Determination of β-Endorphin and Fragments Thereof in Human Plasma Using High-Performance Liquid Chromatography and a Multiple Radioimmunoassay System

A method for the determination of -endorphin and -endorphin fragments in human plasma was developed. -Endorphin-related peptides were extracted from plasma using octadecasilyl-silica cartridges. Extracts were subjected to re versed-phase high-performance liquid chromatography (HPLC). Extracts as well as HPLC column eluates were assayed using a multiple radioimmunoassay system; several antibodies directed against various distinct regions of the -endorphin molecule were employed. Using this method, evidence for the presence of multiple -endorphin fragments in the plasma of healthy young volunteers (under normal conditions) was obtained.     

Prediction of Human Brain Penetration of P-glycoprotein and Breast Cancer Resistance Protein Substrates Using In Vitro Transporter Studies and Animal Models

Four P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates with human cerebrospinal fluid (CSF) concentrations and preclinical neuropharmacokinetics were used to assess in vitro–in vivo extrapolation of brain penetration in preclinical species and the ability to predict human brain penetration. Unbound brain (C_b,u), unbound plasma (C_p,u), and CSF compound concentrations (C_CSF) were measured in rats and nonhuman primates (NHPs), and the unbound partition coefficients (C_b,u/C_p,u and C_CSF/C_p,u) were used to assess brain penetration. The results indicated that for P-gp and BCRP dual substrates, brain penetration was severally impaired in all species. In comparison, for P-gp substrates that are weak or non-BCRP substrates, improved brain penetration was observed in NHPs and humans than in rats. Overall, NHP appears to be more predictive of human brain penetration for P-gp substrates with weak or no interaction with BCRP than rat. Although C_CSF does not quantitatively correspond to C_b,u for efflux transporter substrates, it is mostly within 3-fold higher of C_b,u in rat and NHP, suggesting that C_CSF can be used as a surrogate for C_b,u. Taken together, a holistic approach including both in vitro transporter and in vivo neuropharmacokinetics data enables a better estimation of human brain penetration of P-gp/BCRP substrates.     

How Minimally Invasive is Microdialysis Sampling? A Cautionary Note for Cytokine Collection in Human Skin and other Clinical Studies

It is common to refer to microdialysis as a minimally invasive procedure, likening it to insertion of an artificial capillary. While a comparison of this type allows the process to be easily visualized by those outside the field, it tends to provide a false impression of the localized perturbation of the tissue space that is caused by catheter insertion. With the increased acceptance of microdialysis sampling as a viable in vivo sampling method, many researchers have begun to use the technique to explore inflammatory and immune-mediated diseases in the skin and other organs. Unfortunately, many of the molecules of interest, particularly chemokines and cytokines, are known to be generated during the inflammatory response to wounding and the subsequent cellular events leading to wound repair. With more than 11,000 reports citing the use of microdialysis sampling, only a few researchers have sought to assess the tissue damage that is incurred by probe insertion. For this reason, caution is warranted when collecting these molecules and inferring a role for them in clinical disease states. This commentary seeks to remind the research community of the confounding effects that signaling molecules related to the wounding response will have on clinical studies. Proper controls must be incorporated into all studies in order to assess whether or not particular molecules are truly related to the disease state under investigation or have been generated as part of the tissue response to the wound incurred by microdialysis catheter implantation.     

Understanding and Predicting Drug Delivery from Hydrophilic Matrix Tablets Using the “Sequential Layer” Model

Purpose. The objectives of this work were (i) to study and understand the physicochemical phenomena which are involved in the swelling and drug release from hydrophilic matrix tablets using the sequential layer model; and (ii) to predict the effect of the initial radius, height and size of the tablets on the resulting drug release profiles. Methods. Tablets were prepared by direct compression, using hydroxypropyl methylcellulose (HPMC) grades with different average molecular weights as matrix-forming polymers. The in vitro release of chlorpheniramine maleate, propranolol HCl, acetaminophen, theophylline and diclofenac sodium was studied in phosphate buffer (pH 7.4) and 0.1 M HCl, respectively. The initial drug loading varied from 1 to 70%, while the radius and height of the tablets varied from 1 to 8 mm. Results. The sequential layer model considers water and drug diffusion with non-constant diffusivities and moving boundary conditions, non-homogeneous polymer swelling, drug dissolution, and polymer dissolution. We showed that this model was able to predict the resulting drug release kinetics accurately in all cases. Conclusions. The sequential layer model can be used to elucidate the swelling and drug release behavior from hydrophilic matrix tablets and to simulate the effect of the device geometry on the drug release patterns. Hence, it can facilitate the development of new pharmaceutical products.     

