Human serum albumin fusion protein as therapeutics for targeting amyloid beta in Alzheimer's diseases.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.

Development of a novel human adrenomedullin derivative: human serum albumin-conjugated adrenomedullin.

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.

Entropy-Driven Quick Loading of Functional Proteins in Nanohydrogels for Highly Efficient Tumor Targeting Therapy.

With the gradual deep understanding of the tumorigenesis and development process, nanodrug are thought to have great prospects for individualized treatment of tumors. To deliver adequate concentration of active ingredients to targeted tissues, proteins are usually used as carriers to avoid clearance by the immune system. Herein, a new strategy is developed for preparation of the protein-functionalized targeting nanodrugs; different kinds of proteins (albumin, horseradish, transferrin, and ricin) can be quickly loaded in polyacrylic acid nanohydrogels (PAA-NGs) without discrimination within 1 min under the strong driving force of entropy; and the loading efficiency can reach 99% with about 50% loading content. Meanwhile, the activity of the released protein can be well retained. After oriented binding of the targeting agent on the surface of the nanocarriers by a unique and facile technique, the protein-loaded nanodrug exhibits excellent tumor cell uptake and targeting effect. The excellent targeting ability from the oriented binding is further proved by comparing with the non-oriented targeting system. With quick loading of the anti-tumor protein of ricin and oriented binding of transferrin protein (Tf), the targeting nanodrug (PAA-BB@Ricin/Tf) shows a remarkable anti-tumor effect. This study proves a new universal delivery and targeting strategy for improving the nanodelivery system, which has great potentials for clinical application.

Programmable half-life and anti-tumour effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity.

Fc-less bispecific T-cell engagers have reached the immuno-oncology market but necessitate continual infusion due to rapid clearance from the circulation. This work introduces a programmable serum half-life extension platform based on fusion of human albumin sequences engineered with either null (NB), wild type (WT) or high binding (HB) FcRn affinity combined with a bispecific T-cell engager. We demonstrate in a humanised FcRn/albumin double transgenic mouse model (AlbuMus) the ability to tune half-life based on the albumin sequence fused with a BiTE-like bispecific (anti-EGFR nanobody x anti-CD3 scFv) light T-cell engager (LiTE) construct [(t½ 0.6 h (Fc-less LiTE), t½ 19 hours (Albu-LiTE-NB), t½ 26 hours (Albu-LiTE-WT), t½ 37 hours (Albu-LiTE-HB)]. We show in vitro cognate target engagement, T-cell activation and discrimination in cellular cytotoxicity dependent on EGFR expression levels. Furthermore, greater growth inhibition of EGFR-positive BRAF mutated tumours was measured following a single dose of Albu-LiTE-HB construct compared to the Fc-less LiTE format and a full-length anti-EGFR monoclonal antibody in a new AlbuMus RAG1 knockout model introduced in this work. Programmable half-life extension facilitated by this albumin platform potentially offers long-lasting effects, better patient compliance and a method to tailor pharmacokinetics to maximise therapeutic efficacy and safety of immuno-oncology targeted biologics.

The Uniqueness of Albumin as a Carrier in Nanodrug Delivery.

Albumin is an appealing carrier in nanomedicine because of its unique features. First, it is the most abundant protein in plasma, endowing high biocompatibility, biodegradability, nonimmunogenicity, and safety for its clinical application. Second, albumin chemical structure and conformation allows interaction with many different drugs, potentially protecting them from elimination and metabolism in vivo, thus improving their pharmacokinetic properties. Finally, albumin can interact with receptors overexpressed in many diseased tissues and cells, providing a unique feature for active targeting of the disease site without the addition of specific ligands to the nanocarrier. For this reason, albumin, characterized by an extended serum half-life of around 19 days, has the potential of promoting half-life extension and targeted delivery of drugs. Therefore, this article focuses on the importance of albumin as a nanodrug delivery carrier for hydrophobic drugs, taking advantage of the passive as well as active targeting potential of this nanocarrier. Particular attention is paid to the breakthrough NAB-Technology, with emphasis on the advantages of Nab-Paclitaxel (Abraxane), compared to the solvent-based formulations of Paclitaxel, i.e., CrEL-paclitaxel (Taxol) in a clinical setting. Finally, the role of albumin in carrying anticancer compounds is depicted, with a particular focus on the albumin-based formulations that are currently undergoing clinical trials. The article sheds light on the power of an endogenous substance, such as albumin, as a drug delivery system, signifies the importance of the drug vehicle in drug performance in the biological systems, and highlights the possible future trends in the use of this drug delivery system.

