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Intact plasma quantification of the large therapeutic lipopeptide bulevirtide.

ABSTRACT: Bulevirtide is a first-in-class entry inhibitor of the hepatitis B and hepatitis delta virus blocking the sodium/bile acid co-transporter NTCP, and was recently approved for the treatment of hepatitis D as a priority medicine (prime) in an accelerated assessment by the European Medicines Agency. It is a very large lipopeptide comprising 47 amino acids in its sequence and a myristoylation at the N-terminus. For support of clinical development, we established highly sensitive plasma quantification assays using 100 μL of plasma, spanning concentrations of 0.1 to 100 ng/mL and 1 to 1000 ng/mL with the option to measure ten-fold diluted samples up to 10,000 ng/mL. Quantification was performed with UPLC-MS/MS measurements after extraction with protein precipitation. Both assays were fully validated according to the pertinent guidelines of the FDA and EMA, including incurred sample reanalyses and cross-validation using clinical study samples. Graphical abstract.

SUBMITTER: Sauter M

PROVIDER: S-EPMC8410713 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Intact plasma quantification of the large therapeutic lipopeptide bulevirtide.

Sauter Max M Blank Antje A Stoll Felicitas F Lutz Natalie N Haefeli Walter E WE Burhenne Jürgen J

Analytical and bioanalytical chemistry 20210520 22

Bulevirtide is a first-in-class entry inhibitor of the hepatitis B and hepatitis delta virus blocking the sodium/bile acid co-transporter NTCP, and was recently approved for the treatment of hepatitis D as a priority medicine (prime) in an accelerated assessment by the European Medicines Agency. It is a very large lipopeptide comprising 47 amino acids in its sequence and a myristoylation at the N-terminus. For support of clinical development, we established highly sensitive plasma quantification ...[more]

PMID: 34018034

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Project description:Background & aimsAccurate HDV RNA quantification is crucial for diagnosis and management of chronic hepatitis delta (CHD), yet a significant variability between assays exists. We compared three methods to quantify HDV RNA levels in untreated and bulevirtide (BLV)-treated patients with CHD.MethodsFrozen plasma from untreated and BLV-treated patients with CHD were tested in a single-center retrospective study using three different assays: Robogene 2.0 HDV RNA Quantification Kit 2.0 (Roboscreen GmbH; limit of detection [LOD] 6 IU/ml on 7500 Fast Real-Time PCR System [Applied Biosystem]), EurobioPlex HDV PCR quantitative kit (Eurobio Scientific; LOD 100 IU/m) on CFX96™ real-time PCR detection system [Bio-Rad]), and AltoStar HDV RT-PCR RUO Kit 1.5 (Altona Diagnostics; estimated LOD <10 IU/ml) on the AltoStar®AM16.ResultsOverall, 431 plasma samples from 130 patients with CHD (69 untreated and 61 BLV-treated) were studied. Compared with Robogene 2.0, EurobioPlex reported higher HDV RNA levels (3.78 [0.70-7.99] vs. 4.69 [2.00-8.19] log IU/ml, p <0.0001), with viremia higher than >0.5 log in 160 (69%). Likewise, HDV RNA levels were higher with AltoStar than with Robogene 2.0 (3.32 [0.70-7.37] vs. 3.91 [0.19-7.54] log IU/ml, p <0.0001), with AltoStar reporting HDV RNA levels >0.5 log in 127 (52%). Although virological response rates (≥2 log decline vs. baseline) at Weeks 24 (Robogene 2.0 vs. EurobioPlex and AltoStar) and 48 (Robogene 2.0 vs. AltoStar) were similar across assays, rates of HDV RNA undetectability significantly differed between the three assays at Weeks 24 and 72 (p = 0.003 and p = 0.02, respectively).ConclusionsHDV RNA levels quantified by EurobioPlex and AltoStar were 1 and 0.5 logs higher than those quantified by Robogene 2.0, respectively. HDV RNA undetectability rates during BLV treatment were assay-dependent.Impact and implicationsManagement and diagnosis of chronic hepatitis delta (CHD) require standardized tests for HDV RNA quantification. Quantification of HDV RNA is significantly influenced by the quantification method, with EurobioPlex detecting approximatively 1 log and AltoStar 0.5 log IU/ml more than Robogene 2.0, respectively. The HDV RNA undetectability rates during BLV monotherapy significantly differed among assays. These findings are of clinical relevance as patients who achieve negative viremia during BLV monotherapy might be entitled to stop therapy successfully.

Project description:AimTo quantify the mitochondrial structure of ECs in intact arteries vs. cultured cells.Methods and resultsCre-stop mito-Dendra2 mice, expressing the fluorescent protein Dendra2 in the mitochondrial matrix only, were used to label EC mitochondria using Cre-recombinase under the control of the VE-cadherin promoter. Conduit arteries, resistance arterioles and veins were fixed, mounted on glass slides and fluorescent images were obtained using a laser scanning confocal microscope (ex 488 nm; em 550 nm). ImageJ was used to calculate form factor (FF) and aspect ratio (AR) of the mitochondrial segments. Mitochondrial fragmentation count (MFC) was calculated by counting non-contiguous mitochondrial particles and dividing by the number of pixels which comprise the mitochondrial network. Primary aortic EC cultures (48 h on culture plates) were generated to compare the mitochondrial structure of cultured ECs vs. intact arteries. Aortic segments were also exposed to high glucose overnight (33 mM) ex vivo, and separate groups of mice were either infused with a high-glucose saline solution (300 mM) via tail vein catheter for 1 h or injected with streptozotocin (STZ; 50 mg/kg) to cause hyperglycaemia. Compared with cultured ECs, the mitochondria of ECs from the intact aorta were more fragmented (MFC: 6.4 ± 2.5 vs. 18.6 ± 9.4, respectively; P < 0.05). The mitochondrial segments of ECs within the aorta were more circular in shape (FF: 3.5 ± 0.75 vs. 1.8 ± 0.30, respectively; P < 0.05) and had less branching (AR: 2.9 ± 0.60 vs. 2.0 ± 0.25, respectively; P < 0.05) compared with cultured ECs. Ex vivo exposure of the intact aorta to high glucose overnight caused mitochondrial fission compared with normal glucose conditions (5 mM; MFC: 25.5 ± 11.1 high glucose vs. 11.0 ± 3.6 normal glucose; P < 0.05). Both 1-h infusion of high glucose saline (MFC: 22.4 ± 4.3) and STZ treatment (MFC: 40.3 ± 14.2) caused mitochondrial fission compared with freshly fixed aortas from control mice (MFC: 18.6 ± 9.4; P < 0.05 vs. high-glucose infusion and STZ treatment).ConclusionsUsing a novel mouse model, we were able to, for the first time, obtain high resolution images of EC mitochondrial structure in intact arteries. We reveal the endothelial mitochondrial network is more fragmented in the intact aorta compared with cultured ECs, indicating that mitochondria assume a more elongated and branched phenotype in cell culture.

Dataset Information

Intact plasma quantification of the large therapeutic lipopeptide bulevirtide.

Publications

Intact plasma quantification of the large therapeutic lipopeptide bulevirtide.

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