Human serotonin transporter gene (SLC6A4) variants: their contributions to understanding pharmacogenomic and other functional G×G and G×E differences in health and disease

Recent major findings from studies of SLC6A4 and its corresponding protein, the serotonin (5-HT) transporter (SERT) in humans, rodents and non-human primates indicate that combinations of SLC6A4 non-coding 5', 3' UTRs and intronic regions plus coding variants acting together can change 5HT transport as much as 40-fold in vitro. In vivo, SLC6A4 variants in humans and other species lead to marked physiological changes, despite mitigating neurodevelopmental adaptations in 5-HT receptors plus compensatory alterations in 5-HT synthesis and metabolism. Polymorphisms in SLC6A4 are associated with differences in emotional, endocrine, and personality characteristics as well as many diseases. This gene, in combinations with gene×gene (G×G) and gene×environment (G×E) interactions nonetheless remains incompletely understood, with some association findings remaining controversial. Considering its primary importance in the regulation and function of the entire serotonergic system (as evidenced by the consequences of SERT-mediated reuptake inhibition by SRIs like fluoxetine in humans and of genetically engineered changes in mice and rats), it seems likely that SLC6A4 and SERT will remain areas of high interest in our field's attempts to better understand and treat 5-HT-related disorders.     

Rationale and Staff Evaluation of Using a “Therapeutic Milieu” for Substance Users Within a Tuberculosis Ward

Approximately 30% of tuberculosis (TB) patients in Israel were treated, in part, in two dedicated hospital wards during the years 2003–2005. A portion of them manifested severe psychosocial conditions. An intervention based on the “Therapeutic Milieu” (TM) model was implemented in the larger ward and included a staff evaluation of this intervention. The concept of TM, based on psychosocial paradigms and behavioral medicine, is aimed at providing a supportive environment for patients. Weekly group patients' meetings and monthly group staff supervisions were performed during 15 months (2003–2005). Forty of the 196 (20%) TB patients, mainly “complex,” and 13 of 20 staff members (65%) attended regularly and discussed how to deal with substance abuse, personality disorders, and immigration-related crises. Out of 40 TB cases, 30 (75%) were also substance users. Ten staff members self-analyzed the impact of this intervention in terms of (1) having given adequate tools for the staff, (2) reducing physical violence, (3) increasing adherence to TB treatment, and (4) more efficient treatment for their substance use. No direct evaluation was done among the TB patients. According to staff members, this intervention had a positive overall impact. However, using Therapeutic Milieu in TB ward hospitalization, as a “window of opportunity,” remains the first step in a longer journey for rehabilitation. The study's limitations are noted.     

The “Real” Number of Washington State Adolescents Using Marijuana,and Why: A Misclassification Analysis

Background: Although causality is difficult to establish, the regular use of marijuana has been associated with many adverse physiological and sociological consequences. There is considerable concern regarding marijuana use among adolescents, as the likelihood of adverse consequences increases significantly for this age group. The most comprehensive data for identifying risk factors for adolescent marijuana use is typically self-report, which may be over- or under-reported for a variety of reasons, including stigmatization, peer-pressure, or fear of being discovered. Objectives: To identify the prevalence of adolescent marijuana use in Washington State, and the associated risk and protective factors, while controlling for and estimating the extent of misreporting, and its determinants. Method: Data came from the 2014 Washington State Healthy Youth Survey. We accounted for missingness using chained multivariate imputation equations, resulting in 33,320 complete observations. Our model was estimated using a maximum likelihood multiple regression designed to control for systematic misclassification in binary dependent variables. Results: Approximately 12% of Washington adolescents claimed to have used marijuana in the past 30?days. Our estimates indicate this figure is likely closer to 18%. Determinants of use included use of other substances, gender, age, and measures of deviant social influences, personality/attitude, school and family bonds, bullying, and acquisition ease. Determinants of misreporting included use of other substances, gender, parental education, and family bonds. Conclusions: Failing to control for misreporting considerably underestimates the prevalence of marijuana use among adolescents. Our model allows us to better identify at-risk adolescents and inform focused prevention efforts.     