Serum albumin: clinical significance of drug binding and development as drug delivery vehicle.

Human serum albumin, the primary transport and reservoir protein in the human circulatory system, interacts with numerous endogenous and exogenous ligands of varying structural characteristics. The mode of binding of drugs to albumin is central to understanding their pharmacokinetic profiles and has a major influence on their in vivo efficacy. Altered drug binding to albumin due to drug-drug interactions or abnormal physiology may result in marked changes in the active drug concentration, thus affecting its pharmacokinetic and pharmacodynamic properties. The propensity of drug-drug interaction to be clinically significant as well as possible exploitation of such interactions for therapeutic purposes is reviewed. Being the major organs of albumin metabolism, any impairment in the liver and kidney functions frequently alter the level of serum albumin, which affects the pharmacokinetic profiles of drugs and may have serious clinical implications. The natural function of serum albumin as a drug carrier is facilitated by its interaction with various cellular receptors. These receptors not only promote the uptake of drugs into cells but are also responsible for the extraordinarily long circulatory half-life of albumin. This property in combination with the presence of multiple ligand binding pockets have led to the emergence of serum albumin as an attractive vehicle for novel drug delivery systems. Here, we provide an overview of various albumin-based drug delivery strategies, classified according to their methods of drug attachment, and highlight their experimental and clinical successes.

Preparation, radiolabeling with 99mTc and 67Ga and biodistribution studies of albumin nanoparticles covered with polymers. / Preparación, radiomarcaje con 99mTc y 67Ga y estudios de biodistribución de nanopartículas de albúmina con recubrimientos poliméricos.

OBJECTIVE: To optimize radiolabeling with 99mTc and 67Ga of albumin nanoparticles coated with 4 differents synthetic polymers and to evaluate their stability in vivo and in vitro, as well as their biodistribution in vivo after intravenous administration. MATERIAL AND METHODS: The nanoparticles were prepared using albumin and NOTA-modified albumin by the desolvation method and coated with 4 different polymers; HPMC, GMN2, GPM2 and GTM2. They were purified, lyophilized and characterized. Radiolabelling with 99mTc was perfomed with 74 MBq of 99mTc sodium pertechnetate, previously reduced with and acid solution of tin chloride at different concentrations (0.003, 0.005, 0.007, 0.01, 0.05 and 0.1mg/ml) and at different times (5, 10, 15, 30 and 60minutes) and temperatures (room temperature, 40°C and 60°C). Radiolabelling with 67Ga was perfomed by incubation of the nanoparticles with 37 MBq of 67Gallium chloride (obtained from commercial gallium-67 citrate) at different times (10 and 30minutes) and temperatures (room temperature, 30°C and 60°C), and posterior purification with microconcentrators. The radiochemical purity was evaluated by TLC. Stability studies of radiolabeled nanoparticles in physiological serum and blood plasma were perfomed. Biodistribution studies of nanoparticles coated with GPM2 polymer were carried out in Wistar rats after intravenous administration of the nanoparticles. Control animals were carried out with 99mTc sodium pertechnetate and 67Ga chloride. To do so, the animals were killed and activity in organs was measured in a gamma counter. RESULTS: 99mTc labeling was carried out optimally with a tin concentration of 0.007mg/ ml for the GPM2 nanoparticles and 0.005mg / ml for the rest of the formulations, with a radiolabelling time of 10minutes at room temperature. In the case of 67Ga the label was optimized at 30° C temperature and 30minutes of incubation. In both cases the radiochemical purity obtained was greater than 97%. The nanoparticles showed high stability in vitro after 48hours of labeling (70% nanoparticles labeled with 99mTc and 90% those labeled with 67Ga). Biodistribution studies of nanoparticles 99mTc -GPM2 and 67Ga -NOTA-GPM2 showed a high accumulation of activity in the liver at 2 and 24hours after intravenous administration. CONCLUSION: The labeling procedure with 99mTc and 67Ga of albumin and albumin modified with NOTA nanoparticles allows obtaining nanoparticles with high labeling yields and adequate in vitro stability, allowing their use for in vivo studies.

