scholarly journals Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has ImprovedIn VitroSerum Stability

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Vinojini B. Nair ◽  
Ross A. D. Bathgate ◽  
Frances Separovic ◽  
Chrishan S. Samuel ◽  
Mohammed Akhter Hossain ◽  
...  
Keyword(s):  
Half Life ◽  
Solid Phase ◽  
Phase Iii ◽  
Phase Iii Clinical Trials ◽  
H2 Relaxin ◽  
Covalent Dimer ◽  
Dimeric Form ◽  
Covalently Linked ◽  
G Protein Coupled

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, itsin vivohalf-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.

scholarly journals Microbiology and Preclinical Review of Omadacycline

2019 ◽  
Vol 69 (Supplement_1) ◽  
pp. S6-S15 ◽  
Author(s):  
James A Karlowsky ◽  
Judith Steenbergen ◽  
George G Zhanel
Keyword(s):  
Legionella Pneumophila ◽  
Animal Studies ◽  
Action Spectrum ◽  
Binding Activity ◽  
Phase Iii ◽  
Phase Iii Clinical Trials ◽  
Clostridioides Difficile ◽  
Semisynthetic Derivative

AbstractOmadacycline is a novel aminomethylcycline antimicrobial and semisynthetic derivative of tetracycline. In vitro, omadacycline displays potent activity against gram-positive and many gram-negative bacteria, including methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococci, vancomycin-resistant Enterococcus, and Enterobacteriaceae. Omadacycline is also active against atypical and anaerobic pathogens, including Legionella pneumophila, Mycoplasma spp., Ureaplasma spp., Bacteroides spp., and Clostridioides difficile. This review outlines the microbiology and preclinical studies of omadacycline, including its mechanism of action; spectrum of activity; protein binding; activity in the presence of surfactant, serum, normal, and pH-adjusted urine, or bacterial biofilms; postantibiotic effect; pharmacodynamic properties; and in vitro and in vivo efficacy. The results of in vitro and in vivo animal studies support the observations made in phase III clinical trials and the clinical development of omadacycline.

GPV Is a Marker of In Vivo Platelet Activation – Study in a Rat Thrombosis Model

2000 ◽  
Vol 83 (02) ◽  
pp. 327-333 ◽  
Author(s):  
Monique Freund ◽  
Pierre Mangin ◽  
David O. Azorsa ◽  
Catherine Schwartz ◽  
Sylvie Moog ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
G Protein ◽  
Platelet Activation ◽  
Half Life ◽  
Thrombin Generation ◽  
Polyclonal Antibodies ◽  
Protease Activated Receptors ◽  
Thrombosis Model ◽  
G Protein Coupled

SummaryThrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life.In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

scholarly journals Conservation of S20 as an Ineffective and Disposable IFNγ-Inducing Determinant of Plasmodium Sporozoites Indicates Diversion of Cellular Immunity

2021 ◽  
Vol 12 ◽  
Author(s):  
Calvin Hon ◽  
Johannes Friesen ◽  
Alyssa Ingmundson ◽  
Diana Scheppan ◽  
Julius C. R. Hafalla ◽  
...  
Keyword(s):  
Genetic Background ◽  
Cell Epitope ◽  
Specific Protein ◽  
Phase Iii ◽  
Mammalian Host ◽  
Pivotal Phase ◽  
Phase Iii Clinical Trials ◽  
Immunogenic Proteins

Despite many decades of research to develop a malaria vaccine, only one vaccine candidate has been explored in pivotal phase III clinical trials. This candidate subunit vaccine consists of a portion of a single Plasmodium antigen, circumsporozoite protein (CSP). This antigen was initially identified in the murine malaria model and shown to contain an immunodominant and protective CD8+ T cell epitope specific to the H-2Kd (BALB/c)-restricted genetic background. A high-content screen for CD8+ epitopes in the H2Kb/Db (C57BL/6)-restricted genetic background, identified two distinct dominant epitopes. In this study, we present a characterization of one corresponding antigen, the Plasmodium sporozoite-specific protein S20. Plasmodium berghei S20 knockout sporozoites and liver stages developed normally in vitro and in vivo. This potent infectivity of s20(-) sporozoites permitted comparative analysis of knockout and wild-type parasites in cell-based vaccination. Protective immunity of irradiation-arrested s20(-) sporozoites in single, double and triple immunizations was similar to irradiated unaltered sporozoites in homologous challenge experiments. These findings demonstrate the presence of an immunogenic Plasmodium pre-erythrocytic determinant, which is not essential for eliciting protection. Although S20 is not needed for colonization of the mammalian host and for initiation of a blood infection, it is conserved amongst Plasmodium species. Malarial parasites express conserved, immunogenic proteins that are not required to establish infection but might play potential roles in diverting cellular immune responses.

scholarly journals Extending the in vivo Half-Life of Adalimumab Fab via Sortase A-Mediated Conjugation of Adalimumab Fab with Modified Fatty Acids

