Background:
Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is ...the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity. Furthermore, soluble ACE2 variants have been proposed as potential therapeutics for kidney disease and hypertensive disorders.
Objective:
Soluble ACE2 variants conjugated to human Fc domains and selected for high-potency viral SARS-CoV-2 neutralization were prepared and evaluated for ACE2 activity in vitro. Lead candidates were then tested for systemic ACE2 activity, stability, and effects on blood pressure and albuminuria in mice with Ang II-induced hypertension.
Methods:
ACE2 activity of 10 soluble ACE2 variants was first assessed in cell-free conditions using a fluorogenic substrate, or by Ang II hydrolysis to Ang-(1-7). Hypertension was induced in male or female mice by implantation of osmotic minipumps containing Ang II. Two lead ACE2 variants were injected intravenously (i.v.) into hypertensive mice, followed by measurements of blood pressure (tail-cuff plethysmography), albuminuria, and tissue ACE2 activity and protein (immunoblots).
Results:
Soluble ACE2-Fc variants demonstrated significant ACE2 enzymatic activity, with kinetics comparable with human recombinant ACE2. In hypertensive mice, single dose i.v. injection of ACE2-Fc variant K (10 mg/kg) significantly decreased systolic blood pressure at 24 hours, with partial lowering sustained to 48 hours, and tendency to reduce albuminuria at 72 hours. By contrast, ACE2-Fc variant I had no effect on blood pressure or albuminuria in hypertensive mice; ACE2-Fc variant K was detected by immunoblotting in plasma, kidney, heart, lung, liver, and spleen lysates 72 hours after injection, associated with significantly increased ACE2 activity in all tissues except kidney and spleen. Angiotensin-converting enzyme 2-Fc variant I had no effect on plasma ACE2 activity.
Conclusions:
Soluble ACE2-Fc variant K reduces blood pressure and tends to lower albuminuria in hypertensive mice. Furthermore, soluble ACE2-Fc variant K has prolonged tissue retention, associated with increased tissue ACE2 activity. The results support further studies directed at the therapeutic potential of soluble ACE2-Fc variant K for cardiovascular and kidney protection.
Overexpression of Integrin Linked Kinase (ILK) in intestinal and mammary epithelial cells results in a highly invasive phenotype, associated with increased levels of expression of the matrix ...metalloproteinase MMP-9. This increase was at the transcriptional level as determined by MMP-9 promoter-CAT reporter assays. Mutations in the two AP-1 binding sites within the MMP-9 promoter completely inhibited the reporter activity. We have previously shown that ILK inhibits glycogen synthase kinase-3 (GSK-3) activity. Transient transfection of wild-type GSK-3beta in ILK-overexpressing cells decreased MMP-9 promoter activity and AP-1 activity, indicating that ILK can stimulate MMP-9 expression via GSK-3beta and AP-1 transcription factor. A small molecule inhibitor of the ILK kinase reduced the in vitro invasiveness of ILK-overexpressing cells as well as the invasiveness of several human brain tumor cell lines. Furthermore, both MMP-9 promoter and AP-1 activities were inhibited by the ILK inhibitor. Invasiveness of ILK-overexpressing cells was also reduced by inhibition of MMP-9. These data demonstrate that ILK can induce an invasive phenotype via AP-1-dependent upregulation of MMP-9.
Purpose
We have recently demonstrated the brain-delivery of an Amyloid-ß oligomer (Aßo)-binding peptide-therapeutic fused to the BBB-crossing single domain antibody FC5. The bi-functional fusion ...protein, FC5-mFc-ABP (KG207-M) lowered both CSF and brain Aß levels after systemic dosing in transgenic mouse and rat models of Alzheimer’s disease (AD). For development as a human therapeutic, we have humanized and further engineered the fusion protein named KG207-H. The purpose of the present study was to carry out comparative PK/PD studies of KG207-H in wild type rat and beagle dogs (middle-aged and older) to determine comparability of systemic PK and CSF exposure between rodent species and larger animals with more complex brain structure such as dogs.
