mean % tail DNA, which is an indicator for DNA damage, in the corneal epithelial cells treated with all compounds exhibited statistically significant increases compared with those in the negative controls of saline at 0.5, 2, 4, and 6 h. There was a difference in the DNA damage response between EtBr and the other two compounds. In the 3% MMS- and 1% 4-NQO-treated eyes, the values of the % tail DNA were the highest at 0.5 h and then decreased gradually. In contrast, in the 1% EtBr-treated eyes, the highest value was noted at 4 h. The results of the in vitro comet assay showed that the % tail DNA increased in all groups. A further increase in the % tail DNA occurred in the EtBr-treated cells even after removing the compound but not in the MMS- and 4-NQO-treated cells.ConclusionRelatively high values of the % tail DNA were maintained from 0.5 to 6 h after administration, suggesting that the optimal sampling time is any one point from 0.5 to 6 h in the comet assay of the corneal surface.

A SYSTEMATIC REVIEW ON SUPERSATURABLE SELF-NANO EMULSIFYING DRUG DELIVERY SYSTEM: A POTENTIAL STRATEGY FOR DRUGS WITH POOR ORAL BIOAVAILABILITY

The most fundamental important extensive constitutive of drug molecules to be available for systemic absorption is aqueous solubility; subsequently, that is the nature of GIT fluid. When the drug molecules become solubilized, it has to reach the systemic circulation via the biological membrane. The solubility problem of many effective pharmaceutical molecules is still one of the major challenges in the formulation of this molecule. Drug molecules that belong to class II have a problem in bioavailability mainly due to low aqueous solubility and the rate-limiting step is the dissolution process and so electing of suitable drug delivery and proper additives are decisive to overcome this major obstruction and promote the fraction that will reach the systemic circulation. Among the different lipid-based systems, the su-SNEDDSs have gained attention because the inclusion of precipitation inhibitors within su-SNEDDSs helps maintain drug supersaturation after dispersion and digestion in the gastrointestinal tract. This enhances the bioavailability of drugs and minimizes the variability of exposure. Nowadays, supersaturable self-nano emulsifying and nano lipid-based drug delivery systems have constrained a substantial concern from pharmaceutical scientists for managing the oral delivery of poorly water-soluble compounds. By following oral administration, self-nano emulsifying drug delivery systems show complex aqueous dispersion and digestion in the GIT and enduring intestinal lymphatic transport, exorbitant pre-absorptive metabolism by gut membrane-bound cytochrome enzymes and preventing P-gp mediated drug efflux. Mostly these processes result in drug supersaturation, which leads to increased absorption or the high drug concentrations may cause precipitation with capricious and variable oral bioavailability. This procession review briefly summarized drug supersaturation obtained from self-nano emulsifying and other lipid-based formulations and this review also delineate the effects of numerous physiological factors and the probable interactions between PIs and lipid, lipase or lipid digested products on the in vivo performance of su-SNEDDS and focuses on reviewing the application of su-SNEDDS in enhancing the solubility and bioavailability of anti-cancer drugs in cancer therapy.

Gap Junction Protein, Connexin 43 is a Key Regulator of Metastasis in Syngeneic Ovarian Cancer Mouse Model

