scholarly journals Tritium labeling of antisense oligonucleotides via different conjugation agents

AAPS Open ◽  
2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Martin R. Edelmann ◽  
Christophe Husser ◽  
Martina B. Duschmalé ◽  
Guy Fischer ◽  
Claudia Senn ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Specific Activity ◽  
Plasma Concentrations ◽  
In Vivo Studies ◽  
Radioactive Label ◽  
Target Interaction ◽  
Time Profiles ◽  
Novel Approach

AbstractA novel approach to tritium-labeled antisense oligonucleotides (ASO) was established by conjugating N-succinimidyl propionate, as well as maleimide-derivatives, to the 3′-end of ASOs targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat1) containing amino- or sulfhydryl-linkers. In vitro stability and Malat1 RNA reduction studies demonstrated that N-ethylmaleimide (NEM) could be used as a stable tag while maintaining the desired target interaction. The corresponding radioactive label conjugation using [3H]-NEM resulted in tritium-labeled ASOs with a high molar specific activity of up to 17 Ci/mmol. Single-dose in vivo studies in mice were carried out to compare [3H]-ASOs with their unlabeled counterpart ASOs, with and without conjugation to N-acetylgalactosamine (GalNAc), for tissue and plasma concentrations time profiles. Despite the structural modification of the labeled ASOs, in vitro target interaction and in vivo pharmacokinetic behaviors were similar to that of the unlabeled ASOs. In conclusion, this new method provides a powerful technique for fast and safe access to tritium-labeled oligonucleotides, e.g., for pharmacokinetic, mass balance, or autoradiography studies. Graphical abstract

scholarly journals In Vitro Characterization of Rabbit Thrombin, Antithrombin III, and Thrombin-Antithrombin III Complex, and Determination of Their Survival Times in Vivo

1977 ◽  
Author(s):  
Christine N. Vogel ◽  
Kingdon S. Henry ◽  
Roger L. Lundblad
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
In Vivo Studies ◽  
Survival Times ◽  
At Iii

Our intention is to study the interaction of rabbit thrombin with antithrombin III (AT-III) in vitro and in vivo. After activation of crude prothrombin with tissue thromboplastin and CaCl2, thrombin was purified and showed two species of thrombin with molecular weights of 36,000 and 39,000 daltons as determined by sodium dodecyl sulfate discontinuous gel electrophoresis. Rabbit AT-III was purified using a heparin agarose column and had a molecular weight of 55,000 daltons. The inhibition of thrombin by AT-III was followed by fibrinogen clotting assays and an AT-III-thrombin complex was observed on gel electrophoresis. For the in vivo studies both thrombin and AT-III were radiolabelled with Na125i using the solid state lactoperoxidase method and retained 99% of the pre-iodinated specific activity. Radiolabelled thrombin and a radiolabelled AT-III-thrombin complex were injected into different rabbits. The rate of removal of both was very similar with a half-life of approximately 9 hours. When radiolabelled AT-III was injected, the half-life was approximately 60 hours. Since the disappearance rate of thrombin more closely approximates that of the preformed AT-III-thrombin complex and is clearly shorter than the turnover rate of AT-III, the possibility is raised that thrombin combines in vivo with a native inhibitor such as AT-III and may in fact be removed from the circulation as a complex rather than as a native molecule.

scholarly journals In Vitro Studies of Synthesis and Release of Factor VIII Related Protein by Endothelial Cells

1977 ◽  
Author(s):  
E. G. D. Tuddenham ◽  
L. W. Hoyer
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Calf Serum ◽  
In Vivo Studies ◽  
Separate Experiment ◽  
Related Protein ◽  
Binding Studies ◽  
Radioactive Label

In vivo studies have shown that many stimuli such as epinephrine, exercise and pregnancy lead to a rise in factor VIII levels. However, the physiologic mechanisms controlling factor VIII levels are poorly, if at all, understood. Since endothelial cells synthesize factor VIII related protein (FVIII:RP) and can be grown in tissue culture, they provide a suitable in vitro model to study synthesis and release of FVIII:RP. Endothelial cells were harvested by collagenase digestion from human umbilical cords and grown in medium 199 supplemented with 20 to 30% pooled human serum. Confluent cultures were washed and then maintained in medium 199 supplemented with 20% fetal calf serum. Release of FVIII:RP into the medium was measured by immunoradiometric assay. Labeled amino acids were added to the medium for studies of FVIII:RP synthesis. Incorporation of radioactive label into FVIII:RP was measured in binding studies using a specific immunoadsorbent. Epinephrine in concentrations from 1 ng to 10 ug per ml had no effect on rate of release of FVIII:RP from cultured endothelial cells, suggesting that the in vivo effect of epinephrine is not due to a direct action on endothelial cells. In a separate experiment,exogenous FVIII:RP was added to the culture medium at a high concentration (2 units FVIII:RP per ml) along with 3H Leucine. A control without exogenous VIII:RP incorporated as much radioactivity into VIII:RP as did the culture with added FVIII:RP. This result suggests that there is no end product inhibition of FVIII:RP synthesis which operates on the endothelial cell.