Method
Beagle dogs were used in this study as they accumulate cerebral Aß with age, as seen in human AD patients, and can serve as a model of sporadic AD. KG207-H (5 to 50 mg/kg) was administered intravenously and serum and CSF samples were serially collected for PK studies and to assess target engagement. KG207-H and Aβ levels were quantified using multiplexed selected reaction monitoring mass spectrometry.
Results
After systemic dosing, KG207-H demonstrated similar serum pharmacokinetics in rats and dogs. KG207-H appeared in the CSF in a time- and dose-dependent manner with similar kinetics, indicating CNS exposure. Further analyses revealed a dose-dependent inverse relationship between CSF KG207-H and Aß levels in both species indicating target engagement.
Conclusion
This study demonstrates translational attributes of BBB-crossing Aβ-targeting biotherapeutic KG207-H in eliciting a pharmacodynamic response, from rodents to larger animal species.
Inhibition of carbonic anhydrase IX in glioblastoma multiforme Amiri, Abdolali; Le, Phuong Uyen; Moquin, Alexandre ...
European journal of pharmaceutics and biopharmaceutics,
December 2016, 2016-Dec, 2016-12-00, 20161201, Letnik:
109
Journal Article
Recenzirano
Odprti dostop
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Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among ...gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.
In vivo biomarker abnormalities provide measures to monitor therapeutic interventions targeting amyloid-β pathology as well as its effects on downstream processes associated with Alzheimer’s disease ...pathophysiology. Here, we applied an in vivo longitudinal study design combined with imaging and cerebrospinal fluid biomarkers, mirroring those used in human clinical trials to assess the efficacy of a novel brain-penetrating anti-amyloid fusion protein treatment in the McGill-R-Thy1-APP transgenic rat model. The bi-functional fusion protein consisted of a blood-brain barrier crossing single domain antibody (FC5) fused to an amyloid-β oligomer-binding peptide (ABP) via Fc fragment of mouse IgG (FC5-mFc2a-ABP). A five-week treatment with FC5-mFc2a-ABP (loading dose of 30 mg/Kg/iv followed by 15 mg/Kg/week/iv for four weeks) substantially reduced brain amyloid-β levels as measured by positron emission tomography and increased the cerebrospinal fluid amyloid-β42/40 ratio. In addition, the 5-week treatment rectified the cerebrospinal fluid neurofilament light chain concentrations, resting-state functional connectivity, and hippocampal atrophy measured using magnetic resonance imaging. Finally, FC5-mFc2a-ABP (referred to as KG207-M) treatment did not induce amyloid-related imaging abnormalities such as microhemorrhage. Together, this study demonstrates the translational values of the designed preclinical studies for the assessment of novel therapies based on the clinical biomarkers providing tangible metrics for designing early-stage clinical trials.
Background
We have developed a blood‐brain barrier (BBB) crossing anti‐amyloid fusion protein KG207 as a potential AD therapeutic. This humanized bi‐functional molecule was generated by fusing an Aß ...oligomer (AßO)‐ binding peptide (ABP) with a BBB carrier FC5 via IgG‐1 Fc fragment. Present study shows that KG207 crosses the BBB in vitro and in vivo (mouse, rat and dog), penetrates target regions of the brain (cortex and hippocampus) and engages parenchymal Aß. KG207 neutralizes AßO‐induced toxicity in vitro and does not stimulate pro‐inflammatory cytokine production in mouse microglia. Studies demonstrated that KG207 was safe up to 300 mg/kg.
