Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Sustained intra-cartilage delivery of interleukin-1 receptor antagonist using cationic peptide and protein-based carriers.

OBJECTIVE: Blocking the interleukin-1 (IL-1) catabolic cascade following joint trauma can be achieved using its receptor antagonist, IL-1Ra. However, its clinical translation for osteoarthritis therapy has been unsuccessful due to its rapid joint clearance and lack of targeting and penetration into deep cartilage layers at therapeutic concentrations. Here, we target the high negative charge of cartilage aggrecan-glycosaminoglycans (GAGs) by attaching cationic carriers to IL-1Ra. IL-1Ra was conjugated to the cartilage targeting glycoprotein, Avidin, and a short length optimally charged cationic peptide carrier (CPC+14). It is hypothesized that electro-diffusive transport and binding properties of IL-1Ra-Avidin and IL-1Ra-CPC+14 will create intra-cartilage depots of IL-1Ra, resulting in long-term suppression of IL-1 catabolism with only a single administration. DESIGN: IL-1Ra was conjugated to Avidin or CPC+14 using site specific maleimide linkers, and confirmed using gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry. Intra-cartilage transport and retention of conjugates was compared with native IL-1Ra. Attenuation of IL-1 catabolic signaling with one-time dose of IL-1Ra-CPC+14 and IL-1Ra-Avidin was assessed over 16 days using IL-1α challenged bovine cartilage and compared with unmodified IL-1Ra. RESULTS: Positively charged IL-1Ra penetrated through the full-thickness of cartilage, creating a drug depot. A single dose of unmodified IL-1Ra was not sufficient to attenuate IL-1-induced cartilage deterioration over 16 days. However, when delivered using Avidin, and to a greater extent CPC+14, IL-1Ra significantly suppressed cytokine induced GAG loss and nitrite release while improving cell metabolism and viability. CONCLUSION: Charge-based cartilage targeting drug delivery systems hold promise as they can enable long-term therapeutic benefit with only a single dose.

The Effect of Avidin on Viability and Proliferation of Colorectal Cancer Cells HT-29.

OBJECTIVE: The aim of this study was to analyze the effect of avidin treatment on cell viability, proliferation and cyclin D1 expression in colorectal cancer cells HT-29. METHODS: Colorectal cancer cell line HT-29 incubated with 50, 100, 150, and 200 µg/mL of avidin concentration during 24, 48, and 72 hours, then the cell viability and proliferation   were analyzed. Each avidin concentration was conducted together with HT-29 cell line without avidin treatment as a control group. The cell viability was measured by MTS assay and the proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay. According to cell viability and proliferation result, we determined the 100 µg/mL avidin concentration for analyzing mRNA and protein of cyclin D1. RESULTS: We demonstrated that the viability and proliferation of HT-29 cells were significantly decreased in all concentration of avidin treatment compared to control.   The cell proliferation showed larger reduction in avidin treatment rather than cell viability. This proves avidin could inhibit proliferation of colorectal cancer cell HT-29 quite well. The expression of cyclin D1, both mRNA and protein, was also significantly decreased after the avidin treatment group compared to control group, it supports the suppression of proliferation result. CONCLUSION: We concluded that avidin treatment could decrease cell viability and proliferation, accompanied by suppression of cyclin D1 expression in colorectal cells HT-29.

Single-Dose Intra-Cartilage Delivery of Kartogenin Using a Cationic Multi-Arm Avidin Nanocarrier Suppresses Cytokine-Induced Osteoarthritis-Related Catabolism.

OBJECTIVE: Kartogenin (KGN) has proven as a both chondrogenic and chondroprotective drug for osteoarthritis (OA) therapy. However, being a small hydrophobic molecule, KGN suffers from rapid joint clearance and inability to penetrate cartilage to reach chondrocytes following intra-articular administration. As such multiple high doses are needed that can lead to off-target effects including stimulation and tissue outgrowth. Here we design charge-based cartilage targeting formulation of KGN by using a multi-arm cationic nano-construct of Avidin (mAv) that can rapidly penetrate into cartilage in high concentrations owing to weak-reversible electrostatic binding interactions with negatively charged aggrecan-glycosaminoglycans (GAGs) and form an extended-release drug depot such that its therapeutic benefit can be reaped in just a single dose. DESIGN: We synthesized 2 novel formulations, one with a releasable ester linker (mAv-OH-KGN, release half-life ~58 h) that enables sustained KGN release over 2 weeks and another with a non-releasable amide linker (mAv-NH-KGN) that relies on mAv's ability to be uptaken and endocytosed by chondrocytes for drug delivery. Their effectiveness in suppressing cytokine-induced catabolism was evaluated in vitro using cartilage explant culture model. RESULTS: A single 100 µM dose of cartilage homing mAv-KGN was significantly more effective in suppressing cytokine-induced GAG loss, cell death, inflammatory response and in rescuing cell metabolism than a single dose of free KGN; multiple doses of free KGN were needed to match this therapeutic response. CONCLUSION: mAv mediated delivery of KGN is promising and can facilitate clinical translation of KGN for OA treatment with only a single dose.

