► First evidence on enantioselective binding of propanocaine to human serum albumin. ► Approximate in vivo conditions allows in vitro data unapproachable from in vivo assays. ► Optimised experimental ...design reduces methodological errors. ► Direct equations enable statistical advantages and robust results. ► Data from two days and two processed fractions provide reliable uncertainty.
Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO–HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers’ identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, logKE1=3.20±0.16 and log KE2=3.40±0.14, medium protein binding percentage, PB=48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES=KE2/KE1=1.5±0.3.
This paper points out the usefulness of biopartitioning micellar chromatography (BMC) using capillary columns as a high-throughput primary screening tool providing key information about the oral ...absorption, skin permeability, and brain–blood distribution coefficients of 15 polyphenols (6 flavones, 2 flavonols, a flavanone, 2 flavan-3-ols, 3 phenolic acids, and a phloroglucinol) in a simple and economical way. For the compounds studied, except vicenin-2, rutin, chlorogenic acid, p-hydroxycinnamic acid, and 4-hydroxybenzoic acid, maximal oral absorption (>90%) can be expected, if there are not solubility problems or metabolic processes. On the other hand, the most retained compounds in BMC, that is, 5-hydroxyflavone, flavone, and flavanone, show the highest brain–blood distribution coefficients and skin permeability coefficients.
The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC‐partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with ...HSA by CE‐frontal analysis was performed. The binding parameters obtained for the first site of interaction were n1 = 0.84; K1 = 9.7 ± 0.3×103 M–1 and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 ± 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 μM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2–8×10–4 M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30°C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.
The study aims to identify the relevance of immunohistochemistry (IHC), copy number aberrations (CNA) and epigenetic disorders in BRCAness breast cancers (BCs). We studied 95 paraffin included BCs, ...of which 41 carried BRCA1/BRCA2 germline mutations and 54 were non hereditary (BRCAX/Sporadic). Samples were assessed for BRCA1ness and CNAs by Multiplex Ligation-dependent Probe Amplification (MLPA); promoter methylation (PM) was assessed by methylation-specific-MLPA and the expression of miR-4417, miR-423-3p, miR-590-5p and miR-187-3p by quantitative RT-PCR. IHC markers Ki67, ER, PR, HER2, CK5/6, EGFR and CK18 were detected with specific primary antibodies (DAKO, Denmark). BRCAness association with covariates was performed using multivariate binary logistic regression (stepwise backwards Wald option).
BRCA1/2
mutational status (
p
= 0.027), large tumor size (
p
= 0.041) and advanced histological grade (
p
= 0.017) among clinic-pathological variables; ER (
p
< 0.001) among IHC markers;
MYC
(
p
< 0.001) among CNA;
APC
(
p
= 0.065),
ATM
(
p
= 0.014) and
RASSF1
(
p
= 0.044) among PM; and miR-590-5p (
p
= 0.001), miR-4417 (
p
= 0.019) and miR-423 (
p
= 0.013) among microRNA expression, were the selected parameters significantly related with the BRCAness status. The logistic regression performed with all these parameters selected ER+ as linked with the lack of BRCAness (
p
= 0.001) and
MYC
CNA,
APC
PM and miR-590-5p expression with BRCAness (
p
= 0.014, 0.045 and 0.007, respectively). In conclusion, the parameters ER expression,
APC
PM,
MYC
CNA and miR-590-5p expression, allowed detection of most BRCAness BCs. The identification of BRCAness can help establish a personalized medicine addressed to predict the response to specific treatments.
The retention of compounds in micellar liquid chromatography (MLC) is governed by hydrophobic and electrostatic forces. For ionic compounds, both interactions should be considered. The present report ...offers a novel retention model that includes the hydrophobicity of compounds and the molar fraction of the charged form of compounds and compares it with other previously reported models. High correlations between the logarithm of capacity factors and these structural parameters were obtained for local anesthetics with different degrees of ionization using a nonionic surfactant solution as mobile phase. Modeling the retention of compounds as a function of physicochemical parameters and experimental variables is established by means of multiple linear regression. In addition, a predictive model for estimating the hydrophobicity of local anesthetics is proposed. Finally, quantitative and qualitative retention−activity relationships in MLC are also investigated for these compounds. An excellent correlation between the capacity factors in MLC and the anesthetic potency of local anesthetics was obtained.
Soil-sorption coefficient (
K
OC) assessment is essential before placing a new chemical substance on the market.
Since experimental
K
OC measurements are tedious, time-consuming and costly, ...alternative approaches to estimating
K
OC from other descriptors have been proposed. We review the use of chromatographic retention (log
k) values, obtained in different types of chromatographic systems, as descriptors to establish predictive log
K
OC-log
k models.
We highlighted critical aspects of such models and performed a comparative study using data in the literature. We compare two strategies to deal with the large variability of experimental log
K
OC data. We contrast the results of this study with the main conclusions reported in the literature.
The retention factor is one of the most universally used parameters in chromatography. The errors associated with the conventional ways to determine the retention factor of compounds in liquid ...chromatography are studied and compared with those corresponding to new approaches. The later avoid the use of extra-column time and hold-up time values, which have proven to be tedious and ambiguous. Simulations and real data, used to examine the accuracy of four different approaches (two classic and two new), suggest that the new approaches could be considered more satisfactory than the classic ones.
Hepatitis C virus (HCV)/human immunodefficiency virus (HIV) coinfection is a major health problem, affecting mostly to individuals with exposure to blood products, as hemophiliacs or intravenous drug ...users, or those exposed to high-risk sexual practices. Genotyping of interleukin 28B (IL-28B) rs12979860 polymorphism is a useful tool for guiding therapeutic decisions in this disease. On the contrary, there is not enough information on the pathogenic role of this polymorphism in the natural history of the disease. The objective of this study is to describe the relationships between the CT/TT genotype of this polymorphism with viral loads and also with a number of biomarkers of liver function in coinfected patients naïve for treatment for HCV. Seventy-five HCV/HIV coinfected patients were retrospectively recruited in our Hospital from 2010 to 2011. Logistic regression analysis adjusting by Age, Sex, HCV viral genotype, HCV viral load, HIV viral load, and CD4 T cells levels revealed the IL-28B rs12979860 (CT/TT) genotype as a protective factor against alanine aminotransferase (ALT) levels (>100 IU/L), aspartate aminotransferase (AST) levels (>75 IU/L), and AST-to-platelet ratio index (APRI) score for liver fibrosis (>1.5) OR, (95% CI), p: ALT 0.026 (0.001-0.576) 0.021; AST 0.001 (0.000-0.297) 0.019; APRI 0.031 (0.002-0.41) 0.008. Stepwise regression analysis considering the same adjusting variables showed the same results. In consequence, the IL-28B rs12979860 (CT/TT) genotype, which is a marker of poor response to HCV treatment, could be mediating on the contrary a certain protective effect against the hepatic damage caused by this virus in patients coinfected by HIV.
