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Physical Chemistry Chemical Physics :... Jun 2016(14)N ultra-wideline (UW), (1)H{(15)N} indirectly-detected HETCOR (idHETCOR) and (15)N dynamic nuclear polarization (DNP) solid-state NMR (SSNMR) experiments, in...
(14)N ultra-wideline (UW), (1)H{(15)N} indirectly-detected HETCOR (idHETCOR) and (15)N dynamic nuclear polarization (DNP) solid-state NMR (SSNMR) experiments, in combination with plane-wave density functional theory (DFT) calculations of (14)N EFG tensors, were utilized to characterize a series of nitrogen-containing active pharmaceutical ingredients (APIs), including HCl salts of scopolamine, alprenolol, isoprenaline, acebutolol, dibucaine, nicardipine, and ranitidine. A case study applying these methods for the differentiation of polymorphs of bupivacaine HCl is also presented. All experiments were conducted upon samples with naturally-abundant nitrogen isotopes. For most of the APIs, it was possible to acquire frequency-stepped UW (14)N SSNMR spectra of stationary samples, which display powder patterns corresponding to pseudo-tetrahedral (i.e., RR'R''NH(+) and RR'NH2(+)) or other (i.e., RNH2 and RNO2) nitrogen environments. Directly-excited (14)N NMR spectra were acquired using the WURST-CPMG pulse sequence, which incorporates WURST (wideband, uniform rate, and smooth truncation) pulses and a CPMG (Carr-Purcell Meiboom-Gill) refocusing protocol. In certain cases, spectra were acquired using (1)H → (14)N broadband cross-polarization, via the BRAIN-CP (broadband adiabatic inversion - cross polarization) pulse sequence. These spectra provide (14)N electric field gradient (EFG) tensor parameters and orientations that are particularly sensitive to variations in local structure and intermolecular hydrogen-bonding interactions. The (1)H{(15)N} idHETCOR spectra, acquired under conditions of fast magic-angle spinning (MAS), used CP transfers to provide (1)H-(15)N chemical shift correlations for all nitrogen environments, except for two sites in acebutolol and nicardipine. One of these two sites (RR'NH2(+) in acebutolol) was successfully detected using the DNP-enhanced (15)N{(1)H} CP/MAS measurement, and one (RNO2 in nicardipine) remained elusive due to the absence of nearby protons. This exploratory study suggests that this combination of techniques has great potential for the characterization of solid APIs and numerous other organic, biological, and inorganic systems.
PubMed: 27314503
DOI: 10.1039/c6cp02855a -
Gene Jan 2015The genetic variants of butyrylcholinesterase (BChE) are the cause of concern in individuals experiencing prolonged apnea on administration of muscle relaxants. In an...
The genetic variants of butyrylcholinesterase (BChE) are the cause of concern in individuals experiencing prolonged apnea on administration of muscle relaxants. In an Indian community called Vysya, a variant (L307P; T920C) was associated with imperceptible plasma BChE. Since BChE has several pharmacological significances in humans, the identification of its variants having altered activity is very important. Previous studies for identifying the mutants in a population were based only on its functional attributes (phenotypic characters) such as esterase activity, dibucaine number and fluoride number. Generally phenotyping method is not considered as the accurate methodology till date though it might be used as a primary screening tool. Molecular biology provides a better technique in identifying these variants. Our aim was to screen this particular community living in South India for the heterozygosity of T920C mutation by phenotypic and genotypic analysis and to find the reliability between the two methods. We analysed 266 individuals for the heterozygosity of T920C. Based on BChE phenotypes, we found that 95% of the individuals are heterozygous. Real-time PCR based genotyping revealed that 96% of individuals are heterozygous. The allele frequency for the mutant allele C was 0.52 which confirmed that the genetic pool of this allele is much higher in Vysya. Also, we observed that genotyping correlates 97% with the phenotype in our study. Further, both phenotype and genotype of age matched other ethnic group do not show any preponderance to this mutation authenticating the vulnerability of Vysyas to this mutation is dominant.
Topics: Adult; Aged; Alleles; Butyrylcholinesterase; Female; Gene Frequency; Genetics, Population; Genotype; Heterozygote; Humans; India; Male; Middle Aged; Mutation; Phenotype; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Young Adult
PubMed: 25447891
DOI: 10.1016/j.gene.2014.11.040 -
Talanta Jul 2016A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect...
