Blood Protein Binding Research Articles

Understanding the Liver’s Role in the Clearance of Aβ40

The clearance of peripheral beta amyloid (Aβ) is a potential target for the treatment of Alzheimer’s disease (AD). The liver has been implicated in the elimination of Aβ from the peripheral circulation. Here, the single-pass uptake of Aβ40 in perfused livers from young and old rats (6 to 10 rats per group) was investigated with the multiple indicator dilution technique. Aβ40 had volumes of distribution between those of the vascular marker Evans Blue and the extracellular marker sucrose. The hepatic extraction of Aβ40 was negligible, explained in part by the small permeability surface area products consistent with a high endothelial barrier to liver uptake. There were no substantial effects of age on any of these results. In vitro experiments with isolated hepatocytes and liver sinusoidal endothelial cells showed only very small amounts of Aβ uptake consistent with low intrinsic clearance. These results indicate that the hepatic clearance of Aβ is capacity-limited, explained by the low-permeability surface area products and hepatocyte uptake. However, this does not preclude an effect of aging in longer-term in vivo studies where age-related changes in liver blood flow and protein binding influence liver clearance.

Oral Drug Dosing After Gastric Bypass and Diet-Induced Weight Loss: Simpler Than We Think? Lessons Learned From the COCKTAIL Study.

This article summarizes the lessons learned from the COCKTAIL study: an open, three-armed, single-center study including patients with obesity scheduled for treatment with Roux-en-Y gastric bypass (RYGB) or nonsurgical calorie restriction, and a normal- to overweight control group. The clinical implications of the results from multiple peer-reviewed articles describing the effects of RYGB, severe caloric restriction, weight loss, and type 2 diabetes on the in vivo activity and protein expression of drug-metabolizing enzymes (cytochrome P450 (CYP) 1A2, 2C9, 2C19, and 3A) and transporters (DMETs; organic anion-transporting polypeptide (OATP) 1B1 and P-glycoprotein (P-gp)) are discussed in the perspective of three clinically relevant questions: (1) How should clinicians get the dose right in patients after RYGB? (2) Will drug disposition in patients with obesity be normalized after successful weight loss? (3) Are dose adjustments needed according to obesity and diabetes status? Overall, RYGB seems to have a lower impact on drug disposition than previously assumed, but clinicians should pay close attention to drugs with a narrow therapeutic range or where a high maximum drug concentration may be problematic. Whether obesity-related alterations of DMETs normalize with substantial weight loss depends on the DMET in question. Obesity and diabetes downregulate the in vivo activity of CYP2C19 and CYP3A (only obesity) but whether substrate drugs should be dose adjusted is also dependent on other factors that influence clearance, that is, liver blood flow and protein binding. Finally, we recommend frequent and individualized follow-up due to high inter- and intraindividual variability in these patients, particularly following RYGB.

Gamma globulin binding of hinokiflavone with anti-osteoporosis effects: A mechanistic study

Oxidative stress can trigger apoptosis and associated reduction of osteoblast activity. Hinokiflavone (HNK) with a biflavonoid-based structure might show potent antioxidant activity. However, before the application of this compound as a promising therapeutic bioactive material, its interaction with blood proteins should be investigated to further reveal its pharmacokinetic and pharmacodynamic properties. Therefore, in this paper, the interaction of HNK with γ-globulin, one of the main blood proteins, was assessed. Then, the antioxidant properties of HNK against H2O2-induced osteoblast cytotoxicity in MC3T3-E1 cells were assessed. It was shown that HNK can potentially bind to γ-globulin mostly with the aid of hydrophilic forces, with a slight effect on the protein structure. It was then determined that HNK significantly recovered cell survival and alkaline phosphatase (ALP) activity and collagen-I content in MC3T3-E1 cells exposed to H2O2. In addition, HNK decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) in MC3T3-E1 cells triggered by H2O2. Finally, HNK was shown to control the apoptosis induction in MC3T3-E1 cells triggered by H2O2 mediated by regulation of Bax, Bcl-2 and caspase-3. Taken together, these data demonstrated that HNK with promising blood protein binding properties can show significant protective effects in MC3T3-E1 cells mediated by inhibition of osteoblast dysfunction, oxidative stress, and apoptosis.

