Assembly of nucleic acid-lipid nanoparticles from aqueous-organic monophases.

Effective, reproducible, and scalable methods for DNA-lipid assembly are important for the success of non-viral vectors in in vivo gene therapy. We hypothesized DNA-lipid assembly would be optimal if started from a liquid monophase where both DNA and lipids separately form molecular or micellar solutions prior to mixing, without preexisting condensed lipid phases, thus allowing DNA-lipid assembly under conditions close to equilibrium. Previously, we found that mixing plasmid DNA, 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), cholesterol and a cationic lipid, 1, 2-dioleoyl-3-(trimethylammonio) propane (DOTAP) in 50% (v/v) aqueous ethanol spontaneously produced an optically transparent solution. Upon ethanol removal, DNA-lipid nanoparticles (Genospheres) were formed. For comparison with well-known technologies, different DNA-lipid particles were prepared by interaction of plasmid DNA and stable or ethanol-destabilized lipid vesicles by combining the components in water or 30% (v/v) aqueous ethanol, respectively. Among the three studied DNA-lipid assembly methods, only Genospheres combined the properties of small size (less than or around 100 nm), high incorporation of both lipid and DNA, high degree of DNA protection (dye accessibility 5-12%), a narrow distribution of particle density and when immuno-targeted, the highest transfection efficiency in HER2-overexpressing cells in vitro. We conclude that the Genosphere assembly methodology offers advantages for the development of effective, scalable and targetable non-viral gene delivery vectors.

In vitro characterization of a novel polymeric-based pH-sensitive liposome system.

This study demonstrates rapid and pH-sensitive release of a highly water-soluble fluorescent aqueous content marker, pyranine, from egg phosphatidylcholine liposomes following incorporation of N-isopropylacrylamide (NIPA) copolymers in liposomal membranes. The pH-sensitivity of this system correlates with the precipitation of the copolymers at acidic pH. In vitro release can be significantly improved by increasing the percentage of anchor in the copolymer and thus favoring its binding to the liposomal bilayer. In the case of liposomes containing a poly(ethylene glycol)-phospholipid conjugate, the insertion of the pH-sensitive copolymer in the liposomal membrane appears to be sterically inhibited. Dye release from these formulations at acidic pH can still be achieved by varying the anchor molar ratio and/or molecular mass of the polymers or by including the latter during the liposome preparation procedure. Removal of unbound polymer results in decreased leakage only when the copolymer is inserted by incubation with preformed liposomes, but can be overcome by preparing liposomes in the presence of polymer. Aqueous content and lipid mixing assays suggest contents release can occur without membrane fusion. The results of this study indicate that the addition of pH-sensitive copolymers of NIPA represents promising strategy for improving liposomal drug delivery.

Different drink cues elicit different physiological responses in non-dependent drinkers.

Different kinds of physiological response to stimuli which have been associated with alcohol ingestion have been observed in human subjects. A literature review shows that when subjects are exposed to alcohol associated stimuli without consuming the drinks then increases in arousal, as indexed by skin conductance and heart rate increases, tend to occur. If subjects consume drinks which have been associated with alcohol ingestion, then decreases in arousal tend to occur. Forty non-dependent drinkers were asked to either consume or hold drinks which either had a history of alcohol association or did not. Interactions were observed between the activities subjects engaged in with the drinks and the degree of alcohol association of the drinks. Presentation of alcohol associated drinks produced smaller increases in arousal than non-alcohol associated drinks if the drinks were consumed but vice versa if the drinks were just held. The results support the conclusions drawn from the literature review and have implications for current theories of conditioned responses to drug cues and the related theories of the motivational processes involved in the regulation of drug intake.

N-isopropylacrylamide copolymers for the preparation of pH-sensitive liposomes and polymeric micelles.

Hydrophobically-modified copolymers of N-isopropylacrylamide bearing a pH-sensitive moiety were investigated for the preparation of pH-responsive liposomes and polymeric micelles. The copolymers having the hydrophobic anchor randomly distributed within the polymeric chain were found to more efficiently destabilize egg phosphatidylcholine (EPC)/cholesterol liposomes than the alkyl terminated polymers. Release of both a highly-water soluble fluorescent contents marker, pyranine, and an amphipathic cytotoxic anti-cancer drug, doxorubicin, from copolymer-modified liposomes was shown to be dependent on pH, the concentration of copolymer, the presence of other polymers such as polyethylene glycol, and the method of preparation. Both polymers were able to partially stabilize EPC liposomes in human serum. These polymers were found to self-assemble to form micelles. The critical association concentration was low (9--34 mg/l) and influenced by the position of the alkyl chains. In phosphate buffered saline, the micelles had a bimodal size distribution with the predominant population having a mean diameter of 35 nm. The polymeric micelles were studied as a delivery system for the photosensitizer aluminum chloride phthalocyanine, (AlClPc), currently evaluated in photodynamic therapy. pH-Responsive polymeric micelles loaded with AlClPc were found to exhibit increased cytotoxicity against EMT-6 mouse mammary cells in vitro than the control Cremophor EL formulation.

What does cue-reactivity have to offer clinical research?

