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.

Impact of different renal function equations on direct oral anticoagulant concentrations.

The purpose of this study is to investigate the correlation between glomerular filtration rate (GFR) estimated by different renal function equations and non-vitamin K antagonist oral anticoagulant concentration. Atrial fibrillation patients who aged ≥ 20 years and used dabigatran, rivaroxaban, or apixaban for thromboembolism prevention were enrolled to collect blood samples and measure drug concentrations using ultra-high-performance liquid chromatography with tandem mass spectrometry. The GFR was estimated using the Cockroft-Gault formula (abbreviated as creatinine clearance, CrCL), Chronic Kidney Disease Epidemiology Collaboration formula (CKD-EPI) featuring both creatinine and cystatin C, and the Modification of Diet in Renal Disease Study equation (MDRD). Multivariate regression was used to investigate the associations of different renal function estimates with drug concentrations. A total of 511 participants were enrolled, including 146 dabigatran users, 164 rivaroxaban users and 201 apixaban users. Compared to clinical trials, 35.4% of dabigatran, 4.9% of rivaroxaban, and 5.5% of apixaban concentrations were higher than the expected range (p < 0.001). CKD-EPI and MDRD estimates classified fewer patients as having GFR < 50 mL/min than CrCL in all 3 groups. Both CrCL and CKD-EPI were associated with higher-than-expected ranges of dabigatran or rivaroxaban concentrations. Nevertheless, none of the renal function equations was associated with higher-than-expected apixaban concentrations. For participants aged ≥ 75 years, CKD-EPI may be associated with higher-than-expected trough concentration of dabigatran. In conclusion, CrCL and CKD-EPI both can be used to identify patients with high trough concentrations of dabigatran or rivaroxaban. Among elderly patients who used dabigatran, CKD-EPI may be associated with increased drug concentration.

Population Pharmacokinetic Analysis and Dosing Optimization Based on Unbound Daptomycin Concentration and Cystatin C in Nonobese Elderly Patients with Hypoalbuminemia and Chronic Kidney Disease.

PURPOSE: This study evaluated the population pharmacokinetics of daptomycin in nonobese elderly patients with hypoalbuminemia and chronic kidney disease (CKD) using the glomerular filtration rate estimated from cystatin C (eGFRcys) and estimated its optimal dose. METHODS: We performed population pharmacokinetic analysis of the unbound concentrations of daptomycin. The probability of target attainment of 90% for achieving an area under the concentration-time curve of unbound daptomycin at steady state/ minimum inhibitory concentration ratio of ≥66.6 was stochastically simulated. RESULTS: In the population pharmacokinetic analysis of 25 patients aged ≥65 years, the two-compartment model using eGFRcys and age as covariates of clearance in central compartment of unbound daptomycin were optimal. The unbound fraction rate (fu) was 0.05-0.14. According to the Monte Carlo simulation, the optimal doses for patients with eGFRcys of 20-60 mL/min and aged 65-95 years were calculated as 200-500 mg q24h. CONCLUSION: These results suggest that establishing the dose using total concentrations may result in under- or overestimation caused by alterations in fu. The optimal dose for nonobese elderly patients with hypoalbuminemia and CKD depends on eGFRcys and age, and a standard dose may be insufficient for some patients.

[Evaluation of nephrocerebral risk with the use of cystatin C in patients with chronic kidney disease]. / Otsenka nefrotserebral'nogo riska s ispol'zovaniem tsistatina S u bol'nykh khronicheskoi bolezn'iu pochek.