Quantitative phosphoproteome analysis reveals differential whey phosphoproteins of bovine milk during lactation

Whey proteins in bovine milk, as the most widely used nutritional components for infant formulae, have been paid more attention. However, the phosphorylation of proteins in bovine whey during lactation has not been thoroughly researched. In this study, a total of 185 phosphorylation sites on 72 phosphoproteins were identified in bovine whey during lactation. 45 differentially expressed whey phosphoproteins (DEWPPs) in colostrum and mature milk were focused on by bioinformatics approaches. Gene Ontology annotation indicated that blood coagulation, extractive space, and protein binding played a key role in bovine milk. The critical pathway of DEWPPs was related to the immune system according to KEGG analysis. Our study investigated the biological functions of whey proteins from a phosphorylation perspective for the first time. The results elucidate and increase our knowledge of differentially phosphorylation sites and phosphoproteins in bovine whey during lactation. Additionally, the data might offer fresh insight into the development of whey protein nutrition.

COMPARATIVE RENAL DISPOSITION OF CREATINE, AND TECHNETIUM DIAGNOSTIC CONTRAST AGENTS IN THE PIGEON (COLUMBA LIVIA).

Clinical assessment of renal function in avian species often involves the measurement of plasma uric acid and blood urea nitrogen, relatively insensitive markers of renal dysfunction and dehydration. In mammals, endogenous creatinine is widely used as an indicator of renal glomerular dysfunction. However, avian species produce primarily creatine. Here, renal creatine, 99mTc99-DTPA (diethylenepentaacetic acid, DTPA) and 99mTc-MAG3 (mercaptoacetyl triglycine, MAG3) renal clearances are characterized in the pigeon avian model by infusing DTPA with inulin and creatine with each tracer and examining the slope of their blood disappearance curves. Clearance curves for inulin and DTPA were parallel, suggesting DTPA is cleared by renal filtration. MAG3 clearance (slope: -2.74 × 105, r2 = 0.97) had a slope almost 10-fold steeper than for DTPA (slope: -6.29 × 104, r2 = 0.90), and orders of magnitude steeper than for creatine (slope: -1.4, r2 = 1.0). These results suggest that DTPA is cleared by glomerular filtration like inulin, while MAG3 is filtered and actively excreted in a manner similar to mammals. In contrast, creatine is filtered and resorbed, has a larger volume of distribution (Vd), or exhibits a greater blood protein binding, making it more complex as a renal marker, when compared with creatinine handling in mammals. The two radiotracers can be readily adapted for use in birds, inviting both qualitative and semiquantitative functional evaluation of avian renal function for research and clinical purposes. The elimination of creatine appears to be more complex requiring further study.

Особенности фармакокинетики антибактериальных антибиотиков при беременности

Представлена современная информация о фармакокинетических особенностях отдельных антибактериальных антибиотиков у беременных, включающая увеличение объема распределения, почечного клиренса и снижение степени связывания с белками крови. Показано, что существует прямая зависимость между антибактериальной активностью препарата и степенью его связывания с белками: чем больше препарата находится в связанном состоянии, тем меньше его антибактериальная активность. Приведены особенности фармакокинетики пенициллинов, цефалоспоринов, аминогликозидов, макролидов у беременных, которые предопределяют необходимость изменения режима дозирования этих препаратов. Рассмотрены аспекты безопасности применения антибиотиков для плода.

The hard protein corona of stealth liposomes is sparse

The protein corona is widely recognized as a key concept in contemporary nanomedicine. In recent years, the interest in the protein corona has reached new heights as a number of reports have suggested that a comprehensive protein corona can form around nanomaterials that previously were thought to be resistant to protein binding. For example, PEGylated stealth liposomes were long thought to be protein repellent, but a number of recent studies have found that a significant protein corona forms around such liposomes in the bloodstream. Prompted by these surprising recent findings, we here present an extensive quantitative study of the binding of blood proteins to standard PEGylated stealth liposomes. To make the study relevant for targeted as well as non-targeted drug delivery systems, the liposomes were prepared both with and without a targeting antibody conjugated to their surface. The prepared liposomes were either incubated in vitro in fetal bovine serum or administered in vivo into the bloodstream of mice. Subsequently, the liposomes were recovered and analyzed using a variety of techniques. There was very little protein binding to the liposomes recovered after in vitro incubation. In contrast, there was more protein binding to the liposomes recovered after in vivo circulation, but a deeper analysis estimated that the bound proteins still only covered a very low fraction of the liposomal surface area. Both the liposomes recovered after in vitro incubation and the liposomes recovered after in vivo circulation completely retained their size and receptor targeting characteristics. Taken collectively, our results thus demonstrate that the hard protein corona of both non-targeted and antibody-targeted stealth liposomes is sparse and does not affect the structure and function of the liposomes.

