7 research outputs found
In Vivo Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1 Analyzed by Ligand-Binding Mass Spectrometry Combined with Quantitation by ELISA
The
in vivo biotransformation of a novel fusion protein tetranectin/apolipoprotein
A1 (TN-ApoA1) was investigated by ligand-binding mass spectrometry
(LB-MS) in support of enzyme-linked immunosorbent assays (ELISA).
The main focus was on catabolites formed by proteolysis of the fusion
protein in rabbit following intravenous administration of lipidated
TN-ApoA1. The drug and its catabolites were isolated from rabbit plasma
by immunocapture with a monoclonal antibody (mAb) binding to the fusion
region of TN-ApoA1. The captured drug and catabolites were released
from the streptavidin-coated magnetic beads, separated by monolithic
RP capillary HPLC, and online detected by high-resolution mass spectrometry.
In addition, the same extract was digested with LysN to confirm or
further narrow down the structure of the found catabolites. Two pharmacologically
active catabolites were identified with conserved fusion region. The
major catabolite [3-285] was formed by truncation of AP at the N-terminus
and the minor catabolite [29-270] by truncations of either side of
the TN-ApoA1 sequence. Since the ELISA determined the sum of TN-ApoA1,
along with its two main catabolites, the individual PK profiles of
all three components could be derived by applying their mass peak
composition for each sampling point. Parent drug accounted for 25%
of drug-related material, whereas that of the catabolites [3-285]
and [29-270] accounted for 66% and 9%, respectively. This result could
be obtained without catabolite specific ELISAs or quantitative LC-MS
assays. It was also confirmed that all relevant functional molecules
of TN-ApoA1 in the plasma samples were quantified by the ELISA, which
provided a good relationship for pharmacokinetic/pharmacodynamic evaluations
Pharmacokinetics of serum rituximab in cynomolgus monkeys.
<p>PK analysis of serum rituximab levels after (<b>A</b>) a single SC dose of rituximab preformulated in rHuPH20 administered at 20Â mg/kg (Individual animal samples with mean score shown (n=3)), and (<b>B</b>) 2 Ă 10 mg/kg doses of SC (preformulated in rHuPH20) or standard IV rituximab, given 7 days apart (Individuals animal samples with mean shown (n=4)). </p
CD20 target coverage and B-cell depletion in Peripheral Blood.
<p>PBMC analysis of (<b>A</b>) CD20 target coverage as determined by flow cytometric staining for free surface CD20 levels on (B cells identified as in Figure 2A) and (<b>B</b>) depletion of those PBMC B-cells at baseline and 2, 9, and 14 days after second dose of subcutaneous or intravenous rituximab as determined by a ratio to CD4+/CD3+ T cells (Individuals animal samples with mean shown (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)); and long-term PBMC analysis of (<b>C</b>) percent free surface CD20 target coverage and (<b>D</b>) percent remaining B-cells out to 63 days; normalized to PBS vehicle (group mean ± SD (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)) with ex-vivo rituximab treatment of PBS vehicle sample to show maximal target coverage.</p
CD20 target coverage and B-cell depletion in lymph nodes.
<p>Lymph node analysis of (<b>A</b>) CD20 target coverage as determined by flow cytometric staining for free surface CD20 levels on (B cells identified as in Figure 2A) and (<b>B</b>) depletion of those lymph node B-cellsat baseline and 9 days after second dose of subcutaneous or intravenous rituximab as determined by a ratio to CD4+/CD3+ T cells (Individuals animal samples with mean shown (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)). </p
CD20 target coverage in B-cells and according to CD21+ status.
<p>Paradigms for flow cytometry staining: (<b>A</b>) CD20-independent identification of B cells using fsc/scc lymphocyte gating followed by CD4/CD3 negative gating and CD40 positive CD16 negative gating shows a progressively increasing specific B cell population; (<b>B</b>) Free surface CD20 levels on B cells (as identified in A) with and without rituximab treatment to show target coverage of CD20 compared to CD4+/CD3+ T cells; (<b>C</b>) Identification of CD21+ and CD21- peripheral blood B-cell subsets (within the CD4-/CD3-/CD16-/CD40+ gated B cells as identified in A) showing CD21, CD40 and CD20 levels of the subsets.</p
Depletion of Peripheral Blood CD21+ and CD21- B-cell subsets.
<p>Short and long-term analysis of PBMC B-cell depletion of (<b>A</b>) CD21+ and (<b>B</b>) CD21- B-cell subsets (as identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080533#pone-0080533-g002" target="_blank">Figure 2C</a>). Individuals animal samples with mean shown for baseline, day 2, day 9 and day 14 post second dose (top) and percent remaining as normalized to PBS vehicle treated group (bottom) (group mean ± SD (n=4 rituximab treated groups, n=3 PBS vehicle treated groups).</p
6âAlkoxy-5-aryl-3-pyridinecarboxamides, a New Series of Bioavailable Cannabinoid Receptor Type 1 (CB1) Antagonists Including Peripherally Selective Compounds
We identified 6-alkoxy-5-aryl-3-pyridinecarboxamides
as potent
CB1 receptor antagonists with high selectivity over CB2 receptors.
The series was optimized to reduce lipophilicity compared to rimonabant
to achieve peripherally active molecules with minimal central effects.
Several compounds that showed high plasma exposures in rats were evaluated
in vivo to probe the contribution of central vs peripheral CB1 agonism
to metabolic improvement. Both rimonabant and <b>14g</b>, a
potent brain penetrant CB1 receptor antagonist, significantly reduced
the rate of body weight gain. However, <b>14h</b>, a molecule
with markedly reduced brain exposure, had no significant effect on
body weight. PK studies confirmed similarly high exposure of both <b>14h</b> and <b>14g</b> in the periphery but 10-fold lower
exposure in the brain for <b>14h</b>. On the basis of these
data, which are consistent with reported effects in tissue-specific
CB1 receptor KO mice, we conclude that the metabolic benefits of CB1
receptor antagonists are primarily centrally mediated as originally
believed