Preparation and investigation of core-shell nanoparticles containing human interferon-α.

Sustained release of active interferon-α (IFN-α) has been achieved from core-shell nanoparticles (NPs) prepared by aqueous precipitation of IFN-α-enriched human serum albumin (HSA-IFN-α) and layer-by-layer (L-b-L) by coating of the IFN-α NPs with poly(sodium-4-styrene) sulphonate (PSS) and chitosan (Chit). The concentration and the pH of HSA solution were optimized during the development of this method. Dynamic light scattering (DLS), zeta-potential, thermal analysis (differential scanning calorimetry (DSC) and termogravimetry (TG)), X-ray diffraction (XRD), IFN-α activity and morphology (transmission electron microscope (TEM)) studies were used to control the preparation and analyse the products. The dissolution kinetics of NPs was measured in vitro over 7 days in Hanson dissolution tester with Millex membrane. In vivo studies in Pannon white rabbit detected steady IFN-α plasma level for 10 days after subcutaneous injection administration of the HSA-IFN-α NPs. The IFN-α plasma concentration was detected by using the enzyme-linked immunosorbent assay (ELISA) method. In the present paper we discuss the preparation method, the optimization steps and the results of in vitro and in vivo release studies. It was established that 76.13% HSA-IFN-α are encapsulated in the core-shell NPs.

Hybrid Protein Nano-Reactors Enable Simultaneous Increments of Tumor Oxygenation and Iodine-131 Delivery for Enhanced Radionuclide Therapy.

It is hard for current radionuclide therapy to render solid tumors desirable therapeutic efficacy owing to insufficient tumor-targeted delivery of radionuclides and severe tumor hypoxia. In this study, a biocompatible hybrid protein nanoreactor composed of human serum albumin (HSA) and catalase (CAT) molecules is constructed via glutaraldehyde-mediated crosslinking. The obtained HSA-CAT nanoreactors (NRs) show retained and well-protected enzyme stability in catalyzing the decomposition of H2 O2 and enable efficient labeling of therapeutic radionuclide iodine-131 (131 I). Then, it is uncovered that such HSA-CAT NRs after being intravenously injected into tumor-bearing mice exhibit efficient passive tumor accumulation as vividly visualized under the fluorescence imaging system and gamma camera. As the result, such HSA-CAT NRs upon tumor accumulation would significantly attenuate tumor hypoxia by decomposing endogenous H2 O2 produced by cancer cells to molecular oxygen, and thereby remarkably improve the therapeutic efficacy of radionuclide 131 I. This study highlights the concise preparation of biocompatible protein nanoreactors with efficient tumor homing and hypoxia attenuation capacities, thus enabling greatly improved tumor radionuclide therapy with promising potential for future clinical translation.

Enhanced dissolution and oral bioavailability of praziquantel by emulsification with human serum albumin followed by spray drying.

The goal of this study was to enhance the oral bioavailability of praziquantel through its conjugation with human serum albumin (HSA). Praziquantel-HSA particles were produced by spray drying an emulsion of an aqueous solution of HSA and a solution of praziquantel in oil. The particles were agglomerates of multiple smooth corrugated particles containing amorphous praziquantel nearly equivalent to the theoretical doses. The solubility of praziquantel in an aqueous medium was enhanced in both the produced particles and the physical mixture. In addition, the dissolution rate in an aqueous medium was enhanced in the case of particles, but not in a physical mixture. Thus, the inclusion of HSA by emulsification followed by spray drying appeared to contribute to the enhanced dissolution rate. In a pharmacokinetic study, the maximum plasma concentration (Cmax) and the area under the concentration-time curve (AUC) for the produced particles (HSA/praziquantel = 1/1 w/w) were approximately two times higher than the corresponding values for raw praziquantel. This increased oral bioavailability of the particles was considered to be due to the enhanced dissolution rate. This process for producing praziquantel-HSA particles could be useful in terms of improving the oral bioavailability of the other hydrophobic drugs.

Development of 99mTc-Labeled Human Serum Albumin with Prolonged Circulation by Chelate-then-Click Approach: A Potential Blood Pool Imaging Agent.