Avidin grafted dextran nanostructure enables a month-long intra-discal retention.

Low back pain is often the direct result of degeneration of the intervertebral disc. A wide range of therapeutics including anti-catabolic, pro-anabolic factors and chemo-attractants that can stimulate resident cells and recruit endogenous progenitors are under consideration. The avascular nature and the dense matrix of this tissue make it challenging for systemically administered drugs to reach their target cells inside the nucleus pulposus (NP), the central gelatinous region of the intervertebral disc (IVD). Therefore, local intra-discal injection of therapeutic drugs directly into the NP is a clinically relevant delivery approach, however, suffers from rapid and wide diffusion outside the injection site resulting in short lived benefits while causing systemic toxicity. NP has a high negative fixed charge density due to the presence of negatively charged aggrecan glycosaminoglycans that provide swelling pressures, compressive stiffness and hydration to the tissue. This negative fixed charge density can also be used for enhancing intra-NP residence time of therapeutic drugs. Here we design positively charged Avidin grafted branched Dextran nanostructures that utilize long-range binding effects of electrostatic interactions to bind with the intra-NP negatively charged groups. The binding is strong enough to enable a month-long retention of cationic nanostructures within the NP following intra-discal administration, yet weak and reversible to allow movement to reach cells dispersed throughout the tissue. The branched carrier has multiple sites for drug conjugation and can reduce the need for multiple injections of high drug doses and minimize associated side-effects, paving the way for effective clinical translation of potential therapeutics for treatment of low back pain and disc degeneration.

Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63.

BACKGROUND: Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin-based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli. METHODS: Fourteen individuals responsive to anti-IgE, nine healthy controls, and five birch pollen-allergic patients, and five nonresponders were studied. Activation experiments included anti-IgE, fMLP, interleukin-(IL)-3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti-CD63, anti-CD203c, and avidin. RESULTS: Stimulation with anti-IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti-IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10-20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti-IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL-3 only upregulates CD203c, while no CD63 or avidin binding is observed. CONCLUSIONS: Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout.

Stable Surface Modification of Mesenchymal Stem Cells Using the Avidin-Biotin Complex Technique.

Mesenchymal stem cells (MSCs) hold promise in cell-based therapies because of their strong tissue repair ability and immunosuppressive effects; however, the therapeutic efficacy of transplanted MSCs is limited due to low survival rates and short-term functioning after transplantation. While the functionalization of MSCs is an ideal way to solve these problems, conventional cell functionalization methods have disadvantages such as cell damage, changes in cellular characteristics, and short-term modification. This unit describes a technique for MSC functionalization by surface modification via the avidin-biotin complex (ABC). This technique provides long-term modification MSC surfaces with biotinylated compounds. This easy method of MSC functionalization will support effective MSC-based therapy. © 2018 by John Wiley & Sons, Inc.

Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation.

The conductance of KATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface KATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.

Long-term drug modification to the surface of mesenchymal stem cells by the avidin-biotin complex method.

Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method). The modification of NanoLuc luciferase (Nluc), a reporter protein, to C3H10T1/2 cells by the ABC method lasted for at least 14 days in vitro without major effects on the cellular characteristics (cell viability, cell proliferation, migration ability, and differentiation ability). Moreover, in vivo, the surface Nluc modification to C3H10T1/2 cells by the ABC method lasted for at least 7 days. Therefore, these results indicate that the ABC method may be useful for long-term surface modification of drugs and for effective MSC-based therapy.

Comparative Study of Osteogenic Activity of Multilayers Made of Synthetic and Biogenic Polyelectrolytes.