2021 ◽  
Author(s):  
Qing-Bin Zhang ◽  
Si-Da Ruan ◽  
Yong Wu ◽  
Jin-Hua Zhang ◽  
Jian-Guang Lu ◽  
...  
Keyword(s):  
Half Life ◽  
Solid Phase ◽  
Inflammatory Diseases ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sortase A ◽  
Recognition Sequence ◽  
Tnf Α

AbstractAdalimumab, a full-length monoclonal antibody, is widely used as an anti-tumor necrosis factor-α (anti-TNF-α) agent. In this article, we aimed to prolong the in vivo half-life of adalimumab antigen-binding fragment (Fab) through Sortase A (SrtA)-mediated conjugation of its Fab with fatty acid (FA). In our study, adalimumab Fab analog was prepared by adding an SrtA recognition sequence (LPETGG) and His6 tag to the heavy chain C-terminal of the Fab via (G4S)3 linker. Four FA motifs with different linkers were designed and synthesized by solid-phase methodology, then conjugated with the Fab analog using SrtA to produce Fab bioconjugates. The successful generation of four Fab bioconjugates (Fab–FA1, Fab–FA2, Fab–FA3, and Fab–FA4) was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and mass spectrometry. Then, the bioactivities and half-life of these Fab bioconjugates were examined using TNF-α-/human serum albumin (HSA)-binding enzyme-linked immunosorbent assay, cytotoxicity assay, and pharmacokinetic study, respectively. All Fab bioconjugates exhibited similar TNF-α-neutralizing activities when compared with the Fab analog, even in the presence of albumin, indicating that there were no apparent influences on the functional site of Fab after FA modification. However, different degrees of affinity for HSA were observed among these Fab–FA bioconjugates, with Fab–FA3 exhibiting the maximal affinity. An in vivo study in mice further revealed remarkably improved pharmacokinetics of Fab– FA3 with a 15.2-fold longer plasma half-life (19.86 hours) compared with that of the Fab analog (1.31 hours). In summary, we have developed a novel long-acting adalimumab Fab bioconjugate, Fab–FA3, with more sustained in vivo activity, which can be used for drug development targeting TNF-α-mediated inflammatory diseases.

scholarly journals Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 364 ◽  
Author(s):  
Mijeong Bak ◽  
Junyong Park ◽  
Kiyoon Min ◽  
Jinhwan Cho ◽  
Jihyoun Seong ◽  
...  
Keyword(s):  
Half Life ◽  
Solid Phase ◽  
Biological Activities ◽  
Recombinant Peptide ◽  
Site Specific ◽  
Therapeutic Peptide ◽  
Therapeutic Peptides ◽  
Long Acting

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A “clickable” non-natural amino acid—p-azido-l-phenylalanine (AzF)—was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide–alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

Ibrexafungerp: A novel oral glucan synthase inhibitor

2019 ◽  
Vol 58 (5) ◽  
pp. 579-592 ◽  
Author(s):  
M R Davis ◽  
M A Donnelley ◽  
G R Thompson
Keyword(s):  
Fungal Pathogens ◽  
Phase Iii ◽  
Tolerability Profile ◽  
Candida Spp ◽  
Glucan Synthase ◽  
Phase Iii Clinical Trials ◽  
In Vivo Animal Models ◽  
Synthase Inhibitor

Abstract Ibrexafungerp is a novel glucan synthase inhibitor currently undergoing phase II and phase III clinical trials. This compound has demonstrated in vitro activity against clinically important fungal pathogens including Candida spp. and Aspergillus spp. It is able to retain activity against many echinocandin-resistant strains of Candida due to differential avidity for the target site compared to echinocandins. In vivo animal models have demonstrated efficacy in murine models of invasive candidiasis, aspergillosis, and pneumocystis. Due to high bioavailability, it can be administered both orally and intravenously. A favorable drug interaction and tolerability profile is observed with this compound. This review summarizes existing data that have either been published or presented at international symposia.

Rome I versus Rome II; Overlap in patients enrolled in tegaserod phase III clinical trials for irritable bowel syndrome (IBS)

2001 ◽  
Vol 120 (5) ◽  
pp. A284-A284
Author(s):  
B NAULT ◽  
S SUE ◽  
J HEGGLAND ◽  
S GOHARI ◽  
G LIGOZIO ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Irritable Bowel Syndrome ◽  
Phase Iii ◽  
Phase Iii Clinical Trials ◽  
Irritable Bowel ◽  
Rome I

Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials

2001 ◽  
Vol 28 (6) ◽  
pp. 620-625 ◽  
Author(s):  
Pierre Falardeau ◽  
Pierre Champagne ◽  
Patrick Poyet ◽  
Claude Hariton ◽  
[Eacute]ric Dupont
Keyword(s):  
Clinical Trials ◽  
Phase Iii ◽  
Phase Iii Clinical Trials ◽  
Naturally Occurring ◽  
Antiangiogenic Drug

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

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