scholarly journals Double-PEGylated Cyclopeptidic Photosensitizer Prodrug Improves Drug Uptake from In Vitro to Hen’s Egg Chorioallantoic Membrane Model

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6241
Author(s):  
Jordan Bouilloux ◽  
Martin Kiening ◽  
Sopie Yapi ◽  
Norbert Lange
Keyword(s):  
Serine Proteases ◽  
Chorioallantoic Membrane ◽  
Fluorescence Emission ◽  
Fold Increase ◽  
Drug Uptake ◽  
Pheophorbide A ◽  
Light Fluence ◽  
Complete Cell ◽  
Almost All

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.

scholarly journals Co-Delivery of CPT-11 and Panobinostat with Anti-GD2 Antibody Conjugated Immunoliposomes for Targeted Combination Chemotherapy

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3211
Author(s):  
Gils Jose ◽  
Yu-Jen Lu ◽  
Jung-Tung Hung ◽  
Alice L. Yu ◽  
Jyh-Ping Chen
Keyword(s):  
Combination Chemotherapy ◽  
Therapeutic Efficacy ◽  
Imaging System ◽  
Accumulation Rate ◽  
Fold Increase ◽  
Drug Uptake ◽  
Mice Model ◽  
Resistant Variant

The consistent expression of disialoganglioside GD2 in neuroblastoma tumor cells and its restricted expression in normal tissues open the possibility to use it for molecularly targeted neuroblastoma therapy. On the other hand, immunoliposomes combining antibody-mediated tumor recognition with liposomal delivery of chemotherapeutics have been proved to enhance therapeutic efficacy in brain tumors. Therefore, we develop immunoliposomes (ImmuLipCP) conjugated with anti-GD2 antibody, for targeted co-delivery of CPT-11 and panobinostat in this study. U87MG human glioma cell line and its drug resistant variant (U87DR), which were confirmed to be associated with low and high expression of cell surface GD2, were employed to compare the targeting efficacy. From in vitro cytotoxicity assay, CPT-11 showed synergism drug interaction with panobinostat to support co-delivery of both drugs with ImmuLipCP for targeted synergistic combination chemotherapy. The molecular targeting mechanism was elucidated from intracellular uptake efficacy by confocal microscopy and flow cytometry analysis, where 6-fold increase in liposome and 1.8-fold increase in drug uptake efficiency was found using targeted liposomes. This enhanced intracellular trafficking for drug delivery endows ImmuLipCP with pronounced cytotoxicity toward U87DR cells in vitro, with 1.6-fold increase of apoptosis rate. Using xenograft nude mice model with subcutaneously implanted U87DR cells, we observe similar biodistribution profile but 5.1 times higher accumulation rate of ImmuLip from in vivo imaging system (IVIS) observation of Cy5.5-labelled liposomes. Taking advantage of this highly efficient GD-2 targeting, ImmuLipCP was demonstrated to be an effective cancer treatment modality to significantly enhance the anti-cancer therapeutic efficacy in U87DR tumors, shown from the significant reduced tumor size in and prolonged survival time of experiment animals as well as diminished expression of cell proliferation and enhanced expression of apoptosis marker proteins in tumor section.

scholarly journals 720. Efficacy of Fosfomycin for Injection (FOS) vs. Piperacillin–Tazobactam (PIP-TAZ) in Adults with Complicated Urinary Tract Infection (cUTI) and Acute Pyelonephritis (AP): ZEUS Study Outcomes in Patients With Reduced Study Drug Susceptibility

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S324-S324
Author(s):  
Keith S Kaye ◽  
Anita F Das ◽  
Paul B Eckburg ◽  
Steven P Gelone ◽  
Jennifer Schranz ◽  
...  
Keyword(s):  
Clinical Cure ◽  
Complicated Urinary Tract Infection ◽  
Study Drug ◽  
Fold Increase ◽  
The United States ◽  
Drug Susceptibility ◽  
Number Of Patients ◽  
Intent To Treat ◽  
Almost All