The Changing Role of “Using” Dreams in Addiction Recovery

The Dream Interview Method of dream interpretation can be applied to dreams depicting alcohol and drug use to elucidate the meaning of such dreams in relation to stages of recovery. This paper summarizes the method and demonstrates its application utilizing seven dreams collected from clients in psychotherapy and participants in dream workshops. The incorporation of such interpreted dreams into substance abuse treatment as well as their relationship to other life issues is discussed. Published by Elsevier Science Inc.     

A New Drug Screening System “MULTI-HPLC” by High Performance Liquid Chromatography Using Multiwavelength UV Detector and Its Clinical Application

We have developed a new drug screening system, MULTI-HPLC, a high performance liquid chromatography (HPLC) system that uses a simultaneous multiwavelength ultraviolet (UV) detector. In this system, HPLC coupled with a 32-channel photodiode-array UV detector can provide multidimensional chromatographic information about compounds and yield identification and quantification of unknown intoxicating agents rapidly and nearly automatically by computer assistance. This detector can provide spectral data. Special attention is given to spectral interpretation techniques (spectrum search function, peak deconvolution, retention prediction program). Possible applications of this system were demonstrated in clinical cases and were found useful and practical, especially in multidrug poisoning. The comprehensive drug screening and quantification within a short turnaround time allow the toxicology laboratory to assume a much more important role in the diagnosis and management of acute poisoning patients.     

Identification and Characterization of the Insecticidal Toxin “Makes Caterpillars Floppy” in Photorhabdus temperata M1021 Using a Cosmid Library

Photorhabdus temperata is an entomopathogenic enterobacterium; it is a nematode symbiont that possesses pathogenicity islands involved in insect virulence. Herein, we constructed a P. temperata M1021 cosmid library in Escherichia coli XL1-Blue MRF` and obtained 7.14 × 10⁵ clones. However, only 1020 physiologically active clones were screened for insect virulence factors by injection of each E. coli cosmid clone into Galleria mellonella and Tenebrio molitor larvae. A single cosmid clone, PtC1015, was consequently selected due to its characteristic virulent properties, e.g., loss of body turgor followed by death of larvae when the clone was injected into the hemocoel. The sequence alignment against the available sequences in Swiss-Prot and NCBI databases, confirmed the presence of the mcf gene homolog in the genome of P. temperata M1021 showing 85% homology and 98% query coverage with the P. luminescens counterpart. Furthermore, a 2932 amino acid long Mcf protein revealed limited similarity with three protein domains. The N-terminus of the Mcf encompassed consensus sequence for a BH3 domain, the central region revealed similarity to toxin B, and the C-terminus of Mcf revealed similarity to the bacterial export domain of ApxIVA, an RTX-like toxin. In short, the Mcf toxin is likely to play a role in the elimination of insect pests, making it a promising model for use in the agricultural field.     