Ovarian cancer is the 7th most common cancer of women and the most lethal gynecological cancer that demands rigorous treatments involving increased radiation cycles and higher drug concentration that triggers toxicity and eventually mortality of patients. Understanding of the disease's progression warrants effective therapeutic inventions. Epithelial Ovarian Cancer cell lines ID8 derived from C57Bl/6 mice and ID8 transfected with Vascular Endothelial Growth Factor‐A (ID8‐VEGF) were used in vitro to generate a Syngeneic Ovarian Cancer Mouse Model. Vascular Endothelial Growth Factor‐A (VEGF‐A/VEGF) is the critical regulator of tumor angiogenesis that supports the transport of nutrients, hormones and O2 to the tumor cells and removal of waste products and CO2. It regulates vascular permeability, actin reorganization, cell migration, enhances tumor growth and progression, which may be associated with aberrant cellular metabolism due to altered gap junction intercellular communication (GJIC) contributing to the tumorigenesis. The transmembrane protein Connexin forms hexameric hemichannels known as connexons and docks with another connexon of adjacent cells to form a functional GJIC. Connexin 43 (Cx43) is one of the most ubiquitously expressed connexins and its C‐terminus interacts with various molecules to regulate many biological functions, including modulation of hemichannel activity, apoptotic signaling, cell cycle regulation and, cytoskeletal rearrangements. Our study aims to understand the effect of VEGF expression on the spatial expression of Cx43 and its nexus with other cellular factors contributing to consequent GJIC formation in the ovarian cancer cells as well as in the mouse model. Our findings suggest that upregulation of VEGF in ID8‐VEGF cell lines is associated with accelerated tumor formation in syngeneic mouse models. A series of biochemical and cellular studies revealed that Sodium Phenylbutyrate(4PBA) treatment induced differential upregulation of Cx43 trafficking to the cell surface, rearrangement of cytoskeletal proteins, cell migration, and the formation of GJIC in ID8 and ID8‐VEGF cells. 4PBA treatment on ID8‐VEGF cells significantly reduced the rate of tumor formation. These differential cellular activities denote that VEGF may directly or indirectly interact with cytoskeletal proteins that regulate Cx43 trafficking and its GJIC formation. Overall, The increased capacity of GJIC formation post‐4PBA treatment is associated with reduction in cell migration in vitro (ID8 and ID8‐VEGF) and tumor formation in vivo (ID8‐VEGF). Interactome studies combining RNASeq and IPA are designed to understand the nexus between several cellular pathways, including oxidative stress in the regulation of Cx43 in the context of VEGF, to dissect the cellular and molecular mechanism of metastasis.

Pulmonary Targeting of Levofloxacin Using Microsphere-Based Dry Powder Inhalation.

The objective of the current study was to develop poly (lactic-co-glycolic acid) (PLGA) microspheres loaded with the anti-tuberculosis (anti-TB) fluoroquinolone, Levofloxacin (LVX), in the form of dry powder inhalation (DPI). LVX-loaded microspheres were fabricated by solvent evaporation technique. Central Composite Design (CCD) was adopted to optimize the microspheres, with desired particle size, drug loading, and drug entrapment efficiency, for targeting alveolar macrophages via non-invasive pulmonary delivery. Structural characterization studies by differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction analysis revealed the absence of any possible chemical interaction between the drug and the polymer used for the preparation of microspheres. In addition, the optimized drug-loaded microspheres exhibited desired average aerodynamic diameter of 2.13 ± 1.24 μm and fine particle fraction of 75.35 ± 1.42%, indicating good aerosolization properties. In vivo data demonstrated that LVX-loaded microspheres had superior lung accumulation, as evident by a two-fold increase in the area under the curve AUC0–24h, as compared with plain LVX. Furthermore, LVX-loaded microspheres prolonged drug residence time in the lung and maintained a relatively high drug concentration for a longer time, which contributed to a reduced leakage in the systemic circulation. In conclusion, inhalable LVX-loaded microspheres might represent a plausible delivery vehicle for targeting pulmonary tuberculosis via enhancing the therapeutic efficacy of LVX while minimizing its systemic off-target side effects.

Nutrient Gradients Mediate Complex Colony-Level Antibiotic Responses in Structured Microbial Populations.

Antibiotic treatments often fail to eliminate bacterial populations due to heterogeneity in how individual cells respond to the drug. In structured bacterial populations such as biofilms, bacterial metabolism and environmental transport processes lead to an emergent phenotypic structure and self-generated nutrient gradients toward the interior of the colony, which can affect cell growth, gene expression and susceptibility to the drug. Even in single cells, survival depends on a dynamic interplay between the drug’s action and the expression of resistance genes. How expression of resistance is coordinated across populations in the presence of such spatiotemporal environmental coupling remains elusive. Using a custom microfluidic device, we observe the response of spatially extended microcolonies of tetracycline-resistant E. coli to precisely defined dynamic drug regimens. We find an intricate interplay between drug-induced changes in cell growth and growth-dependent expression of resistance genes, resulting in the redistribution of metabolites and the reorganization of growth patterns. This dynamic environmental feedback affects the regulation of drug resistance differently across the colony, generating dynamic phenotypic structures that maintain colony growth during exposure to high drug concentrations and increase population-level resistance to subsequent exposures. A mathematical model linking metabolism and the regulation of gene expression is able to capture the main features of spatiotemporal colony dynamics. Uncovering the fundamental principles that govern collective mechanisms of antibiotic resistance in spatially extended populations will allow the design of optimal drug regimens to counteract them.