Polyelectrolyte multilayer (PEM) coatings on biomaterials are applied to tailor adhesion, growth, and function of cells on biomedical implants. Here, biogenic and synthetic polyelectrolytes (PEL) are used for layer-by-layer assembly to study the osteogenic activity of PEM with human osteosarcoma MG-63 cells in a comparative manner. Formation of PEM is achieved with biogenic PEL fibrinogen (FBG) and poly-l-lysine (PLL) as well as biotinylated chondroitin sulfate (BCS) and avidin (AVI), while poly(allylamine hydrochloride) (PAH) and polystyrene sulfonate (PSS) represent a fully synthetic PEM used as a reference system here. Surface plasmon resonance measurements show highest layer mass for FBG/PLL and similar for PSS/PAH and BCS/AVI systems, while water contact angle and zeta potential measurements indicate larger differences for PSS/PAH and FBG/PLL but not for BCS/AVI multilayers. All PEM systems support cell adhesion and growth and promote osteogenic differentiation as well. However, FBG/PLL layers are superior regarding MG-63 cell adhesion during short-term culture, while the BCS/AVI system increases alkaline phosphatase activity in long-term culture. Particularly, a multilayer system based on affinity interaction like BCS/AVI may be useful for controlled presentation of biotinylated growth factors to promote growth and differentiation of cells for biomedical applications.

Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

Avidin was covalently conjugated to the surface of calcium phosphate nanoparticles, coated with a thin silica shell and terminated by sulfhydryl groups (diameter of the solid core about 50nm), with a bifunctional crosslinker connecting the amino groups of avidin to the sulfhydryl group on the nanoparticle surface. This led to a versatile nanoparticle system where all kinds of biotinylated (bio-)molecules can be easily attached to the surface by the non-covalent avidin-biotin-complex formation. It also permits the attachment of different biomolecules on the same nanoparticle (heteroavidity), creating a modular system for specific applications in medicine and biology. The variability of the binding to the nanoparticle surface of the was demonstrated with various biotinylated molecules, i.e. fluorescent dyes and antibodies. The accessibility of the conjugated avidin was demonstrated by a fluorescence-quenching assay. About 2.6 binding sites for biotin were accessible on each avidin tetramer. Together with a number of about 240 avidin tetramer units per nanoparticle, this offers about 600 binding sites for biotin on each nanoparticle. The uptake of fluorescently labelled avidin-conjugated calcium phosphate nanoparticles by HeLa cells showed the co-localization of fluorescent avidin and fluorescent biotin, indicating the stability of the complex under cell culture conditions. CD11c-antibody functionalized nanoparticles specifically targeted antigen-presenting immune cells (dendritic cells; DCs) in vitro and in vivo (mice) with high efficiency. STATEMENT OF SIGNIFICANCE: Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging.

Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

A luminescent cyclometalated gold(iii)-avidin conjugate with a long-lived emissive excited state that binds to proteins and DNA and possesses anti-proliferation capacity.

Here we describe a luminescent cyclometalated Au(iii)-avidin conjugate that exhibits a 520 nm emission with a lifetime of 1.8 µs in PBS solution in open air. The conjugate stains proteins and DNA and can inhibit cancer cell proliferation.

Observations of the "egg white injury" in ants.

A key determinant of the relationship between diet and longevity is the balance of protein to carbohydrate in the diet. Eating excess protein relative to carbohydrate shortens lifespan in solitary and social insects. Here we explored how lifespan and behavior in ants was affected by the quality of protein ingested and the presence of associated antinutrients (i.e. compounds that interfere with the absorption of nutrients). We tested diets prepared with either egg white protein only or a protein mixture. Egg white contains an anti-nutrient called avidin. Avidin binds to the B vitamin biotin, preventing its absorption. First, we demonstrate that an egg-white diet was twice as deleterious as a protein-mixture diet. Second, we show that ingestion of egg-white diet drastically affected social behavior, triggering elevated levels of aggression within the colony. Lastly, we reveal that by adding biotin to the egg white diet we were able to lessen its detrimental effects. This latest result suggests that ants suffered biotin deficiency when fed the egg white diet. In conclusion, anti-nutrients were known to affect health and performance of animals, but this is the first study showing that anti-nutrients also lead to severe changes in behavior.

Electrostatic interactions enable rapid penetration, enhanced uptake and retention of intra-articular injected avidin in rat knee joints.

Intra-articular (i.a.) drug delivery for local treatment of osteoarthritis remains inadequate due to rapid clearance by the vasculature or lymphatics. Local therapy targeting articular cartilage is further complicated by its dense meshwork of collagen and negatively charged proteoglycans, which can prevent even nano-sized solutes from entering. In a previous in vitro study, we showed that Avidin, due to its size (7 nm diameter) and high positive charge (pI 10.5), penetrated the full thickness of bovine cartilage and was retained for 15 days. With the goal of using Avidin as a nano-carrier for cartilage drug delivery, we investigated its transport properties within rat knee joints. Avidin penetrated the full thickness of articular cartilage within 6 h, with a half-life of 29 h, and stayed inside the joint for 7 days after i.a. injection. The highest concentration of Avidin was found in cartilage, the least in patellar tendon and none in the femoral bone; in contrast, negligible Neutravidin (neutral counterpart of Avidin) was present in cartilage after 24 h. A positive correlation between tissue sGAG content and Avidin uptake (R(2) = 0.83) confirmed the effects of electrostatic interactions. Avidin doses up to at least 1 µM did not affect bovine cartilage explant cell viability, matrix catabolism or biosynthesis.

