Correlated cholinomimetic-stimulated beta-endorphin and prolactin release in humans

Previous studies, both in animals and humans, have demonstrated that the intravenous or intraventricular administration of endogenous opioids and opiates produce dose dependent increases in plasma concentrations of prolactin. Notably, in humans, intravenous infusion of centrally active cholinomimetic drugs, such as physostigmine or arecoline, may produce significant increases in plasma concentrations of prolactin and beta-endorphin immunoreactivity. In three separate studies, conducted collaboratively between the National Institute of Mental Health and the University of California at San Diego, physostigmine and arecoline associated increases in plasma concentrations of beta-endorphin immunoreactivity were highly correlated with increases in plasma prolactin concentrations. These results are of interest because centrally active cholinomimetic drugs have been variously reported either to have no effect, to increase, or to inhibit anterior pituitary prolactin release. We propose that cholinergic stimulation of hypothalamic beta-endorphin may represent an interesting example of peptidergic modulation of primary neurochemical effects on hypothalamic-pituitary hormonal regulation.     

Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.     

CSF and Blood Oxytocin Concentration Changes following Intranasal Delivery in Macaque

Oxytocin (OT) in the central nervous system (CNS) influences social cognition and behavior, making it a candidate for treating clinical disorders such as schizophrenia and autism. Intranasal administration has been proposed as a possible route of delivery to the CNS for molecules like OT. While intranasal administration of OT influences social cognition and behavior, it is not well established whether this is an effective means for delivering OT to CNS targets. We administered OT or its vehicle (saline) to 15 primates (Macaca mulatta), using either intranasal spray or a nebulizer, and measured OT concentration changes in the cerebral spinal fluid (CSF) and in blood. All subjects received both delivery methods and both drug conditions. Baseline samples of blood and CSF were taken immediately before drug administration. Blood was collected every 10 minutes after administration for 40 minutes and CSF was collected once post-delivery, at the 40 minutes time point. We found that intranasal administration of exogenous OT increased concentrations in both CSF and plasma compared to saline. Both delivery methods resulted in similar elevations of OT concentration in CSF, while the changes in plasma OT concentration were greater after nasal spray compared to nebulizer. In conclusion our study provides evidence that both nebulizer and nasal spray OT administration can elevate CSF OT levels.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.     

Accumulation of spectrin and calpain-cleaved spectrin breakdown products in CSF after traumatic brain injury in rats

The introduction of acetylcholine esterase inhibitors for symptomatic treatment of Alzheimer's disease, and the promise of drugs that may delay disease progression, has created a great need for reliable diagnostic tools. However, current criteria for the clinical diagnosis of AD are largely based on the exclusion of other dementia disorders and disease markers are lacking. Since biochemical changes in the brain are reflected in the cerebrospinal fluid (CSF), the search for diagnostic tools for AD has been directed toward CSF markers. CSF markers for AD should reflect the central pathogenic processes of the disorder, i.e. the mismetabolism of β-amyloid (Aβ) and the hyperphosphorylation of tau. Several studies have found that the CSF level of Aβ42 is decreased, and the CSF levels of total tau and phosphorylated tau are increased in AD as compared with normal controls. Thus, the sensitivity of these changes in AD is high. But changes in CSF-Ab42 and CSF-tau have been found in other neurodegenerative disorders and therefore, the specificity seems to be moderately high. Other potential markers that may increase the clinical diagnostic accuracy include the CSF/serum albumin ratio (for identification of blood–brain barrier damage related to disturbances in the small intracerebral vessels), CSF-sulfatide (for identification of ongoing demyelination related to white matter changes and CSF-neurofilament light protein (NFL) [for identification of ongoing axonal (tau and NFL) degeneration]. Use of the summarized information from analyses of several CSF biochemical markers, from the clinical examination, and from brain imaging (SPECT, CT/MRI) may increase the accuracy of the clinical diagnosis.     

Towards a high resolution separation of human cerebrospinal fluid

Human cerebrospinal fluid is an ultrafiltrate of plasma that is largely produced by the choroid plexus. It consists of a mixture of anorganic salts, various sugars, lipids and proteins from the surrounding brain tissues. The predominant proteins in cerebrospinal fluid are isoforms of serum albumin, transferrin and immunoglobulins, representing more than 70% of the total protein amount. A rough overview of the protein compounds of human cerebrospinal fluid including their respective concentrations is given by Blennow et al. [Eur. Neurol. 33 (1993) 129]. In contrast, the aim of this work is to display the detailed protein composition of CSF by two-dimensional gel electrophoresis and to identify both high and low concentrated proteins using different mass spectrometry techniques. This extensive overview of proteins in human cerebrospinal fluid will be highly relevant for clinical research. Furthermore, the comparison of 2D gels will help to analyze the standard protein variability in CSF of healthy persons and detect specific protein variations of patients with various neurological diseases (e.g., Alzheimer's disease, Huntington's chorea). Sample preparation for two-dimensional gel electrophoresis must include concentration and desalting steps such as precipitation and ultrafiltration due to the high amount of salts, sugars and lipids and the low total amount of protein of 0.3-0.7 microg/microl present in human CSF. Up to now we were able to identify more than 480 spots from suchlike generated 2D gels using MALDI- and ESI-mass spectrometry.     