Technetium-99m-labeled human serum albumin (99mTc-HSA) has been utilized as a blood pool imaging agent in the clinic for several decades. However, 99mTc-HSA has a short circulation time, which is a critical shortcoming for a blood pool imaging agent. Herein, we developed a novel 99mTc-labeled HSA with a long circulation time using click chemistry and a chelator, 2,2'-dipicolylamine (DPA), (99mTc-DPA-HSA). Specifically, we examined the feasibility of copper-free strain-promoted alkyne-azide cycloaddition (SPAAC) for the incorporation of HSA to the [99mTc (CO)3(H2O)3]+ system by adopting a chelate-then-click approach. In this strategy, a potent chelate system, azide-functionalized DPA, was first complexed with [99mTc (CO)3(H2O)3]+, followed by the SPAAC click reaction with azadibenzocyclooctyne-functionalized HSA (ADIBO-HSA) under biocompatible conditions. Radiolabeling efficiency of azide-functionalized DPA (99mTc-DPA) was >98%. Click conjugation efficiency of 99mTc-DPA with ADIBO-HSA was between 76 and 99% depending on the number of ADIBO moieties attached to HSA. In whole-body in vivo single photon emission computed tomography images, the blood pool uptakes of 99mTc-DPA-HSA were significantly enhanced compared to those of 99mTc-HSA at 10 min, 2, and 6 h after the injection ( P < 0.001, 0.025, and 0.003, respectively). Furthermore, the blood activities of 99mTc-DPA-HSA were 8 times higher at 30 min and 10 times higher at 3 h after the injection compared to those of conventional 99mTc-HSA in ex vivo biodistribution experiment. The results exhibit the potential of 99mTc-DPA-HSA as a blood pool imaging agent and further illustrate the promise of the pre-labeling SPAAC approach for conjugation of heat-sensitive biological targeting vectors with [99mTc (CO)3(H2O)3]+.

A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 µm for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 µm for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 (t½ = 22.4 h) compared with its WT counterpart (t½ = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.

Evaluation of the Biological Behavior of a Gold Nanocore-Encapsulated Human Serum Albumin Nanoparticle (Au@HSANP) in a CT-26 Tumor/Ascites Mouse Model after Intravenous/Intraperitoneal Administration.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

Pharmacokinetics of protein and peptide conjugates.

Protein and peptide conjugates have become an important component of therapeutic and diagnostic medicine. These conjugates are primarily designed to improve pharmacokinetics (PK) of those therapeutic or imaging agents, which do not possess optimal disposition characteristics. In this review we have summarized preclinical and clinical PK of diverse protein and peptide conjugates, and have showcased how different conjugation approaches are used to obtain the desired PK. We have classified the conjugates into peptide conjugates, non-targeted protein conjugates, and targeted protein conjugates, and have highlighted diagnostic and therapeutic applications of these conjugates. In general, peptide conjugates demonstrate very short half-life and rapid renal elimination, and they are mainly designed to achieve high contrast ratio for imaging agents or to deliver therapeutic agents at sites not reachable by bulky or non-targeted proteins. Conjugates made from non-targeted proteins like albumin are designed to increase the half-life of rapidly eliminating therapeutic or imaging agents, and improve their delivery to tissues like solid tumors and inflamed joints. Targeted protein conjugates are mainly developed from antibodies, antibody derivatives, or endogenous proteins, and they are designed to improve the contrast ratio of imaging agents or therapeutic index of therapeutic agents, by enhancing their delivery to the site-of-action.

The carboxyl-terminal region of human coagulation factor X as a natural linker for fusion strategies.