scholarly journals CITCO as an Adjuvant Facilitates CHOP-Based Lymphoma Treatment in hCAR-Transgenic Mice

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2520
Author(s):  
Ritika Kurian ◽  
William Hedrich ◽  
Bryan Mackowiak ◽  
Linhao Li ◽  
Hongbing Wang
Keyword(s):  
Transgenic Mice ◽  
Constitutive Androstane Receptor ◽  
Plasma Concentrations ◽  
Survival Rates ◽  
Pharmacokinetic Profile ◽  
In Vivo Studies ◽  
Chop Regimen ◽  
Transgenic Mouse Line

Non-Hodgkin’s lymphoma (NHL) is a malignant cancer originating in the lymphatic system with a 25–30% mortality rate. CHOP, consisting of cyclophosphamide (CPA), doxorubicin, vincristine, and prednisone, is a first-generation chemotherapy extensively used to treat NHL. However, poor survival rates among patients in advanced stages of NHL shows a need to improve this standard of care treatment. CPA, an integral component of CHOP, is a prodrug that requires CYP2B6-mediated bioactivation to 4-hydroxy-CPA (4-OH-CPA). The expression of CYP2B6 is transcriptionally regulated by the constitutive androstane receptor (CAR, NRi13). We have previously demonstrated that the induction of hepatic CYP2B6 by CITCO, a selective human CAR (hCAR) agonist, results in CHOP’s enhanced antineoplastic effects in vitro. Here, we investigate the in vivo potential of CITCO as an adjuvant of CPA-based NHL treatment in a hCAR-transgenic mouse line. Our results demonstrate that the addition of CITCO to the CHOP regimen leads to significant suppression of the growth of EL-4 xenografts in hCAR-transgenic mice accompanied by reduced expression of cyclin-D1, ki67, Pcna, and increased caspase 3 fragmentation in tumor tissues. CITCO robustly induced the expression of cyp2b10 (murine ortholog of CYP2B6) through hCAR activation and increased plasma concentrations of 4-OH-CPA. Comparing to intraperitoneal injection, oral gavage of CITCO results in optimal hepatic cyp2b10 induction. Our in vivo studies have collectively uncovered CITCO as an effective facilitator for CPA-based NHL treatment with a pharmacokinetic profile favoring oral administration, promoting CITCO as a promising adjuvant candidate for CPA-based regimens.

M945. Nonhepahinic Mucopolisacarid. In Vivo Studies(Humans)

1979 ◽  
Author(s):  
K. Giuliani ◽  
E. Stwarcer
Keyword(s):  
Plasma Concentrations ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Healthy Individuals ◽  
Enhanced Activity ◽  
Post Infusion ◽  
Level 1 ◽  
Made In

It has been reported,in in vivo studies (doga, rabbita), that the antithrombotic protection of heparin is clearly related to AntiXa enhanced activity,than to a particular KCCT level(1)(2)In this work, 20 healthy individuals were studied. KCCT, TT, AntiXa (Denaon-Bonnar) deter minations were made. In vitro studies of M945 actiona on these tests, at different plasma concentrations of the drug (0.02-0.2UI/al) were performed.M945 waa then SC administered, 100UI/kg weight, and blood aamples collected each two houra, and KCCT, TT, AntiXa studied.In vitro samples showed no differences in KCCT, TT, and Anti Xa activity, that the ones expected when using heparin. In 16 humana, in post infusion studie s, it was seen that nearly no changea on KCCT or TT occured,vhile AntiAa waS enhanced in its activity, up to 90" (more than corresponding valuea for 0.2UI/al of heparin in plaamal.Further work with similar drugs to M945, should open the poasibility of safe antithrombotic treatments in patients with anticoagulant contraindications.1. Szwarcer E, Giuliani R, vIII World Congr Cardiol, Abatr, 1, 1159, pg 381, 19782. Chiu HM, Hirsh J, Yung WL, Regoeczi E, Gent H, Elood, Vol 49, N82, 171, (feb), 1977

scholarly journals Protection of pancreatic islets using theranostic silencing nanoparticles in a baboon model of islet transplantation