Urinary metallothionein as a new index of renal dysfunction in “itai-itai” disease patients and other Japanese women environmentally exposed to cadmium

The significance of elevated excretion of metallothionein in urine of women living in cadmium-polluted and non-polluted areas of Japan was studied with respect to renal dysfunction. The relationships between the concentrations of metallothionein in urine and those of other non-specific urinary indices of renal dysfunction, i.e., total protein, glucose, ₂-microglobulin, retinol-binding protein, -amino nitrogen and proline were examined. In addition, the relationships between urinary metallothionein and urinary cadmium and copper were also evaluated. It was found that the logarithm of the metallothionein concentration in urine was significantly correlated with the logarithm of the concentrations of each of the above parameters. When subjects with signs of renal dysfunction, including itai-itai disease patients and patients suspected of the disease, were compared with subjects with normal renal functions, as a group, the former excreted significantly higher concentrations of metallothionein in their urine than the latter. The results suggest that the elevated excretion of metallothionein is not only an index of excessive cadmium exposure, but also of renal dysfunction caused by chronic exposure to this metal.Presented in part at the 66th Annual Meeting of the Federation of American Societies for Experimental Biology, New Orleans, April, 1982     

Patterns of Use,Acute Subjective Experiences,and Motivations for Using Synthetic Cathinones (“Bath Salts”) in Recreational Users

Given the variety and potential toxicity of synthetic cathinones, clinicians and educators would benefit from information about patterns of and motivations for use, frequency of psychosocial consequences, and experience of acute subjective effects. We administered a comprehensive, web-based survey to 104 recreational users of synthetic cathinones. Sixty percent of respondents consumed synthetic cathinones once or more per month, usually snorting or swallowing these drugs, typically at home, usually with others, customarily during the evening and nighttime hours, and often in combination with another drug such as alcohol or marijuana. Acute subjective effects attributed to synthetic cathinones were similar to those of other psychostimulants, including increased energy, rapid heartbeat, racing thoughts, difficulty sleeping, euphoria, decreased appetite, open-mindedness, and increased sex drive. Reported reasons for using synthetic cathinones included its stimulating effects, curiosity, substitution for another drug, and being at a party/music event. Respondents had experienced an average of six negative consequences of using synthetic cathinones during the previous year (e.g., tolerance, neglecting responsibilities, personality change). In combination with previously published investigations, these findings increase our understanding of the reported rationales and outcomes of recreational use of synthetic cathinones.     

“Anxiolytic” and “anxiogenic” benzodiazepines and β-carbolines: effects on aggressive and social behavior in rats and squirrel monkeys

Ethopharmacological studies on the behavior of socially housed rats and squirrel monkeys were conducted to explore the role of the benzodiazepine GABA_A-coupled ionophore receptor complex in aggressive and social interactions. Benzodiazepine receptor (BZR) antagonists, ZK 93426 (1–10 mg/kg) and flumazenil (3–10 mg/kg), the partial agonist, ZK 91296 (1–10 mg/kg) and the partial inverse agonists RO 15-4513 (0.3–10 mg/kg), were administered to (1) squirrel monkeys prior to 1 h focal observations within established social groups or to (2) resident male rats before confrontations with a naive male intruder in their home cage for 5 min. Aggression was modified in a similar manner in both species, although squirrel monkeys were more sensitive to BZR challenges. Specifically, resident male rats showed dose dependent reductions in attack bites directed at intruder males that were significant at the highest dose of ZK 93426 (10 mg/kg). In squirrel monkeys, ZK 93426 (3 and 10 mg/kg) reduced aggressive grasps, threats and displays, as well as reducing the duration of being the target of aggression from untreated group members (1–10 mg/kg). The BZR partial agonist, ZK 91296 and the antagonist, flumazenil produced few effects on social behavior, low and high intensity aggression and motor activity in both species. Flumazenil (10–30 mg/kg) and ZK 91296 (10 mg/kg), but not ZK 93426, produced significant increases in foraging and feeding behaviors in squirrel monkeys. The hyperphagic effects of ZK 91296 and flumazenil, that are typical of BZR agonists compounds, were not observed in rats. Similarly, the inverse agonist-like reductions in social interactions produced by ZK 93426 (3–10 mg/kg) were observed only in squirrel monkeys. The partial inverse agonist Ro 15-4513 reduced aggression in rats, but low doses (1 mg/kg) produced tremors or seizures in 80% of the monkeys tested. Decreases in aggressive and social behaviors are often interpreted to reflect anxiogenic drug properties, whereas increased feeding has been associated with anxiolytic actions. The concurrent emergence of these apparent opposites suggests independent actions on social and alimentary functions.