Effect of avidin-like proteins and biotin modification on mesenchymal stem cell adhesion.

The avidin-biotin system is a highly specific reaction that has been used in a wide range of biomedical applications, including surface modification and cell patterning. We systematically examined a number of avidin derivatives as the basis for a simple and cost effective tissue culture polystyrene substrate surface modification for human stem cell culture. Non-specific adhesion between human mesenchymal stem cells and various avidin derivatives, media conditions, and subsequent biotinylation reactions was quantified. We observed significant non-specific cell adhesion to avidin and strepthavidin, indicating that previous observations using this system may be artifactual. Seeding of cells in serum free media, blocking with bovine serum albumin, and the use of the avidin derivative neutravidin were all necessary for elimination of background adhesion. Neutravidin conjugated with biotinylated bsp-RGD(15) peptide provided the most robust cell adhesion, as well as the greatest increase in cell adhesion over background levels.

Effects of cecal oxytetracycline infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts.

The objective of this 49-d experiment was to test effects of cecal oxytetracycline (OTC) infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts. Twenty-eight crossbred gilts with an initial age of 160 d and BW of 120 kg were surgically fitted with a T-cannula in the terminal ileum, a cecal fistula, and an indwelling catheter in the anterior vena cava, and allotted to 7 dietary treatments. Treatments with the basal semipurified (SP) diet fed at 1.86 kg/d were: SP-1, negative control; SP-2, positive control with 270 µg of biotin/kg; SP-3, with spray-dried egg albumen (EA, 100 g/d) and OTC (2.56 g/d by cecal infusion); and SP-4, with EA, OTC, and 700 µg of biotin/kg. Treatments with the basal corn-soybean meal (CS) diet fed at 1.80 kg/d were: CS-1, negative control; CS-2, with EA and OTC; and CS-3, with EA, OTC, and 700 µg of biotin/kg. Response criteria were: fecal bacteria counts; plasma concentrations of biotin, glucose, and urea N (PUN); liver pyruvate carboxylase (PC) activity; kidney and epithelial tissue histology; ileal and fecal biotin concentrations; ileal and total tract N and energy utilization; daily gilt observation; and BW gain. Blood samples were collected every 7 d with serial samples collected on d 49. Total urine collections and fecal grab samples were made twice daily from d 44 to 49. Gilts were killed on d 50 and liver, kidney, and skin samples were collected. No gilts had symptoms of biotin deficiency. There were no treatment differences in BW gain, plasma glucose concentrations, liver PC activity, kidney and epithelial tissue histology, or fecal bacteria counts. Ileal and total tract N and energy digestibilities (%) did not differ among treatments within the same protein source, with greater (P ≤ 0.05) values for gilts on the SP treatments than the CS treatments. However, N retained/N absorbed and N retained/N intake (%) were less (P ≤ 0.05) and PUN concentrations were greater (P ≤ 0.05) for SP treatments with cecal OTC infusion. The overall fecal biotin concentration mean was 2.6-fold greater than the overall ileal biotin concentration mean. In conclusion, no gilts in the current experiment became biotin deficient because the biotin requirements were met primarily by microbial synthesis and absorption of biotin from the distal small intestine and large intestine, with corn and soybean meal contributing endogenous biotin. Therefore, supplementation of diets for gilts entering the breeding herd with 100% of the current NRC biotin requirement for sows is adequate.

Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa.

EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (k(on) > 10(7)M(-1)s(-1)), a high affinity (K(I) = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (K(D) = 30nM) and activated the serpin to rapidly (k(ass) ∼ 10(6)M(-1)s(-1)) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.

18F -PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, 18F -labelling and an in-vitro assessment of its binding with Neutravidin™-trastuzumab pre-treated cells.

UNLABELLED: In terms of nuclear decay 18F is the most ideal PET nuclide but its short t(½) precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as Neutravidin™-biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with Neutravidin™-conjugated antibodies. METHODS: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with 18F in acidified aqueous solution. Cells expressing high (SKBr3), medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated with Neutravidin™-conjugated trastuzumab, washed, and then incubated with 18F -PEG-biotin. RESULTS: The 18F -fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel 18F -PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with Neutravidin™-conjugated trastuzumab. CONCLUSION: Biotin can be functionalised with boroaryl and readily 18F -radiolabelled in aqueous solution and will bind to cells pre-incubated with Neutravidin™-antibody conjugates.