Discovery of 18F-JK-PSMA-7, a PET Probe for the Detection of Small PSMA-Positive Lesions.

Prostate-specific membrane antigen (PSMA), expressed by most prostate carcinomas (PCa), is a promising target for PCa imaging. The application of PSMA-specific 18F-labeled PET probes such as 18F-DCFPyL and 18F-PSMA-1007 considerably improved the accuracy of PCa tumor detection. However, there remains a need for further improvements in sensitivity and specificity. The aim of this study was the development of highly selective and specific PSMA probes with enhanced imaging properties, in comparison with 18F-DCFPyL, 18F-PSMA-1007, and 68Ga-PSMA-11. Methods: Eight novel 18F-labeled PSMA ligands were prepared. Their cellular uptake in PSMA-positive LNCaP C4-2 and PSMA-negative PC-3 cells was compared with that of 18F-DCFPyL. The most promising candidates were additionally evaluated by small-animal PET in healthy rats using PSMA-positive peripheral ganglia as a model for small PCa lesions. PET images of the ligand with the best outcome, 18F-JK-PSMA-7, were compared with those of 18F-DCFPyL, 18F-PSMA-1007, and 68Ga-PSMA-11 with respect to key image-quality parameters for the time frame 60-120 min. Results: Compared with 18F-DCFPyL, 18F-JK-PSMA-7 demonstrated increased PSMA-specific cellular uptake. Although target-to-background ratios of 18F-DCFPyL and 18F-PSMA-1007 were comparable, this parameter was higher for 18F-JK-PSMA-7 and lower for 68Ga-PSMA-11. Image acutance was significantly higher for 18F-JK-PSMA-7 and 18F-PSMA-1007 than for 18F-DCFPyL and 68Ga-PSMA-11. Image resolution was similar for all 4 tracers. 18F-PSMA-1007 demonstrated significantly higher blood protein binding and bone uptake than the other tracers. Conclusion:18F-JK-PSMA-7 is a promising candidate for high-quality visualization of small PSMA-positive lesions. Excellent preclinical imaging properties justify further preclinical and clinical studies of this tracer.

Nonclinical pharmacokinetics and in vitro metabolism of H3B-6545, a novel selective ERα covalent antagonist (SERCA).

H3B-6545, a novel selective estrogen receptor (ER)α covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα, is in clinical development for the treatment of metastatic breast cancer. Preclinical studies were conducted to characterize the pharmacokinetics and metabolism of H3B-6545 in rat and monkeys. The clearance and metabolic profiles of H3B-6545 were studied using rat, monkey and human hepatocytes, and reaction phenotyping was done using recombinant human cytochrome P450 enzymes. Blood stability, protein binding, and permeability were also determined in vitro. Pharmacokinetics of H3B-6545 was assessed after both intravenous and oral dosing. A nonclinical PBPK model was developed to assess in vitro-in vivo correlation of clearance. H3B-6545 had a terminal elimination half-life of 2.4h in rats and 4.0h in monkeys and showed low to moderate bioavailability, in line with the in vitro permeability assessment. Plasma protein binding was similar across species, at 99.5-99.8%. Nine metabolites of H3B-6545 were identified in hepatocyte incubations, none of which were unique to humans. Formation of glutathione-related conjugate of H3B-6545 was minimal in vitro. H3B-6545, a CYP3A substrate, is expected to be mostly cleared via hepatic phase 1 metabolism. Hepatocyte clearance values were used to adequately model the time-concentration profiles in rat and monkey. We report on the absorption and metabolic fate and disposition of H3B-6545 in rats and dogs and illustrate that in vitro-in vivo correlation of clearance is possible for targeted covalent inhibitors, provided reactivity is not a predominant mechanism of clearance.