Abstract Background FOS is being pursued for US registration in cUTI/AP. Safety and efficacy of FOS vs. PIP-TAZ were demonstrated in the noninferiority ZEUS trial in hospitalized patients with cUTI/AP. Although FOS resistance has been observed in several in vitro studies, resistance rates in clinical settings have remained relatively stable despite >40 years of clinical use of FOS outside of the United States. Here we report outcomes in patients who developed reduced susceptibility to study drug (FOS or PIP-TAZ) after enrollment in ZEUS. Methods Patients received IV FOS 6g q8h or PIP-TAZ 4.5g q8h for 7 days (no oral switch allowed). The primary endpoint was overall success (clinical cure + microbiologic eradication) in microbiologic modified intent-to-treat (m-MITT) population at test-of-cure (TOC; Day 19–21). Reduced susceptibility to FOS or PIP-TAZ was defined as a ≥4-fold increase from baseline in minimum inhibitory concentration (MIC) at Day 5, end of treatment (EOT; Day 7–8), TOC, or late follow-up (LFU; Day 26 ± 2). Microbiologic eradication/persistence of baseline and postbaseline pathogens was confirmed post hoc by pulsed-field gel electrophoresis (PFGE). Results In all m-MITT patients, overall success/clinical cure/microbiologic eradication rates (with PFGE) at TOC were 69.0/90.8/70.7% (FOS) and 57.3/91.6/60.1% (PIP-TAZ). Reduced study drug susceptibility was identified in 7/184 (3.8%) FOS and 8/178 (4.5%) PIP-TAZ patients; all had monomicrobial infections (Table 1). Of these patients, almost all were aged ≥50 years (93%), male (73%), white (100%), and had a screening diagnosis of cUTI (93%). At TOC, 7/7 FOS patients and 7/8 PIP-TAZ patients had microbiologic persistence but all patients were clinical cures; these responses were all sustained through LFU (Table 1). Conclusion In the ZEUS study, few patients had urine isolates with reduced postbaseline susceptibility to either FOS or PIP-TAZ. No trend was observed in isolate species associated with decreased susceptibility to FOS or PIP-TAZ, including various Enterobacteriaceae species and Pseudomonas aeruginosa. Despite microbiologic persistence at TOC in a small number of patients, all of these patients were clinical cures at TOC and sustained cures at LFU. Disclosures All authors: No reported disclosures.

scholarly journals In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion.

1991 ◽  
Vol 115 (4) ◽  
pp. 1107-1112 ◽  
Author(s):  
L Ossowski ◽  
G Clunie ◽  
M T Masucci ◽  
F Blasi
Keyword(s):  
Chorioallantoic Membrane ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Surface Receptor ◽  
In Vivo Model ◽  
Specific Cell ◽  
Upa Receptor ◽  
Surface Receptor

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

Gene transfer of endothelial NO-synthase restores migratory capacity of endothelial progenitor cells from patients with coronary artery diseases

2007 ◽  
Vol 30 (4) ◽  
pp. 96
Author(s):  
Michael R. Ward ◽  
Qiuwang Zhang ◽  
Duncan J. Stewart ◽  
Michael J.B. Kutryk
Keyword(s):  
Risk Factors ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Fold Increase ◽  
No Synthase ◽  
Endothelial Progenitor ◽  
Migratory Capacity ◽  
Acute Mi ◽  
Cad Risk