Fusion with human serum albumin (HSA), which represents a well-established technique to extend half-life of therapeutic proteins, commonly exploits intervening peptide linkers as key components. Here, we explored the human coagulation factor X (FX) carboxyl-terminal region, previously demonstrated by us to be dispensable for secretion and coagulant activity, as a natural linker for fusion purposes. To test our hypothesis, we compared direct FX-HSA fusion with the designed FX-HSA fusion proteins mimicking the recombinant activated factor VII (rFVIIa)-HSA or factor IX (FIX)-HSA chimeras, both strongly dependent from artificial linkers. Three constructs were produced by direct tandem fusion (FX-HSA) and through flexible (glycine/serine; FX-GS-HSA, mimicking rFVIIa-HSA) or cleavable (incorporating the FX activation site; FX-CL-HSA, mimicking FIX-HSA) linkers. The FX-HSA was efficiently secreted and displayed prolonged plasma persistence in mice. All chimeras possessed remarkable pro-coagulant activity, comparable to FX for FX-HSA (88.7 ±â€¯6.0%) and FX-CL-HSA (98.0 ±â€¯16.4%) or reduced for FX-GS-HSA (55.8 ±â€¯5.4%). Upon incubation with activators, FX-HSA and FX-CL-HSA displayed a correct activation profile while the FX-GS-HSA activation was slightly defective. In fluorogenic-based assays, FX-HSA showed normal activity over time and a specific amidolytic activity (1.0 ±â€¯0.12) comparable to that of FX. Overall, the FX-HSA features indicate that the FX carboxyl-terminal region represents an intrinsic sequence allowing direct tandem fusion. Our results provide the first experimental evidence for i) a coagulation factor fusion protein with biological properties independent from artificial linkers, ii) the suitability of FX carboxyl-terminal region as a natural linker for fusion purposes.

Hybrid Nanomaterials of Conjugated Polymers and Albumin for Precise Photothermal Therapy.

Heretofore, conjugated polymers (CPs) attract considerable attention in photothermal therapy (PTT). Although various CPs with different structures have been reported, the suboptimal circulation persistence and biodistribution limit their efficacy in tumor treatment. Human serum albumin (HSA), an endogenous plasma protein, has been widely functioned as a carrier for therapeutic agents. Herein, we construct nanocomplex C16 pBDP@HSA nanoparticles (NPs) from hydrophobic 4,4-difluoro-4-bora-3 a,4 a-diaza- s-indacene (BODIPY)-containing CPs and HSA, which exhibit robust stability in physiological conditions and excellent photothermal activity upon irradiation. The high photothermal conversion efficiency of 37.5%, higher than that of other reported PTT agents such as gold nanorods, phosphorus quantum dots, and 2D materials, results in the potent photocytotoxicity toward cancer cells. Simultaneously, C16 pBDP@HSA NPs' capabilities of near-infrared fluorescence and photoacoustic imaging can provide guidance to the PTT. The outstanding inhibition of tumor growth results from great photothermal activity, the benefited accumulation in tumor, and optimal timing of treatment. To the best of our knowledge, this is the first study which combines the BODIPY-based CPs and HSA in one nanostructure and finds application in cancer treatment. Moreover, this article also offers a new strategy for other insoluble macromolecules to explore more biomedical applications.

An exploratory study of enantioselective behavior of Sol-Gel encapsulated human serum albumin using frontal analysis.

Adsorption behavior of pure enantiomers and racemic mixtures of nonsteroidal anti-inflammatory drugs (ibuprofen and naproxen) on human serum albumin (HSA) was evaluated. The HSA was immobilized by Sol-Gel technique and this biomaterial was used in a chromatographic system where frontal analysis experiments were performed at pH 7.4 and temperatures of 25°C and 37°C. The association constants for enantiomers of the drugs were determined by linear adjustment for data corrected just for dead volume. In uncorrected data for non-specific retention, an inverse ratio between the number of sites and the value of the association constant was found. The participation of non-specific retention was estimated by non-linear regression and the values of association constants (Kass), which were determined considering this information, are comparable to some values reported by other methods at 37°C: 1.4 x105 and 5.7 x104 for Ibuprofen (IBU) R and S, respectively, and 2.3 x105 and 1.8x105 for naproxen (NX) R and S, respectively.

Hemoglobin-albumin cluster: physiological responses after exchange transfusion into rats and blood circulation persistence in dogs.