Pharmacological characterisation of a tool αvβ1 integrin small molecule RGD-mimetic inhibitor

Compound 8 is a selective αvβ1 small molecule inhibitor that has been used in pre-clinical studies to identify and characterise the αvβ1 integrin as a potential target in fibrotic disease. In this study we further investigated the selectivity and pharmacokinetics of compound 8 to determine a link between the levels of αvβ1 engagement required to achieve in vivo pharmacodynamic efficacy. The selectivity of compound 8 for the arginyl-glycinyl-aspartic acid and β1 integrins was measured using purified integrin protein preparations in radioligand binding studies with both labelled ([3H]compound 8) and unlabelled versions. The pharmacokinetic profile of compound 8 was completed in in vitro blood protein binding assays and in in vivo studies using male C57BL/6 mouse following i.v. dosing. The high selectivity of compound 8 for αvβ1 over the other αv integrins was confirmed, however a reduced selectivity was demonstrated for the β1 integrin family, with high affinity observed for α4β1 (comparable to αvβ1), moderate affinity for α2β1, α3β1 and α8β1, and low affinity for α5β1 and α9β1. Compound 8 was shown to be cleared quickly from the blood with a short half-life of 0.5 h. In conclusion, the data in this study suggest that compound 8 has the potential to engage a number of integrins in vivo beyond αvβ1, that raises a degree of uncertainty regarding its mechanism of action in models of fibrotic disease.

Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: an in vitro and in vivo biochemical, biomechanical, and histological study.

PurposeSpinal interbody fusion cages are designed to provide immediate stabilization for adjoining vertebrae and ideally enable bony ingrowth to achieve successful integration. For such an implant, cells must be able to attach, move, grow, and differentiate on its surface. These cellular interactions are dependent on how the implant surface enables the coating and binding of blood and tissue fluid proteins that support cell adhesion. The purpose of this study was to evaluate the in vitro and in vivo osteoblast cell–implant surface interactions that result in osseointegration onto a surface composed of plasma-sprayed titanium on a polyetheretherketone (PEEK) substrate or titanium-coated PEEK (Ti-PEEK) (PlasmaporeXP®) as compared to uncoated PEEK implants.Materials and methodsThe influence of the Ti-PEEK surface modification on the biochemical, biomechanical, and histological properties at the bone–implant interface is demonstrated both in vitro using simulated bone-forming cell culture experiments and in vivo using a 12- and 24-week ovine implant model.ResultsOsteoblast-like cells attached to the Ti-PEEK surface upregulated early bone-forming activity as measured by an increase in transcription and translation of ALP and BMP-2 when compared to cells on PEEK. Similarly, a significant increase in new bone formation, bony apposition, and pullout strength was demonstrated on Ti-PEEK implants when compared to PEEK implants at 12 and 24 weeks in an ovine implant in vivo model.ConclusionThe study shows that the Ti-PEEK surface demonstrated enhanced osseointegrative properties compared to PEEK both in vitro and in vivo.

Discussions on the hepatic well-stirred model: Re-derivation from the dispersion model and re-analysis of the lidocaine data

Roberts and Rowland explained the well-stirred model as an extreme case of the dispersion model when the dispersion number is infinity. However, the inconsistency within and discrepancy between the theoretical explanation and experimental observations of the well-stirred model are discovered. In this paper, the well-stirred model is examined from both mathematical and physiological points of view. The well-stirred model is re-derived from the convection-elimination equation using the finite difference method. The well-stirred model is a subset of the parallel-tube model when the dispersion number is zero and concentration gradient is ignored. The relative errors and sensitivities of the well-stirred and the parallel-tube models are compared using numerical simulations. The well-stirred model should not be used to estimate in vivo apparent intrinsic clearance from hepatic extraction ratio for high extraction ratio drugs and predicting availability is not recommended for high extraction ratio drugs with either the well-stirred or the parallel-tube models. Based on our theoretical derivation and numerical simulations, we found lidocaine data analysis in Pang and Rowland's 1977 paper was based on an unfair comparison of these two models at two different efficiency number ranges. The high sensitivity of the parallel-tube model at a relatively lower efficiency number range versus the lack of sensitivity of the well-stirred model at an extremely high efficiency number range leads one to believe that the well-stirred model fit the data of high extraction ratio drugs such as lidocaine better. However, the intrinsic clearance values estimated by the parallel-tube model are much more consistent with both in vitro data and those estimated by the dispersion model than those by the well-stirred model. In addition, a key assumption used in the data analysis that the intrinsic clearance is independent of changes in blood flow may not be true. Both theoretical discussions and experimental results indicate that apparent intrinsic clearance and intrinsic clearance could be affected by blood flow and protein binding. Moreover, the lidocaine data analysis in 1977 paper could be control condition dependent and misleading when it is applied to data from other drugs, such as diazepam and diclofenac. The distinction between the well-stirred and parallel-tube models is a result of the mathematical treatment of concentration gradient rather than the flow pattern or extent of physical mixing. The well-stirred model is not likely better than the parallel-tube model. One should be cautious of using the well-stirred model for comparing in vitro intrinsic clearance and estimated in vivo “intrinsic clearance” for high clearance drugs.