2020 ◽  
Author(s):  
Ada Admin ◽  
Thomas Pomposelli ◽  
Ping Wang ◽  
Kazuhiro Takeuchi ◽  
Katsunori Miyake ◽  
...  
Keyword(s):  
Islet Transplantation ◽  
In Vivo Studies ◽  
Pancreatic Islet Transplantation ◽  
Insulin Requirements ◽  
Novel Approach ◽  
Apoptotic Effect ◽  
Novel Strategy ◽  
Interfering Rna

The long-term success of pancreatic islet transplantation (Tx) as a cure for type 1 diabetes remains limited. Islet loss after Tx related to apoptosis, inflammation and other factors continues to limit its efficacy. In this project we demonstrate a novel approach aimed at protection of islets prior to Tx in non-human primates (NHP, baboons) by silencing a gene (caspase 3) responsible for induction of apoptosis. This was done using small interfering RNA (siRNA, siCas-3) conjugated to magnetic nanoparticles (MN). In addition to serving as carriers for siCas-3 these nanoparticles also act as reporters for magnetic resonance imaging so islets labeled with MN-siCas-3 can be monitored in vivo after Tx. In vitro studies showed the anti-apoptotic effect of MN-siCas-3 on islets in culture resulting in a minimal islet loss. For in vivo studies donor baboon islets were labeled with MN-siCas-3 and infused into recipient diabetic subjects. A dramatic reduction in insulin requirements was observed in animals transplanted even with a marginal number of labeled islets compared to controls. By demonstrating the protective effect of MN-siCas-3 in the challenging NHP model, this study proposes a novel strategy to minimize the number of donor islets required from either cadaver or living donor.

scholarly journals Amphipathic Dendritic Poly-peptides Carrier to Deliver Antisense Oligonucleotides Against Multi-drug Resistant Bacteria in Vitro and in Vivo

2021 ◽  
Author(s):  
Zhou Chen ◽  
Yue Hu ◽  
Xinggang Mao ◽  
Dan Nie ◽  
Hui Zhao ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Multidrug Resistant ◽  
Great Promise ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
Histidine Residues ◽  
Novel Approach ◽  
Global Health Issue

Abstract Background: Outbreaks of infection due to multidrug-resistant bacteria, especially gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by multidrug-resistant bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity.Results: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo.Conclusions: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated essential features, namely, amphipathicity, 8–10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Synthesis, characterization and evaluation of collagen scaffolds crosslinked with aminosilane functionalized silver nanoparticles: in vitro and in vivo studies

2015 ◽  
Vol 3 (15) ◽  
pp. 3032-3043 ◽  
Author(s):  
Abhishek Mandal ◽  
Santhanam Sekar ◽  
N. Chandrasekaran ◽  
Amitava Mukherjee ◽  
Thotapalli P. Sastry
Keyword(s):  
Silver Nanoparticles ◽  
Clinical Applications ◽  
In Vivo Studies ◽  
Cross Linking ◽  
Collagen Scaffolds ◽  
Novel Approach

This work presents a novel approach for functionalization of silver nanoparticles and cross-linking them with collagen to form FSCSC scaffolds suitable for clinical applications.

Positron emission tomography (PET) with 89Zr-labeled trastuzumab (89Zr-trastuzumab): Monitoring HER2 expression in HER2-positive gastric cancer in vivo.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 35-35
Author(s):  
Y. Y. Janjigian ◽  
N. T. Villegas ◽  
J. P. Holland ◽  
M. A. Shah ◽  
V. Divilov ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Specific Activity ◽  
Gastric Cancer Cell Line ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Organ Distribution ◽  
Her2 Positive ◽  
Bibw 2992