Comparative proteomic analysis of intra- and interindividual variation in human cerebrospinal fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.     

Genes and Pathways Co-associated with the Exposure to Multiple Drugs of Abuse,Including Alcohol,Amphetamine/Methamphetamine,Cocaine, Marijuana,Morphine, and/or Nicotine: a Review of Proteomics Analyses

Drug addiction is a chronic neuronal disease. In recent years, proteomics technology has been widely used to assess the protein expression in the brain tissues of both animals and humans exposed to addictive drugs. Through this approach, a large number of proteins potentially involved in the etiology of drug addictions have been identified, which provide a valuable resource to study protein function, biochemical pathways, and networks related to the molecular mechanisms underlying drug dependence. In this article, we summarize the recent application of proteomics to profiling protein expression patterns in animal or human brain tissues after the administration of alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine/heroin/butorphanol, or nicotine. From available reports, we compiled a list of 497 proteins associated with exposure to one or more addictive drugs, with 160 being related to exposure to at least two abused drugs. A number of biochemical pathways and biological processes appear to be enriched among these proteins, including synaptic transmission and signaling pathways related to neuronal functions. The data included in this work provide a summary and extension of the proteomics studies on drug addiction. Furthermore, the proteins and biological processes highlighted here may provide valuable insight into the cellular activities and biological processes in neurons in the development of drug addiction.     

小分子药物靶标寻找方法研究进展

许多微生物的次生代谢物属于小分子活性化合物,在医疗及农业领域发挥着重要的作用。在基因组学、蛋白质组学与生物信息学等技术的推动下,一些新的小分子药物靶标寻找方法应运而生了,这些新的方法主要是基于细胞中基因或蛋白质的表达量、蛋白质的亲和性、稳定性等各种特性进行靶标寻找的。小分子药物靶标寻找方法的发展加快了阐明小分子药物作用机理的历程,也为发现新的靶标资源以便于进一步筛选活性更高的药物提供了技术保障。     

Effects of debrisoquin on CSF and plasma HVA concentrations in man

Data from animal studies indicate neuroleptic drugs act via their properties as antagonists of CNS dopamine (DA) receptors and this finding has led to the suggestion that alterations in CNS DA neuronal function are associated with psychotic disorders. Clinical investigations of this hypothesis, however, have been hindered by the lack of the availability of a direct and relatively easily obtained index of CNS DA neuronal activity. The work reported here was aimed at the development of such an index. Using a double blind design, human male subjects were given either placebo or debrisoquin, which is a monoamine oxidase inhibitor which does not penetrate brain. On the baseline day (no debrisoquin) and after 6 and 13 days of drug administration blood samples were obtained. In addition, for some patients CSF specimens were obtained via lumbar puncture on the baseline day and after 13 days of drug administration. It was found that debrisoquin produced a highly significant decrease in plasma homovanillic acid (HVA) concentrations whereas the concentrations of HVA in CSF were unchanged. In addition, it was found that the correlation between CSF and plasma HVA prior to debrisoquin was non-significant (r = 0.39, p = N.S., N = 10) whereas after 13 days of debrisoquin treatment the correlation was highly significant (r = 0.95, p less than .01, N = 7). These findings suggest that the administration of debrisoquin produces a situation in which plasma HVA reflects CNS HVA production, and as such debrisoquin may be a useful tool for the clinical investigator who is interested in studying relationships in human subjects between CNS DA neuronal system function and psychopathological states or other disorders which may be mediated via brain DA systems.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

Automated on-like dialysis, trace enrichment and high-performance liquid chromatography Inhibition of interaction with the dialysis membrane and disruption of protein binding in the determination of clozapine in human plasma

Problems related to interaction of drugs with the dialysis membrane and to protein binding must be overcome in order to develop automated methods for drug analysis based on on-line dialysis, trace enrichment and HPLC. In order to study these problems, clozapine and its active metabolite N-desmethylclozapine were chosen as model compounds because they were found to interact with the dialysis membrane, and clozapine is highly protein bound. Addition of a cationic surfactant, dodecylethyldimethyl ammonium bromide, to the donor solution and to the plasma samples was found to inhibit interaction of the drugs with surfaces. The protein binding in plasma was disrupted prior to dialysis by lowering the pH with hydrochloric acid and the plasma proteins were solubilised with glycerol. The results obtained were used to develop a fully automated method for the determination of clozapine and N-desmethylclozapine in human plasma. More than 100 samples could be analysed within 24 h. The limit of detection in human plasma was 0.050 μmol/1 for clozapine and 0.055 μmol/1 for N-desmethylclozapine. Linearity was found for drug concentrations between 0.25–3 μmol/1. The relative standard deviations were between 1.2–6.7% and the method was applicable for therapeutic drug monitoring.     