Intravenous DOPG liposomes do not augment pH gradient liposome supported peritoneal dialysis in treatment of acute intravenous amitriptyline intoxication in rats

Introduction: Liposomes are nanospheres of lipid bilayer which can bind drug either in the phospholipid membrane or in the central aqueous droplet. Liposomes loaded with a drug can facilitate drug delivery, and empty liposomes can sequester certain drugs to reduce aqueous drug concentrations in experimental intoxication. Therapeutic dialysis in intoxication is limited by drug volumes of distribution and blood protein binding. In rats intoxicated with intravenous amitriptyline, we examined adding intravenous membrane-binding 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) liposomes to liposome supported peritoneal dialysis (LSPD) in which liposomes in dialysate were augmented with an acidic core. The hypothesis was that a “gradient of liposome affinity” would increase LSPD amitriptyline concentrations.Methods: Seven control and five intravenous DOPG liposome pre-treated rats were injected with amitriptyline, followed 10 min later by a 10-min LSPD dwell.Results: DOPG liposomes increased blood amitriptyline concentrations by 50%; control (median [IQR] 1250 [951–1356] nmol/L; intravenous DOPG 1702 [1602–1864] nmol/L, p = 0.032). However, there was no corresponding increase in LSPD dialysate amitriptyline concentrations at the end of the dwell; control (median [IQR] 430 [334–2180] nmol/l, intravenous DOPG 414 [387–483] nmol/L p = 0.75).Conclusions: Pre-treatment with DOPG liposomes decreased amitriptyline volume of distribution but had no significant effect on elimination of amitriptyline by LSPD.

The pharmacokinetics of racemic MDPV and its (R) and (S) enantiomers in female and male rats

BackgroundThese studies investigated the serum pharmacokinetic (PK) profile of racemic (3,4)-methylenedioxypyrovalerone [(R,S)-MDPV)] and its (R)- and (S)-enantiomers in female and male Sprague Dawley rats. MethodsIntravenous (R,S)-MDPV (3 and 5.6mg/kg) and single enantiomer of (R)- and (S)-MDPV (1.5mg/kg) were administered to both sexes for PK studies. Intraperitoneal (ip) bioavailability was determined at 3mg/kg (R,S)-MDPV. Locomotor activity studies were conducted after ip treatment with saline and 0.3-5.6mg/kg of (R,S)-MDPV. ResultsPK values after iv (R,S)-MDPV showed a significant (p<0.05) sex-dependent differences in the volume of distribution at steady state (Vdss) for (R)- and (R,S)-MDPV at both (R,S)-MDPV doses. The female S/R enantiomeric ratios for area under the concentration time curve (AUCinf) and clearance were significantly lower and higher, respectively, than values determined in males. Importantly, there was no evidence of in vivo inversion of (R)-MDPV or (S)-MDPV to its antipode. There were, however, significant sex-dependent differences in volume of distribution after administration of the (R)-enantiomer. Bioavailability studies of ip (R,S)-MDPV showed greater variability and significantly greater bioavailability in male rats. Accordingly, there was a significantly greater maximal distance traveled measurement in male rats at a 3.0mg/kg dose. ConclusionPK sex differences in (R,S)-MDPV and enantiomers were most apparent in volume of distribution, which could be caused by differences in drug blood and tissue protein binding. The increased magnitude and variance in ip bioavailability in male compared to female rats could lead to sex-dependent differences in the pharmacological action caused by active enantiomer (S)-MDPV.