Method
Recombinant KG207 was produced in CHO cells. BBB‐permeability was assessed using in vitro BBB (formed by rat or human brain endothelial cells) and in vivo (rat, mouse and dog) models. AßO binding was determined by ELISA. Following iv injection, serum, CSF and brain levels of KG207 and Aß were assessed by nanoLC‐ MRM, ELISA and Western blot methods. Aß toxicity studies were done in human neuroblastoma (SH‐SY5Y) cells and rat primary cortical neuronal cells. Following exposure to KG207, cytokine levels in BV2 microglia were measured using Millipore Luminex assay kit. Safety studies were done in Sprague Dawley rats at 30, 100 and 300 mg/kg.
Result
KG207 retained both Aß‐oligomer binding activity and BBB‐permeability in vitro. When injected iv into rats and mouse, KG207 rapidly appeared in the CSF and brain parenchyma (cortex and hippocampus) indicating active transport of ABP across BBB by FC5 in vivo. In AD transgenic mice, KG207 treatment showed a significant reduction of brain Aß levels. KG207 significantly reduced AßO‐induced toxicity in both human neuroblastoma cells and primary cortical neurons in vitro. KG207 blocked AßO binding to cellular proteins in vitro. KG207 did not activate BV2 mouse microglia and induce pro‐inflammatory cytokines. No adverse effects were seen in rats injected with up to 300 mg/kg, including neurotoxicity.
Conclusion
Collectively, these results indicate that KG207 can effectivey cross the blood‐brain barrier, penetrate the brain and facilitate Aß clearance in vivo. In vitro data suggest that KG207 can safely clear Aß without eliciting pro‐inflammatory cytokine secretion by microglia.
Abstract
Background
We have developed a blood‐brain barrier (BBB) crossing anti‐amyloid fusion protein KG207 as a potential AD therapeutic. This humanized bi‐functional molecule was generated by ...fusing an Aß oligomer (AßO)‐ binding peptide (ABP) with a BBB carrier FC5 via IgG‐1 Fc fragment. Present study shows that KG207 crosses the BBB
in vitro
and
in vivo
(mouse, rat and dog), penetrates target regions of the brain (cortex and hippocampus) and engages parenchymal Aß. KG207 neutralizes AßO‐induced toxicity
in vitro
and does not stimulate pro‐inflammatory cytokine production in mouse microglia. Studies demonstrated that KG207 was safe up to 300 mg/kg.
Method
Recombinant KG207 was produced in CHO cells. BBB‐permeability was assessed using
in vitro
BBB (formed by rat or human brain endothelial cells) and
in vivo
(rat, mouse and dog) models. AßO binding was determined by ELISA. Following iv injection, serum, CSF and brain levels of KG207 and Aß were assessed by nanoLC‐ MRM, ELISA and Western blot methods. Aß toxicity studies were done in human neuroblastoma (SH‐SY5Y) cells and rat primary cortical neuronal cells. Following exposure to KG207, cytokine levels in BV2 microglia were measured using Millipore Luminex assay kit. Safety studies were done in Sprague Dawley rats at 30, 100 and 300 mg/kg.
Result
KG207 retained both Aß‐oligomer binding activity and BBB‐permeability
in vitro
. When injected iv into rats and mouse, KG207 rapidly appeared in the CSF and brain parenchyma (cortex and hippocampus) indicating active transport of ABP across BBB by FC5
in vivo
. In AD transgenic mice, KG207 treatment showed a significant reduction of brain Aß levels. KG207 significantly reduced AßO‐induced toxicity in both human neuroblastoma cells and primary cortical neurons
in vitro
. KG207 blocked AßO binding to cellular proteins
in vitro
. KG207 did not activate BV2 mouse microglia and induce pro‐inflammatory cytokines. No adverse effects were seen in rats injected with up to 300 mg/kg, including neurotoxicity.
Conclusion
Collectively, these results indicate that KG207 can effectivey cross the blood‐brain barrier, penetrate the brain and facilitate Aß clearance
in vivo
.
In vitro
data suggest that KG207 can safely clear Aß without eliciting pro‐inflammatory cytokine secretion by microglia.
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