AIM: To study a role of cystatin C in the nephrocerebral risk in chronic kidney disease at the initial stage of the disease. MATERIAL AND METHODS: One hundred and twenty-eight patients (63 men and 65 women) with chronic kidney disease (CKD) were examined at the pre-dialysis stage of the disease. All patients underwent a complex clinical and laboratory examination with determination of the lipid spectrum, uric acid, fibrinogen, calcium and cystatin C, and subsequent calculation of the glomerular filtration rate (GFR). To assess structural changes in carotid arteries, ultrasound dopplerography was performed. Depending on the thickness of the intima-media (TIM), the entire sample is divided into CKD groups with no signs of carotid atherosclerosis (SC), n=70 and on CKD with SC, n=58. RESULTS: Patients of the second group (CKD with SC), had higher body mass index (p<0.05), systolic (p<0.05) and central (p<0.05) arterial pressure (BP) and blood cystatin C (p<0.05). In the same group, there was a significant decrease in the concentration of high-density lipoprotein cholesterol (p<0.05) compared with those of the first group (CKD). The age of patients and the content of cystatin C (p<0.05) influenced the increase in TIM. Significant positive correlations between cystatin C content and TIM, systolic and diastolic blood pressure (p<0.05), and a negative correlation cystatin C content and GFR were noted in patients of the second group. CONCLUSION: The increase in the level of cystatin C in blood plasma in CKD indicates the development of structural changes in the carotid arteries, the increase in the levels of systolic and central arterial pressure, the decrease in the concentration of HDL cholesterol, which is associated with significant inhibition of GFR.

Estimation of Glomerular Filtration Rate in Patients With Cirrhosis by Using New and Conventional Filtration Markers and Dimethylarginines.

BACKGROUND & AIMS: Equations used to estimate glomerular filtration rate (GFR) are not accurate in patients with cirrhosis. We aimed to develop a new equation to estimate the GFR in subjects with cirrhosis and compare its performance with chronic kidney disease epidemiology collaboration (CKD-EPI) cystatin C and creatinine-cystatin C equations, which were derived in populations without cirrhosis. METHODS: From 2010 through 2014, we measured GFR in 103 subjects with cirrhosis based on non-radiolabeled iothalamate plasma clearance. We measured blood levels of creatinine, cystatin C, ß-trace protein, ß2-microglobulin, L-arginine, and symmetric and asymmetric dimethylarginines simultaneously with GFR. Multivariate linear regression analysis was performed to develop models to estimate GFR. Overall accuracy, defined by the root mean square error (RMSE) of our newly developed model to estimate GFR, was compared with that of the CKD-EPI equations. To obtain an unbiased estimate of our new equation to estimate GFR, we used a leave-one-out cross-validation strategy. RESULTS: After we considered all the candidate variables and blood markers of GFR, the most accurate equation we identified to estimate GFR included serum levels of creatinine and cystatin C, as well as patients' age, sex, and race. Overall, the accuracy of this equation (RMSE = 22.92) was superior to that of the CKD-EPI cystatin C equation (RMSE = 27.27, P = .004). Among subjects with cirrhosis and diuretic-refractory ascites, the accuracy of the equation we developed to estimate GFR (RMSE = 19.36) was greater than that of the CKD-EPI cystatin C (RMSE = 27.30, P = .003) and CKD-EPI creatinine-cystatin C equations (RMSE = 23.37, P = .004). CONCLUSIONS: We developed an equation that estimates GFR in subjects with cirrhosis and diuretic-refractory ascites with greater accuracy than the CKD-EPI cystatin C equation or CKD-EPI creatinine-cystatin C equation.

[Evaluation of the renal function in type 2 diabetes: clearance calculation or cystatin C?]. / Évaluation de la fonction rénale chez le diabétique de type 2 : calcul des clairances ou cystatine C ?

Screening for diabetic nephropathy is usually done by albuminuria/24h and the use of creatinine clearance. The objective of this study was to evaluate the renal function in Type 2 diabetes by using different formulas of creatinine clearance and to assess the contribution of cystatin C; 83 adults with type 2 diabetes (23 men and 60 women) and 83 adult controls (40 men and 43 women) were studied. Biochemical parameters were determinated on Coba 6000™ (Roche diagnostics). Diabetics showed a significant increase in blood glucose, cholesterol, triglycerides, LDLc, the ApoB, Lp(a), urea, uric acid, creatinine and cystatin C and lower HDLc. Cystatin was increased in patients with degenerative complications and in hypertensive patients. We found strong correlations of cystatin C with creatinine (r = 0.9454), urea (r = 0.8999) and uric acid (r = 0.8325). We found a significant exponentially increase of creatinine and cystatin C from one stage to another. Cystatin C has a strong association with MDRD (r = 0.8086) and CG (r = 0.7915) and a low one with creatinine clearance (r = 0.1044). In conclusion, the use of cystatin C for screening and early treatment of incipient diabetic nephropathy appears to be adequate. CG and MDRD formulas still hold their place, in regards to the classical determination of creatinine clearance, to monitor patients.