Fabrication of core–shell nanofibers for controlled delivery of bromelain and salvianolic acid B for skin regeneration in wound therapeutics

The physiological and pathological complexity of the wound healing process makes it more challenging to design an ideal tissue regeneration scaffold. Precise scaffolding with high drug loading efficiency, efficient intracellular efficacy for therapeutic delivery, minimal nonspecific cellular and blood protein binding, and maximum biocompatibility forms the basis for an ideal delivery system. This paper describes a combinational multiphasic delivery system, where biomolecules are delivered through the fabrication of coaxial electrospinning of different biocompatible polymers. The ratio and specificity of polymers for specific biofunction are optimized and the delivery system is completely characterized with reference to the mechanical property and structural integrity of bromelain (debridement enzyme) and salvianolic acid B (pro-angiogenesis and re-epithelialization). The in vitro release profile illustrated the sustained release of debriding protease and bioactive component in a timely fashion. The fabricated scaffold showed angiogenic potential through in vitro migration of endothelial cells and increased new capillaries from the existing blood vessel in response to an in ovo chicken chorioallantoic membrane assay. In addition, in vivo studies confirm the efficacy of the fabricated scaffold. Our results therefore open up a new avenue for designing a bioactive combinational multiphasic delivery system to enhance wound healing.

Effects of embedded surfactants on the surface properties of PDMS; applicability for autonomous microfluidic systems

Controlled surface modification of the PDMS (polydimethylsiloxane) was developed and studied in this work to develop autonomous capillary-driven microfluidic system to be applied in bioanalytical devices. The characteristics of the PDMS surfaces were modified by embedding adequate surfactant molecules in the polymer matrix to be moved onto the free surface by diffusion. The change of the surface characteristics was studied considering the expected performance in autonomous biomicrofluidic applications and the influence on non-specific human blood protein binding also. The method was evaluated from technological aspects also, as the integrability of the microfluidic system, considering the previously published results critically. Compositions were defined to be adequate for fabrication autonomous capillary system with enhanced transport efficiency and moderated non-specific protein adsorption.

A case report in cardiovascular magnetic resonance: the contrast agent matters in amyloid

BackgroundCardiac amyloidosis is a progressive but underdiagnosed and underappreciated cause of heart failure. In the last few years, cardiovascular magnetic resonance (CMR) has become the gold standard for non invasive diagnosis of cardiac amyloidosis with the characteristic subendocardial late gadolinium enhancement.Case presentationWe describe a case of a patient who, in the process of aligning protocols for a trial between different centers, had a paired study with two different contrast agents, Dotarem® and MultiHance®. MultiHance® surprisingly failed to demonstrate the characteristic imaging pattern, showing only non specific late gadolinium enhancement at the inferior right ventricular insertion point and different myocardial extracellular volume fraction compared to the one obtained with Dotarem®. MultiHance® is used by many centres, because its partial blood protein binding is a strength for MR angiography, but late gadolinium enhancement, particularly non-ischemic, appears to be compromised.ConclusionsThis case report suggests that contrast agents should be selected with caution, especially with new therapies lining up for amyloid and CMR being used as exploratory end point in clinical trials.

Probing Bio-Nano Interactions between Blood Proteins and Monolayer-Stabilized Graphene Sheets.

Meeting proteins is regarded as the starting event for nanostructures to enter biological systems. Understanding their interactions is thus essential for a newly emerging field, nanomedicine. Chemically converted graphene (CCG) is a wonderful two-dimensional (2D) material for nanomedicine, but its stability in biological environments is limited. Systematic probing on the binding of proteins to CCG is currently lacking. Herein, we report a comprehensive study on the interactions between blood proteins and stabilized CCG (sCCG). CCG nanosheets are functionalized by monolayers of perylene leading to significant improvement in their resistance to electrolyte salts and long-term stability, but retain their core structural characteristics. Five types of model human blood proteins including human fibrinogen, γ-globulin, bovine serum albumin (BSA), insulin, and histone are tested. The main driving forces for blood protein binding involve the π-π interacations between the π-plane of sCCG and surface aromatic amonic acid (sAA) residues of proteins. Several key binding parameters including the binding amount, Hill coefficient, and binding constant are determined. Through a detailed analysis of key controlling factors, we conclude that the protein binding to sCCG is determined mainly by the protein size, the number, and the density of the sAA.