A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect four local anesthetics procainamide (PAH), tetracaine (TCH), proparacaine (PCH) and cinchocaine (CIN) simultaneously. An europium (III)-doped prussian blue analogue film (Eu-PB) modified platinum electrode was prepared and applied to improve the detection sensitivity. The parameters including additives, concentration and pH of the running buffer, separation voltage and detection potential that affect CE separation and ECL detection were optimized in detail. The four local anesthetics were baseline separated and detected within 10min under the optimized conditions. The detection limits (LOD) of PAH, TCH, PCH and CIN are 5.5×10(-8), 9.6×10(-8), 2.5×10(-8) and 3.5×10(-8)molL(-1) (S/N=3), respectively. RSDs of the migration time for four analytes range from 1.2% to 2.5% within intraday and from 2.4% to 4.9% in interday, RSDs of the peak area for four analytes are from 1.7% to 3.3% within intraday and from 2.2% to 5.6% in interday, respectively. The limits of quantitation (LOQ) (S/N=10) for PAH, TCH, PCH and CIN in human urine sample are 5.9×10(-7), 9.2×10(-7), 8.3×10(-7) and 5.0×10(-7)molL(-1), separately. The recoveries (n=3) of four analytes in human urine are from 87.6% to 107.7% with less than 5.9% in RSDs. The developed method was used to determine four local anesthetics in human urine samples and investigate the interaction between PAH and human serum albumin (HSA). The number of binding sites and the binding constant of PAH with HSA were calculated to be 1.03 and 2.4×10(4)Lmol(-1), respectively.
Topics: Anesthetics, Local; Electrophoresis, Capillary; Humans; Luminescent Measurements; Procainamide; Serum Albumin, Human
PubMed: 27154684
DOI: 10.1016/j.talanta.2016.03.093 -
Evidence-based Complementary and... 2021Linn. (CQ) is a medicinal plant with good evidence for the treatment of hemorrhoids, listed in the Thai National List of Herbal Products in the oral dosage form. (Wall...
BACKGROUND
Linn. (CQ) is a medicinal plant with good evidence for the treatment of hemorrhoids, listed in the Thai National List of Herbal Products in the oral dosage form. (Wall ex. DC.) R. K. Jansen. (AP) is a medicinal plant with a local anesthetic effect.
OBJECTIVE
To investigate the potential of rectal suppositories containing CQ and AP extracts to alleviate symptoms of hemorrhoids compared with the commercialized rectal suppository containing hydrocortisone and cinchocaine.
MATERIALS AND METHODS
Hemorrhoid outpatients ( = 105) with different severity grades (I, II, or III) from eight hospitals in northern Thailand were included in this study. Hemorrhoid severity was graded by proctoscopy associated with either anal pain or bleeding related to hemorrhoids or both. The patients were randomly allocated to two groups: CQ-AP group ( = 52) or the commercialized rectal suppository group ( = 53). One suppository was rectally administered twice daily in the morning and at bedtime for seven days. Evaluations were performed by physicians on days 1, 4, and 8 of the study. The primary endpoints were bleeding and prolapse size, while the secondary endpoint was anal pain.
RESULTS
Baseline demographics, lifestyle, constipation, number of prolapses, grade of hemorrhoid severity, and duration of experiencing hemorrhoids were comparable in both groups of patients. The effects of CQ-AP and the commercialized rectal suppository on bleeding, prolapse size, and anal pain were comparable. The patients in both groups were satisfied with both products at comparable levels and stated a preference for further use in the case of hemorrhoids recurrence. In terms of safety, the patients in the commercialized rectal suppository group experienced a higher incidence of adverse events, including anal pain and bleeding.
CONCLUSION
Rectal suppositories containing a combined extract of CQ and AP show potential in alleviating hemorrhoidal symptoms with a good safety profile.
PubMed: 34765003
DOI: 10.1155/2021/5605323 -
European Journal of Medicinal Chemistry Sep 2020Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children....
Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC = 1.238 μM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.
Topics: Animals; Antiviral Agents; Dibucaine; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enterovirus A, Human; Enterovirus Infections; Enzyme Inhibitors; Humans; Mice; Mice, Inbred Strains; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; RNA Helicases; Structure-Activity Relationship; Viral Proteins
PubMed: 32619885
DOI: 10.1016/j.ejmech.2020.112310 -
PloS One 2015Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of...
Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.
Topics: Biological Assay; Butyrylcholinesterase; Butyrylthiocholine; Cholinesterase Inhibitors; Humans; Indicator Dilution Techniques; Pesticides
PubMed: 26444431
DOI: 10.1371/journal.pone.0139480 -
Biochemical Pharmacology Dec 2014Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in...
Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a Ki=1.35 mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.