MPEG-PDLLA Micelles Potentiate Docetaxel for Intraperitoneal Chemotherapy in Ovarian Cancer Peritoneal Metastasis.

Ovarian cancer is the second most common cause of gynecological cancer death in women. It is usually diagnosed late and accompanied by peritoneal metastasis. For ovarian cancer with peritoneal metastasis, intraperitoneal (IP) chemotherapy can maintain a high drug concentration in the abdominal cavity and reduce local and systemic toxicity. Recently, docetaxel (DTX) has shown broad-spectrum antitumor activity against various malignant tumors, including ovarian cancer with peritoneal metastasis. However, DTX has limited clinical applications due to its poor water solubility, predisposition to hypersensitivity, fluid retention, and varying degrees of neurotoxicity. In this study, we prepared methoxy-poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PDLLA) micelles loaded with DTX and developed an alternative, less toxic, more effective DTX formulation, without Tween 80, and evaluated its pharmacokinetics in the abdominal cavity and its efficacy in ovarian cancer with peritoneal metastasis. The mean diameter of DTX-mPEG-PDLLA was about 25 nm, and the pharmacokinetics of BALB/c mice via IP showed that the plasma exposure of DTX-mPEG-PDLLA was about four times lower than that of DTX. Importantly, DTX-mPEG-PDLLA was significantly more effective than DTX and prolonged the survival period in a SKOV-3 ovarian cancer peritoneal metastasis model. Moreover, the apoptosis rate was significantly increased in vitro. Based on these findings, it is expected that DTX-mPEG-PDLLA can enhance efficacy against ovarian cancer peritoneal metastasis, while reducing toxic side effects, and has the potential to be used in the clinical treatment of peritoneal metastatic cancer.

Otitis media-induced cochlear immune response and opportunistic ototoxicity

Lipopolysaccharide (LPS), an essential component of the bacterial endotoxin, activates tissue macrophages and triggers the release of inflammatory cytokines. In animal models, intratympanic (i.t.) injection of LPS is known to simulate acute otitis media (AOM) and modifies the structure and function of the inner ear. However, whether LPS-induced AOM modulates the uptake of ototoxic aminoglycosides in vivo is unclear. Here, we established an AOM mouse model to investigate the gentamicin uptake in the inner ear and the change of cochlear inflammation for pertaining ototoxicity mechanisms. We found that LPS-induced AOM switched on the cochlear inflammatory response, including macrophage infiltration and upregulation of pro-inflammation cytokines, and significantly enhanced the cochlear uptake of gentamicin. Potential mechanisms of enhanced drug uptake may include increased strial permeability due to acute middle-ear inflammation, resulting in higher drug concentration in the stria vascularis and the endolymph, and subsequently higher drug uptake by hair cells. Other possible mechanisms, such as the activation of specific candidate channels and endocytosis followed by inflammation, need to be further interrogated. In sum, this study improves our understanding of drug trafficking in the pathological cochlea and provides a reference for drug treatment of AOM while protecting the inner ear function.

Development of a Method of Liquid Chromatography Coupled with Tandem Mass Spectrometry for Simultaneous Determination of Linezolid and Tedizolid in Human Plasma.

It is important to select appropriate antibiotics for infection control. Linezolid and tedizolid are newly developed and synthesized oxazolidinone antibacterial agents. It has been pointed out that there is a relationship between a high plasma concentration of the target drug and incidence of adverse effects, although it has been reported that neither linezolid nor tedizolid requires dose adjustment according to renal function. Due to the high incidence of adverse effects, both are often switched. Precise plasma concentration control by therapeutic drug monitoring (TDM) is desirable for reducing the adverse effects of both drugs and obtaining a better therapeutic effect. In this study, we aimed to establish a method for simultaneous quantification of linezolid and tedizolid in human plasma using LC coupled with tandem mass spectrometry. Sample preparation was performed by a simple operation with acetonitrile. Linezolid and tedizolid were separated by an octadecylsilyl column using a gradient elution of acetonitrile in aqueous 0.1% formic acid solution and were detected in the positive ion electrospray mode with multiple reaction monitoring. Quantification of linezolid and tedizolid ranged from 0.5 to 50 and 0.5 to 20 µg/mL, respectively. The intra-day and inter-day precision and accuracy of data were assessed and found to be acceptable. The developed method was successfully applied to measurement of the concentrations of linezolid and tedizolid. This simple method, which can simultaneously quantify both drug concentrations for daily TDM, could contribute to safer treatment of patients.