A core-shell protein cluster comprising hemoglobin and human serum albumins, hemoglobin-albumin cluster (Hb-HSA3), was designed and synthesized for use as an artificial O2 carrier and red blood cell (RBC) substitute. For initial preclinical safety evaluation of the Hb-HSA3 solution, we observed blood compatibility in vitro, physiological responses after exchange transfusion into rats and blood circulation lifetime in dogs. Dilution of human whole blood with Hb-HSA3 showed an appropriate decrease in blood cell number, proportional to the mixing volume ratio. Time courses in the circulation parameters and blood gas parameters after 20% exchange transfusion with Hb-HSA3 in anesthetized rats were almost identical to those observed in a sham group (without infusion) and an HSA group (with HSA administration) for 6 h. Serum biochemical tests of the withdrawn blood indicated safety of the protein cluster. Furthermore, fluorescent Hb-HSA3 was infused into beagle dogs to assess blood retention. Fluorescence measurements of the blood samples enabled us to ascertain the cluster half-life within the intravascular space. Histopathologic inspections of the vital organs imply no abnormality in tissues. All these results indicate sufficient initial preclinical safety of Hb-HSA3 as an alternative material for use in RBC transfusion.

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement.

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method. mNeonGreen or mCherry fusion to the N-terminal of the recombinant human albumin (rHA) variants null-binder (rHA NB), wild-type (rHA WT), high-binder I (rHA HBI), and high-binder II (rHA HBII) did not generally interfere with hFcRn interaction determined by Biolayer Interferometry. Co-localisation of the albumins with endosomal, but not lysosomal, markers was shown by confocal microscopy for high, but not low, hFcRn binders in a human microvascular endothelial hFcRn overexpressing cell line (HMEC-1 FcRn) suggestive of endosomal compartmentalisation. Furthermore, a cellular recycling assay revealed increased recycling of albumin fusions for the high binding variants (mNeonGreen WT; ~1, mNeonGreen HBI; 5.26-fold higher, and mNeonGreen HBII; 5.77-fold higher) in the hFcRn overexpressing cell line. In vivo experiments demonstrated a direct in vitro recycling/in vivo half-life correlation with a longer circulation for the mCherry fusions engineered with high hFcRn affinity that was highest with the HBII variant of 30.1 h compared to 18.2 h for the mCherry WT. This work gives the first direct evidence for an FcRn-driven endosomal cellular recycling pathway for recombinant albumin fusions that correlates with half-life extension controlled by the affinity to hFcRn; promoting a versatile method to tune the pharmacokinetics of albumin fusion-based therapeutics not met by current technologies.

Biodistribution, Pharmacokinetics and Efficacy of 188Re(I)-Tricarbonyl-Labeled Human Serum Albumin Microspheres in an Orthotopic Hepatoma Rat Model.

BACKGROUND/AIM: The biodistribution, pharmacokinetics and therapeutic evaluation of 188Re-human serum albumin microspheres (188Re-HSAM) by labeling with 188Re(I)-tricarbonyl ion (188Re(OH2)3(CO)3)+) were investigated in a GP7TB orthotopic hepatoma rat model. MATERIALS AND METHODS: Male F344 rats received intrahepatic inoculations with GP7TB 1 mm3 cubes. The efficacy of 188Re-HSAM was examined following a single-dose treatment via the intraarterial route. Rats were monitored for survival until death. RESULTS: The labeling efficiency of the 188Re-HSAM was about 80%. After intraarterial administration of 188Re-HSAM, radioactivity in tumors accumulated from 18.41±3.48 %ID/g at 1 h to 12.43±4.70 %ID/g at 24 h. The tumor/liver ratios ranged from 3.03 at 1 h to 1.89 at 72 h. The major uptake organs of 188Re-HSAM were liver (73.35%ID to 48.92%ID), tumor (10.54%ID to 3.51%ID) and kidney (7.48 %ID to 0.14%ID). The T1/2λz of 188Re-HSAM was 259.34 h after intraarterial injection. The AUC(0→96 h) of 188Re-HSAM was 0.69 h*% ID/g. In the efficacy study, the median survival time for the rat (n=6), that received normal saline was 80 d. The median survival times for the mice treated with 10 mCi (n=4), 5.2 mCi (n=6) and 2.9 mCi (n=3) of 188Re-HSAM were 130 d (p=0.003), 106 d (p=0.002) and 83.5 d (p=0.617), respectively. The increase in life span of 10 mCi, 5.2 mCi and 2.9 mCi of 188Re-HSAM were 62.5%, 32.5% and 4.4%, respectively. CONCLUSION: Administration of 188Re-HSAM demonstrated better survival time and therapeutic efficacy at the higher dose in the GP7TB hepatoma model. These results suggested that intraarterial administration of 188Re-HSAM could provide a benefit and promising strategy for delivery of radiotherapeutics in oncology applications.