Pharmacokinetics of single domain antibodies and conjugated nanoparticles using a hybrid near infrared method

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under basal circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents

(Pro)renin receptor-mediated myocardial injury, apoptosis, and inflammatory response in rats with diabetic cardiomyopathy

Excessive activation of the renin-angiotensin system (RAS) in diabetic cardiomyopathy (DCM) provokes a series of structural and functional abnormalities, and causes ventricular remodeling and heart failure in diabetes. (Pro)renin receptor (PRR) is a component of the RAS and has been reported to be up-regulated in some cardiovascular diseases. Furthermore, PRR blockade in some cardiovascular diseases, such as myocardial infarction and hypertension, has been demonstrated to reverse their pathogenesis. However, there have been few studies about the function of PRR in the pathogenesis of DCM. In this study, we hypothesized that PRR is involved in the pathogenesis of DCM and mediates myocardial injury in DCM. To explore the role of PRR in DCM, we evaluated the effects of PRR overexpression and knockdown on the DCM phenotype in vivo and in vitro. The results show that PRR overexpression exacerbates myocardial injury and the inflammatory response in rats with DCM. Conversely, PRR knockdown alleviates myocardial fibrosis, apoptosis, and the inflammatory response, reversing the cardiac dysfunction in rats with DCM. In cell experiments, PRR overexpression also up-regulated the protein expression of collagen I and fibronectin, aggravated the inflammatory response, and increased the production of reactive oxygen species (ROS), while PRR knockdown had the opposite effect. Thus, PRR mediates myocardial injury, apoptosis, and the inflammatory response, likely through a PRR/extracellular signal-regulated kinase/ROS pathway

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under “basal” circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

Abstract Background The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency. Methods A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology. Results The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect. Conclusion The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications

Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration

Abstract Magnetic resonance imaging (MRI) is a widely used non-invasive methodology for both preclinical and clinical studies. However, MRI lacks molecular specificity. Molecular contrast agents for MRI would be highly beneficial for detecting specific pathological lesions and quantitatively evaluating therapeutic efficacy in vivo. In this study, an optimized Magnetization Prepared—RApid Gradient Echo (MP-RAGE) with 2 inversion times called MP2RAGE combined with advanced image co-registration is presented as an effective non-invasive methodology to quantitatively detect T1 MR contrast agents. The optimized MP2RAGE produced high quality in vivo mouse brain T1 (or R1 = 1/T1) map with high spatial resolution, 160 × 160 × 160 µm3 voxel at 9.4 T. Test–retest signal to noise was > 20 for most voxels. Extremely small iron oxide nanoparticles (ESIONPs) having 3 nm core size and 11 nm hydrodynamic radius after polyethylene glycol (PEG) coating were intracranially injected into mouse brain and detected as a proof-of-concept. Two independent MP2RAGE MR scans were performed pre- and post-injection of ESIONPs followed by advanced image co-registration. The comparison of two T1 (or R1) maps after image co-registration provided precise and quantitative assessment of the effects of the injected ESIONPs at each voxel. The proposed MR protocol has potential for future use in the detection of T1 molecular contrast agents