Clinical and Technical Phosphoproteomic Research

An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS) is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative techniques need to be combined to achieve good phosphopeptide recovery and good up- and-down phospho-regulation protein studies.     

Pharmaceutical and pharmacological approaches for bioavailability enhancement of etoposide

Etoposide, a semi-synthetic derivative of podophyllotoxin, is one of the most active and useful antineoplastic agent used routinely in firstline combination chemotherapy of testicular cancer, small-cell lung cancer and non-Hodgkin’s lymphoma. Etoposide displays narrow therapeutic index, erratic pharmacokinetics and dose individualization that needs to be achieved for overcoming inter- and intra-patient variability (25–80%), so as to maintain proper drug exposure within a therapeutic range. Etoposide posses high plasma protein binding (97%) and is degraded via complex metabolic pathways. The main pharmacokinetic determinants of etoposide are still not completely defined in order to optimize the pharmaco-therapeutic parameters including dose, therapeutic schedule and route of administration. Much research has been done to determine drug–drug and herb–drug interactions for improving the bioavailability of etoposide. The present article gives insight on pharmaceutical and pharmacological attempts made from time to time to overcome the erratic inter- and intra-patient variability for improving the bioavailability of etoposide.     

When a TRP goes bad: Transient receptor potential channels in addiction

Drug addiction is a psychiatric disease state, wherein a drug is impulsively and compulsively self-administered despite negative consequences. This repeated administration results in permanent changes to nervous system physiology and architecture. The molecular pathways affected by addictive drugs are complex and inter-dependent on each other. Recently, various new proteins and protein families have been discovered to play a role in drug abuse. Emerging players in this phenomenon include TRP (Transient Receptor Potential) family channels, which are primarily known to function in sensory systems. Several TRP family channels identified in both vertebrates and invertebrates are involved in psychostimulant-induced plasticity, suggesting their involvement in drug dependence. This review summarizes various observations, both from studies in humans and other organisms, which support a role for these channels in the development of drug-related behaviors.     

Preferential targeting of apoptosis in tumor versus normal cells

Elimination of cancer cells by early apoptosis is preferred over other forms of cell growth inhibition. Apoptosis directly leads to tumor regression and reduces risks of selecting more aggressive and/or drug-resistant phenotypes that are often responsible for tumor regrowth and treatment failure. Although DNA damage by anticancer drugs is commonly recognized as an apoptotic stimulus, there is enormous variability in the magnitude and timing of such effects. Especially potent and rapid apoptosis seems to be a hallmark of various alkylating anticancer drugs that are regarded as DNA-reactive agents but are observed to react mainly with cellular proteins. Our studies with such dual-action drugs (irofulven, oxaliplatin) suggest that not only DNA damage, but also protein damage, contributes to apoptosis induction. DNA damage is well known to initiate death-signaling pathways leading to mitochondrial dysfunction. Protein damage, in turn, can distort cell redox homeostasis, which facilitates apoptosis execution. Such dual effects can be particularly lethal to tumor cells, which tend to function under pro-oxidative conditions. In contrast to tumor cells that are highly susceptible, normal cells show marginal apoptotic responses to the dual action drugs. This protection of normal cells might reflect their greater ability to buffer pro-oxidative changes and quickly restore redox homeostasis, despite substantial drug uptake and macromolecular binding. Importantly, by targeting the death process at multiple points, DNA- and protein-damaging drugs can be less vulnerable to various bypass mechanisms possible with single targets. The reviewed studies provide a proof of concept that differential apoptosis targeting in cancer versus normal cells can be a basis for tumor selectivity of anticancer drugs.     

Antibody buffering in the brain

The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.     

Drug–drug plasma protein binding interactions of ivacaftor

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR‐protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co‐administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co‐administered CF drugs for human serum albumin (HSA) and α₁‐acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site‐selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug–drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug–drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug–drug interactions of ivacaftor with co‐administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. Copyright © 2015 John Wiley & Sons, Ltd.     

Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.     

Lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluid from multiple sclerosis patients

Lipid peroxidation (LPx) products were measured as thiobarbituric acid-reactive substances (TS) and lipid-soluble fluorescent pigments (FP) in both plasma and CSF from MS patients and controls. Although no significant changes were found in MS plasma, we report here for the first time increases in both TS and FP in MS CSF (p<0.05 and p<0.01, respectively, compared with patients with other neurological diseases), indicating that increased LPx in CNS may be a feature of MS. Levels of transferrin were normal but caeruloplasmin (CP), a major antioxidant plasma protein, was significantly raised in MS patients (p<0.01) and this may represent an adaptive response to increased oxidative challenge. Neither of these proteins was detectable in CSF using radial immunodiffusion. There was no significant correlation between the severity or duration of the disease nor the period since the last relapse and either LPx products of CP suggesting that the changes observed in this work are not simply the direct result of demyelination and tissue damage.