CHARACTERIZATION AND OPTIMIZATION OF NEW CHEMICAL ENTITY ANTI-PROTEIN MISFOLDING AGENTS

Background and Hypothesis: Both beta amyloid and tau produce neurotoxic protein misfolding co-conspirators most often implicated in the molecular causation of AD. Moreover, since the clinical presentation of AD is heterogeneous (e.g. extrapyramidal symptoms in some people with AD and dementia symptoms in some people with PD), the clinical overlap amongst neurodegenerative disorders implies that AD is more of a syndrome than a disease and that the suppression of other aberrantly misfolded protein (e.g. alpha synuclein) may afford additional therapeutic benefits. Accordingly, we are seeking to design and develop brain-penetrable small molecule new chemical entities as anti-protein misfolding agents targeting three proteins: beta amyloid 40 and 42; tau and alpha synuclein. Based upon extensive in silico modeling, a family of novel compounds has been identified. A representative compound is TRV101. Methods: Efficacy of TRV101 was measured in a variety of in vitro assays including Thioflavin T beta amyloid aggregation, Thioflavin S tau aggregation, and biotin-beta amyloid 42 oligomerization. TRV101 was incubated with full length tau and the resultant solution was examined by electron microscopy. Additionally, data was collected on blood brain penetrance, protein binding, and longevity in mouse liver microsomes. Results: TRV101 showed anti-aggregation activity against both beta amyloid and tau. TRV has drug like properties, has high brain penetrance, and is stable in various ADME tests. Conclusion: We have developed a new class of compounds capable of inhibiting aggregation of both beta amyloid and tau proteins. Our small molecules are pharmacokinetically stable and are able to reach the target tissue. Our compounds are currently being tested in animal models of both beta amyloid and tau pathologies. In Vivo PK (Mouse, p.o.)  AUC (brain) = 8692 hr*ng/ mL  T1/2 = 5.4h  Cmax = 1217 ng/mL (brain)  Bioavailability = 78.3 %  B/P = 1 MLM T1/2 > 60 min Clint = 0.008 mL/min/mg HLM T1/2 = 57 min Clint = 0.024 mL/min/mg Physical Chemistry Properties  LogD = 3.15  MW: 446 g/mol  tPSA = 60.6 Protein binding  98.9 % (mouse)  97.6 % (human) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 A b s o rb a n c e ( 4 5 0 n m ) In vitro assays Thioflavin S aggregation assay – Thioflavin S fluoresces when in the presence of aggregated tau. The reaction is initiated by addition of heparin and the reaction is followed by fluorescence until the signal change plateaus. Test compounds are measured by change in fluorescence compared to a vehicle control. TRV101 inhibits tau-441 aggregation with an IC50 of 2.5μM Oligomer formation assay – Biotinylated amyloid-β is allowed to self-associate in the presence of compound, vehicle, and 0.1% Tween20. The reaction is stopped by addition of 0.1% Tween-20 and the sample is applied to a neutravidin ELISA plate. Oligomers are detected by addition of streptavidin-HRP, which will only detect unbound biotin label, i.e., oligomers. TRV101 inhibits biotinylated amyloidβ (1-40) oligomerization with an IC50 of 5μM Animal models APP/PS1 (amyloid-β model), n=13 The mice were dosed at 30mg/kg (p.o., qd) commencing at 6 weeks of age with behavioral testing (2d-RAWM and fear conditioning) at ~3.5 months of age followed by ex vivo LTP analysis performed at the CA3/CA1 synapse in the hippocampus.

English

The binding of anxiolytic drugs to blood proteins is a process brought out by molecular affinity. The process occurs in a reversible form, for that reason such union is regarded as temporal. However, the release or the displacement of the drug from the protein due to the interaction with other drug with the same or greater affinity may lead to serious consequences. The displaced drug does not only produce an increase in the therapeutic but also in the adverse effects by increasing its unbound, free-form concentration in blood. The consequences of the interaction between substances with anxiolytic effects and blood proteins are reviewed herein. This group of drugs generally shows extensive affinity to blood proteins, thus characterizes them as compounds with high risk of presenting adverse clinical effect as a consequence of its interaction with other drugs or to changes in the protein blood protein binding. For this reason, a considerable number of patients abandon their treatment as an example in which risk overwhelms therapeutic benefit. Key words: Anxiolytic, benzodiazepine, interactions, protein binding.