Kinetics of Blood-Brain Barrier Transport of Monoclonal Antibodies Targeting the Insulin Receptor and the Transferrin Receptor
Biologic drugs are large molecule pharmaceuticals that do not cross the blood-brain barrier (BBB), which is formed by the brain capillary endothelium. Biologics can be re-engineered for BBB transport as IgG fusion proteins, where the IgG domain is a monoclonal antibody (MAb) that targets an endogenous BBB transporter, such as the insulin receptor (IR) or transferrin receptor (TfR). The IR and TfR at the BBB transport the receptor-specific MAb in parallel with the transport of the endogenous ligand, insulin or transferrin.
The kinetics of BBB transport of insulin or transferrin, or an IRMAb or TfRMAb, can be quantified with separate mathematical models. Mathematical models to estimate the half-time of receptor endocytosis, MAb or ligand exocytosis into brain extracellular space, or receptor recycling back to the endothelial luminal membrane were fit to the brain uptake of a TfRMAb or a IRMAb fusion protein in the Rhesus monkey.
Model fits to the data also allow for estimates of the rates of association of the MAb in plasma with the IR or TfR that is embedded within the endothelial luminal membrane in vivo. The parameters generated from the model fits can be used to estimate the brain concentration profile of the MAb over time, and this brain exposure is shown to be a function of the rate of clearance of the antibody fusion protein from the plasma compartment.
Keywords: blood–brain barrier; brain drug delivery; insulin receptor; mathematical model; monoclonal antibody; transferrin receptor.
Canine IVM With SOF Medium, Insulin–Transferrin-Selenium, and Low O 2 Tension Improves Oocyte Meiotic Competence and Decreases Reactive Oxygen Species Levels
Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48-72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA-2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS.
Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 μl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups.
Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.
Keywords: SOF medium; canine; in vitro maturation; insulin-transferrin-selenium; oxygen tension.
Mathematical Models of Blood-Brain Barrier Transport of Monoclonal Antibodies Targeting the Transferrin Receptor and the Insulin Receptor
We develop and analyze mathematical models for receptor-mediated transcytosis of monoclonal antibodies (MAb) targeting the transferrin receptor (TfR) or the insulin receptor (IR), which are expressed at the blood-brain barrier (BBB). The mass-action kinetic model for both the TfR and IR antibodies were solved numerically to generate predictions for the concentrations of all species in all compartments considered. Using these models, we estimated the rates of MAb endocytosis into brain capillary endothelium, which forms the BBB in vivo, the rates of MAb exocytosis from the intra-endothelial compartment into brain extracellular space, and the rates of receptor recycling from the endothelial space back to the luminal endothelial plasma membrane.
Our analysis highlights the optimal rates of MAb association with the targeted receptor. An important role of the endogenous ligand, transferrin (Tf) or insulin, in receptor-mediated-transport (RMT) of the associated MAb was found and was attributed to the five order magnitude difference between plasma concentrations of Tf (25,000 nM) and insulin (0.3 nM). Our modeling shows that the very high plasma concentration of Tf leads to only 5% of the endothelial TfR expressed on the luminal endothelial membrane.
Keywords: biologics; blood-brain barrier; brain drug delivery; insulin receptor; monoclonal antibody; transferrin receptor.
Supplementation of insulin–transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro.
Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations.
After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number.
10mL ITS Insulin-Transferrin-Se |
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| 25-800-CR | Scientific Laboratory Supplies | EACH | 54.15 EUR |
Insulin-Transferrin-Selenium ITS, Insulin-Transferrin-Selenium Supplement (100X), in Earle's Balanced Salts. |
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| ITL03-10ML | Caisson Labs | 10 ml | 134.4 EUR |
Insulin-Transferrin-Selenium-Ethanolamine ITSE, Insulin-Transferrin-Selenium-Ethanolamine Supplement (100X), in Earle's Balanced Salts. |
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| ITL02-10ML | Caisson Labs | 10 ml | 135.6 EUR |
Insulin-Transferrin-Selenium-Pyruvate ITSP, Insulin-Transferrin-Selenium-Pyruvate Supplement (100X), in Earle's Balanced Salts. |
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| ITL04-10ML | Caisson Labs | 10 ml | 134.4 EUR |
Insulin-Transferrin-Selenium Plus (100X), Animal Free |
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| CA202-010 | GenDepot | 10ml | 141.6 EUR |
Insulin-Transferrin-Selenium Max (100X), Animal Free |
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| CA203-010 | GenDepot | 10ml | 141.6 EUR |
Insulin-Transferrin-Selenium Solution I (1000X), Animal Free |
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| CA200-005 | GenDepot | 5x1ml | 235.2 EUR |
Insulin-Transferrin-Selenium Solution II (100X), Animal Free |
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| CA201-010 | GenDepot | 10ml | 141.6 EUR |
Insulin-Transferrin-Selenium-Pyruvate ITSP, Insulin-Transferrin-Selenium-Pyruvate Supplement (100X), in Earle's Balanced Salts. W/O calcium and magnesium. |
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| ITL01-10ML | Caisson Labs | 10 ml | 134.4 EUR |
Transferrin |
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| PR27219 | Neuromics | 100 mg | 295.2 EUR |
Transferrin Antibody |
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| DF13383 | Affbiotech | 100ul | 420 EUR |
Transferrin antibody |
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| 70R-7559 | Fitzgerald | 1 mg | 386.4 EUR |
Transferrin antibody |
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| 70C-CR6072GAP | Fitzgerald | 1 mg | 249.6 EUR |
Transferrin antibody |
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| 70R-10681 | Fitzgerald | 1 ml | 447.6 EUR |
Transferrin antibody |
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| 70R-15371 | Fitzgerald | 100 ug | 392.4 EUR |
Transferrin antibody |
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| 70R-15486 | Fitzgerald | 100 ug | 392.4 EUR |
transferrin fragment |
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| A1093-1 | ApexBio | 1 mg | 129.6 EUR |
transferrin fragment |
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| A1093-10 | ApexBio | 10 mg | 408 EUR |
transferrin fragment |
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| A1093-25 | ApexBio | 25 mg | 547.2 EUR |
transferrin fragment |
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| A1093-5 | ApexBio | 5 mg | 268.8 EUR |
Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.