Kinetics of plasmatic cytokines and cystatin C during and after hemodialysis in septic shock-related acute renal failure.

INTRODUCTION: Cystatin C could be a relevant residual glomerular filtration rate marker during hemodialysis (HD), and a high cytokine plasma (p) rate is associated with an increase in mortality during sepsis. To the best of our knowledge, cytokines and cystatin C kinetics during and after HD during sepsis have never been studied. In this study, we described p cytokines and cystatin C variations during and after hemodialysis in septic-shock patients with acute kidney injury (AKI). METHODS: Ten patients, from two tertiary ICUs, with septic shock-related AKI, according to RIFLE class F, were studied. In this prospective observational study, blood samples were collected at the start, after 1 hour, 2 hours, and at the end of HD with a polymethymethacrylate (PMMA) hemodialyzer (D0, D1, D2, and endD), and 30, 60, 90, 120, and 180 min after HD (postD0.5, postD1, postD1.5, postD2, and postD3). We measured p interleukins (IL)-6, IL-8, IL-10, cystatin C, and albumin. Results are expressed as variations from D0 (mean +/- SD). RESULTS: During HD, p[IL-6] did not vary significantly, whereas p[IL-8] and p[IL-10] reductions by D1 were 31.8 +/- 21.2% and 36.3 +/- 26%, respectively (P < 0.05 as compared with D0). At postD3, p[IL-8] and p[IL-10] returned to their initial values. p[Cystatin C] was significantly reduced from D1 to postD1, with a maximal reduction of 30 +/- 6.7% on D2 (P < 0.05). Norepinephrine infusion rate decreased from D0 to postD3 (0.65 +/- 0.39 to 0.49 +/- 0.37 microg/kg/min; P < 0.05). CONCLUSIONS: HD allows a transient and selective decrease in p cytokines, which are known as being correlated with mortality during septic shock. Because of a significant decrease in p cystatin C during HD, this should not be considered as an accurate marker for residual glomerular filtration rate during septic acute renal failure when receiving HD with a PMMA hemodialyzer.

Cystatin C uptake in the eye.

BACKGROUND: As a secreted protein, cystatin C is assumed to play its role in the extracellular compartment, where it can inhibit virtually all cysteine proteases of families C1 (cathepsin B, L, S) and C13 (mammalian legumain-related proteases). Since many of its potential target enzymes in the eye reside in intracellular compartments, we sought evidence for a cellular uptake of the inhibitor in ocular tissues. METHODS: Fluorescence-labeled human cystatin C was injected intravitreally into normal rat eyes. Ocular tissues were subsequently examined using ELISA, fluorescence microscopy, and immunohistochemistry. Cystatin C uptake was additionally studied in an in vitro retina model. RESULTS: Cystatin C administered intravitreally in vivo is taken up into cells of the corneal endothelium and epithelium, the epithelial cells lining the ciliary processes, and into cells in the neuroretina (mostly ganglion cells) and the retinal pigment epithelium. The uptake is demonstrable also in vitro and was, in the neuroretina, found to be a high-affinity system, inhibited by cooling the specimens or by adding the microfilament polymerization inhibitor, cytochalasin D, to the medium. CONCLUSIONS: There is an active, temperature-dependent uptake system for cystatin C into several cell types in the cornea, ciliary body, and retina. The cell types that take up cystatin C are generally the same that contain endogenous cystatin C, suggesting that much or all cystatin C seen intracellularly in the normal eye may have been taken up from the surrounding extracellular space. The uptake indicates that the inhibitor may exert biological functions in intracellular compartments. It is also possible that this uptake system may regulate the extracellular levels of cystatin C in the eye.