35 Background: The ToGA study established HER2 is a target in the treatment of gastric cancer. Trastuzumab pharmacokinetics and organ distribution is varied in each patient and is heavily affected by the extent of tumor load (Oude Munnink, JCO 2010). 89Zr-trastuzumab HER2 PET can be used to image that variability and may aid in detection and staging of HER2-positive tumors. We are implementing 89Zr-radiolabeled trastuzumab PET in vivo for imaging of HER2-positive gastric cancer and for future non-invasive assessment of HER2 inhibition with a dual irreversible HER1/HER2 inhibitor, BIBW-2992. Methods: 89Zr (t1/2 = 3.17 days) was prepared via the 89Y(p,n)89Zr transmutation with high radiochemical yields (1.52±0.11 mCi/μAh) and purity (>99.99%). Trastuzumab was functionalized with the tris-hydroxamate chelate, desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature. 89Zr-trastuzumab PET experiments in athymic nu/nu mice bearing sub-cutaneous NCI-N87 (HER2+) and/or SNU1 (HER2-) tumors were conducted. NCI-N87 gastric cancer cells were treated with BIBW-2992. Results: 89Zr-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity of 2.82±0.05 mCi/mg. In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±7. In vivo biodistribution experiments revealed high and specific uptake in HER2-positive tumors after 72 h (85.2±11.1% ID/g) with retention of activity for over 120 h. No uptake was seen in HER2-negative gastric cancer xenografts. In vitro, BIBW-2992 demonstrates dose dependent growth inhibition in the HER2+ gastric cancer cell line. Conclusions: 89Zr-trastuzumab provides quantitative and highly-specific delineation of HER2-positive gastric cancer. In vivo studies of BIBW2-2992 in gastric cancer with 89Zr-trastuzumab HER2 PET response assessment are underway. A Phase I study of 89Zr-trastuzumab PETin HER2-positive patients is to open at MSKCC imminently. No significant financial relationships to disclose.

Studies in Man and Experimental Animals of a Low Molecular Weight Heparin Fraction

1981 ◽  
Vol 45 (03) ◽  
pp. 214-218 ◽  
Author(s):  
D P Thomas ◽  
R E Merton ◽  
W E Lewis ◽  
T W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Specific Activity ◽  
Ex Vivo ◽  
High Specific Activity ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Low Molecular Weight

SummaryIn vitro and in vivo studies were carried out on a commercially prepared low molecular weight heparin fraction. By APTT assay the fraction had a specific activity of half that of unfractionated mucosal heparin, yet retained full potency by anti-Xa assay (both clotting and chromogenic substrate). When administered intravenously to human volunteers, the anti-Xa/APTT ratio remained the same as it was in vitro. However, after subcutaneous injection, the ratio increased and anti-Xa activity could not be fully neutralized ex vivo by PF4. The fraction was as effective as unfractionated heparin in preventing experimental serum-induced thrombosis, suggesting that a heparin fraction with high specific activity by anti-Factor Xa assay compared to APTT activity may be an effective drug for the prophylaxis of venous thrombosis.

Impaired aldosterone production by long-term infusion of atrial natriuretic factor

1990 ◽  
Vol 258 (1) ◽  
pp. E51-E56
Author(s):  
M. Nagano ◽  
E. L. Bravo
Keyword(s):  
Angiotensin Ii ◽  
Atrial Natriuretic Factor ◽  
Plasma Concentrations ◽  
In Vivo Studies ◽  
In Vitro Production ◽  
Natriuretic Factor ◽  
Pressor Responses ◽  
Atrial Natriuretic

This study assessed the effect of chronic infusions of atrial natriuretic factor (ANF) on in vivo and in vitro production of aldosterone. Vehicle (saline) or rat ANF-(99-126) was intravenously infused at 100 ng.kg-1.h-1 for 5 consecutive days into male New Zealand White rabbits. At 5 days plasma ANF was 18 +/- 4.1 pg/ml in vehicle-infused and 48.5 +/- 9.0 in ANF-infused rabbits (P less than 0.01). Plasma renin activity was significantly less in ANF-infused rabbits (2.99 +/- 0.35 vs. 0.77 +/- 0.12 ng.ml-1.h-1, P less than 0.01); however no differences were observed in the basal plasma concentrations of aldosterone, corticosterone, potassium, or hematocrit. In in vivo studies, chronically administered ANF attenuated plasma aldosterone, but not pressor, responses to acutely infused angiotensin II given at doses of 4, 16, and 64 ng.kg-1.min-1 for 20 min each. In in vitro experiments, collagenase-dispersed adrenal capsular cells from ANF-infused rabbits exhibited significantly reduced maximal responses to adrenocorticotropic hormone, angiotensin II, and potassium. These results suggest that chronic small increases in circulating ANF can blunt selectively adrenocortical responses to aldosterone secretagogues without affecting pressor responses to angiotensin II.

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