Autologous endothelial progenitor cells (EPCs) have been used extensively in the development of cell-based therapy for acute MI. However, EPCs isolated from patients with CAD and/or CAD risk factors have reduced regenerative activity compared to cells from healthy subjects. As in endothelial cells, endothelial NO synthase (eNOS) expression and subsequent NO production are believed to be critical determinants of EPC function. Recently, the ability of EPCs to migrate in vitro in response to chemotactic stimuli has been shown to predict their regenerative capacity in clinical studies. Therefore, we hypothesized that the regenerative function of EPCs from patients with or at high risk for CAD will be enhanced by overexpression of eNOS, as assessed by migratory capacity. Methods: EPCs were isolated from the blood of human subjects with CAD risk factors (>15% Framingham risk score; FRS) (± CAD) by Ficoll gradient separation and differential culture. Following 3 days in culture, cells were transduced using lentivirus vectors containing either eNOS or GFP (sham) at an MOI of 3. The cells were cultured for an additional 5 days before being used in functional assays. Cell migration and chemotaxis in response to VEGF (50 ng/mL) and SDF-1 (100 ng/mL) were assessed using a modified Boyden Chamber assay. Results: Transduction at an MOI of 3 led to a ~90-100-fold increase in eNOS mRNA expression and a 5-6 fold increase in eNOS protein expression, as assessed by qRT-PCR and Western Blotting. Moreover, there was a significant improvement in the migration of EPCs following eNOS transduction compared to sham-transduced EPCs in response to both VEGF (44.3 ± 8.4 vs. 31.1 ± 4.6 cells/high power field; n=10, p < 0.05) and SDF-1 (51.9 ± 11.1 vs. 34.5 ± 3.3 cells/HPF; n=10, p < 0.05). Conclusions: These data show that the reduced migration capacity of EPCs isolated from patients with CAD and/or CAD risk factors can be significantly improved through eNOS overexpression in these cells. Thus, eNOS transduction of autologous EPCs may enhance their ability to restore myocardial perfusion and function following acute MI. We intend to further explore the regenerative potential of eNOS-transduced EPCs using various in vitro and in vivo models.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions

2020 ◽  
Vol 27 (29) ◽  
pp. 4840-4854 ◽  
Author(s):  
Chrysoula-Evangelia Karachaliou ◽  
Hubert Kalbacher ◽  
Wolfgang Voelter ◽  
Ourania E. Tsitsilonis ◽  
Evangelia Livaniou
Keyword(s):  
Mammalian Cells ◽  
Therapeutic Potential ◽  
Prothymosin Alpha ◽  
Disease States ◽  
Leading Role ◽  
Tissue Extracts ◽  
Biologic Response ◽  
Health And Disease ◽  
Almost All

Prothymosin alpha (ProT&#945;) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProT&#945; is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as “alarmins”. Antibodies and immunochemical techniques for ProT&#945; have played a leading role in the investigation of the biological role of ProT&#945;, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProT&#945; (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions

2020 ◽  
Vol 21 (5) ◽  
pp. 438-450
Author(s):  
Ramya Ramchandran ◽  
Swetha Ramesh ◽  
Anviksha A ◽  
RamLal Thakur ◽  
Arunaloke Chakrabarti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pathogenic Fungi ◽  
Fold Increase ◽  
Production Yield ◽  
Yield Enhancement ◽  
Bioactive Metabolites ◽  
Candida Auris ◽  
Media Formulation ◽  
Static Conditions

Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

Potential of Nanoparticles in Combating Candida Infections

2019 ◽  
Vol 16 (5) ◽  
pp. 478-491 ◽  
Author(s):  
Faizan Abul Qais ◽  
Mohd Sajjad Ahmad Khan ◽  
Iqbal Ahmad ◽  
Abdullah Safar Althubiani
Keyword(s):  
Targeted Delivery ◽  
Recent Progress ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Fold Increase ◽  
Antifungal Drugs ◽  
Low Toxicity ◽  
Candida Infections ◽  
Made In

Aims: The aim of this review is to survey the recent progress made in developing the nanoparticles as antifungal agents especially the nano-based formulations being exploited for the management of Candida infections. Discussion: In the last few decades, there has been many-fold increase in fungal infections including candidiasis due to the increased number of immunocompromised patients worldwide. The efficacy of available antifungal drugs is limited due to its associated toxicity and drug resistance in clinical strains. The recent advancements in nanobiotechnology have opened a new hope for the development of novel formulations with enhanced therapeutic efficacy, improved drug delivery and low toxicity. Conclusion: Metal nanoparticles have shown to possess promising in vitro antifungal activities and could be effectively used for enhanced and targeted delivery of conventionally used drugs. The synergistic interaction between nanoparticles and various antifungal agents have also been reported with enhanced antifungal activity.

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