Topics: Aged; Butyrylcholinesterase; Butyrylthiocholine; Cholinesterase Inhibitors; Female; Heterozygote; Humans; Isoquinolines; Male; Mivacurium; Molecular Dynamics Simulation; Neuromuscular Nondepolarizing Agents; Pedigree; Point Mutation; Succinylcholine
PubMed: 25264279
DOI: 10.1016/j.bcp.2014.09.014 -
Clinica Chimica Acta; International... Oct 2015The CALIPER program has previously reported a comprehensive database of pediatric reference intervals for 63 biochemical and immunochemical markers. Here,...
Complex reference value distributions and partitioned reference intervals across the pediatric age range for 14 specialized biochemical markers in the CALIPER cohort of healthy community children and adolescents.
BACKGROUND
The CALIPER program has previously reported a comprehensive database of pediatric reference intervals for 63 biochemical and immunochemical markers. Here, covariate-stratified reference intervals were determined for a number of special assays not previously reported.
METHODS
A total of 1917 healthy children and adolescents were recruited and serum concentrations of 14 biochemical markers were measured using the Abbott Architect ci4100 system. Age and gender partitions were statistically determined, outliers removed and reference intervals calculated using CSLI C28-A3 guidelines.
RESULTS
Many analytes showed dynamic changes in concentration requiring at least 3 age partitions. Unique intervals were required within the first year of life for: pancreatic amylase, C-peptide, ceruloplasmin, insulin, β-2-microglobulin, cystatin C, dehydroepiandrosterone sulfate (DHEA-S), and α-1-glycoprotein. Cholinesterase, cholinesterase-dibucaine number, and immunoglobulin E required only 2 age partitions and α-1-antitrypsin required only one. Anti-CCP and anti-TPO levels were below the detection limit of the assay. Some analytes including insulin and DHEA-S required additional gender partitions for specific age groups.
CONCLUSIONS
Complex profiles were observed for endocrine and special chemistry markers, requiring establishment of age- and gender-specific reference intervals. These updated reference intervals will allow improved laboratory assessment of pediatric patients but should be validated for each analytical platform and local population as recommended by CLSI.
Topics: Adolescent; Aging; Biomarkers; Blood Chemical Analysis; Child; Child, Preschool; Databases, Factual; Female; Growth and Development; Health; Humans; Infant; Infant, Newborn; Male; Reference Values; Residence Characteristics
PubMed: 26310965
DOI: 10.1016/j.cca.2015.08.020 -
Biomedicine & Pharmacotherapy =... May 2017There is a compelling need for prolonged local anesthetic that would be used for analgesia with a single administration. However, due to the low molecular weight of...
PURPOSE
There is a compelling need for prolonged local anesthetic that would be used for analgesia with a single administration. However, due to the low molecular weight of local anesthetics (LA) (lidocaine, bupivacaine, procaine, dibucaine, etc), they present fast systemic absorption.
METHODS
The aim of the present study was to develop and evaluate bupivacaine lipid-polymer hybrid nanoparticles (BVC LPNs), and compared with BVC loaded PLGA nanoparticles (BVC NPs). Their morphology, particle size, zeta potential and drug loading capacity were evaluated. In vitro release study, stability and cytotoxicity were studied. In vivo evaluation of anesthetic effects was performed on animal models.
RESULTS
A facile nanoprecipitation and self-assembly method was optimized to obtain BVC LPNs, composed of PLGA, lecithin and DSPE-PEG, of ∼175nm particle size. Compared to BVC NPs, BVC LPNs exhibited prolonged in vitro release in phosphate-buffered saline (pH=7.4). Further, BVC LPNs displayed enhanced in vitro stability in 10% FBS and lower cytotoxicity (the concentration of BVC ranging from 1.0μM to 20μM). In addition, BVC LPNs exhibited significantly prolonged analgesic duration.
CONCLUSION
These results demonstrate that the LPNs could function as promising drug delivery system for overcoming the drawbacks of poor stability and rapid drug leakage, and prolonging the anesthetic effect with slight toxicity.
Topics: Anesthetics, Local; Animals; Bupivacaine; Cell Survival; Cells, Cultured; Delayed-Action Preparations; Drug Delivery Systems; Drug Stability; Lactic Acid; Lipids; Male; Mice; Mice, Inbred BALB C; Nanoparticles; Pain Measurement; Particle Size; Phosphatidylethanolamines; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Solubility
PubMed: 28267672
DOI: 10.1016/j.biopha.2017.01.175 -
Contact Dermatitis Oct 2019
Topics: Adrenal Cortex Hormones; Anesthetics, Local; Dermatitis, Allergic Contact; Dermatitis, Atopic; Dibucaine; Female; Humans; Middle Aged; Patch Tests; Phenylephrine
PubMed: 30977131
DOI: 10.1111/cod.13290