This paper examines the application of the cue-reactivity paradigm as a means of studying alcohol dependence in clinical populations. Three main areas of application will be examined: cue-reactivity as a means of understanding the nature of alcohol dependence; cue-reactivity as a predictor of relapse; and cue-reactivity as a method of studying treatment effects. The study of cue exposure and cue-reactivity has a long history but it is only relatively recently that the potential of cue-reactivity as a means of understanding and treating addictive behaviours has been studied in depth. The principal advantage of cue-reactivity over other existing paradigms to study addictive behaviour is in having a solid basis in widely studied general theories of behaviour. Cue-reactivity also provides a means of measuring and unpacking the concept of craving. Craving has long been believed to represent the underlying basis for addictive behaviour, and in the majority of research studies craving has been conceptualized and measured in relatively simplistic ways. Craving has generally been viewed as a unitary phenomenon and measured using self-reported questionnaires. Such approaches have had limited explanatory value, particularly in recent psychopharmacology research. There is clearly a need to develop new paradigms to study the effects of pharmacological agents aimed at attenuating drinking behaviour. It is in this area that cue-reactivity currently offers the greatest potential. In particular, the cue-reactivity paradigm draws an important distinction between cue mediated craving and withdrawal craving. This can be viewed as similar to the distinction between generalised anxiety and anxiety provoked by phobic stimuli. However, while much is now known about the phenomenon of cue-reactivity, several aspects require further elucidation and research investment.

Theories of drug craving, ancient and modern.

This paper reviews the principal theoretical models of drug craving and provides some directions for future research. The main models are classified broadly into three categories: (1) phenomenological models; based on clinical observation and description; these have been influential in classification systems of addictive disorders and in the development of pharmacological therapies; (2) conditioning models: based on conditioning theory; these have been influential in the development of cue exposure treatments; (3) cognitive theories; based on cognitive social learning theory: these have been influential in the development of cognitive therapies of addiction. It is concluded that no one specific theory provides a complete explanation of the phenomenon of craving. However, theories of craving grounded in general theories of human behaviour offer greatest promise, and generate more specific and testable research hypotheses. Theories that do not require craving to be present for relapse to occur have more empirical support than those that provide simplistic causal explanations. The cue-reactivity model shows promise in the exploration of the relationship between craving and relapse. However, further attention to the phenomenology of craving could help to advise the future measurement and study of drug craving, particularly in the context of research in which drugs are available to human subjects, with adequate ethical safeguards. There is a need for further study of the temporal dynamics of craving and consensus in the field on the most appropriate methods of measurement. Finally, new psychotherapies such as cue exposure and pharmacotherapies that aim to attenuate drinking behaviour, such as naltrexone and acamprosate, provide opportunities to improve understanding of the nature and significance of craving. However, the relatively uncritical assumption that craving is the underlying basis of addiction and represents the most appropriate target for treatment is challenged.

Alcohol interventions: do the best things come in small packages?

Several extensive reviews have highlighted the effectiveness of brief alcohol interventions. The same reviews were pessimistic about the role of more intensive, specialist treatments. It is argued here that the research evidence should be interpreted with caution. There are problems of generalizability of the research, and studies focusing on brief interventions in the primary health care field are largely not comparable with clinical trials conducted in the specialist setting. The efficacy of brief interventions as a routine mass intervention approach has been exaggerated. Even after extensive research, little is known of the effective ingredients and the most effective methods of delivery. Reviews of brief interventions have been overly selective, and meta analysis in this area is problematic. It is argued that such reviews lead to overgeneralization and turn attention away from promising specialist treatment approaches. More research is needed into identifying the target group most likely to benefit from brief interventions, cost effectiveness, and into shared care and stepped care approaches, before embarking on a major shift in treatment policy towards brief interventions.

New drug treatments for alcohol problems: a critical appraisal.

This review considers the novel drug treatments that have been suggested to help prevent relapse or attenuate drinking in people with alcohol problems. The evidence from randomized controlled trials for the efficacy of some of the main candidates: acamprosate, naltrexone, bromocriptine, selective serotonin re-uptake inhibitors and buspirone, was examined. Important methodological problems which may have introduced bias were detected in many of the trials. These included failure to test the integrity of the double blind, excluding or estimating outcome in early withdrawals and the comparison of groups on multiple outcome measures with selective reporting of results. In addition, the generalizability of some studies was limited by the procedures used for sample selection. In view of the potential adverse effects of drug treatment it is concluded that the evidence is not strong enough to support the introduction of any of these substances into routine clinical practice at present. The review also emphasizes the importance of methodological rigour to maximize objectivity in treatment evaluation research.

The quality of alcohol treatment research: an examination of influential controlled trials and development of a quality rating system.

BACKGROUND: The importance of evidence-based practice has stimulated interest in the methodology of clinical trials. Various weaknesses of evaluation research in the alcohol field have been indicated previously. This study set out to develop a comprehensive system for the assessment of the methodological quality of outcome research for treatment of alcohol misuse and to apply the system to well-known trials in the area. METHODOLOGY: A sample of the most highly cited controlled trials of interventions for alcohol misuse was selected using the Science Citation Index. Thirty methodological criteria were formulated and a scoring system devised. Two raters applied this system to the sample of trials. Reliability testing was performed and used to refine the criteria. RESULTS: Inter-rater reliability of the overall quality score was initially 0.85 and 0.92 after review and re-rating. Internal consistency was also high (0.87). Quality score correlated with year of publication. Certain areas of methodology were poorly addressed in the sample, including specification of main outcomes, documentation of recruitment and selection procedures, testing of blinding, analysis of withdrawals and reporting of results. DISCUSSION: The methodology of this sample of trials was frequently deficient in ways which might bias results or compromise generalizability. It is recommended that the system of quality assessment described here is used to evaluate existing research and to inform the design of future studies.

Research perspectives on alcohol craving: an overview.

This overview of the Addiction supplement on 'Research Perspectives on Alcohol Craving' has three objectives. The first is to familiarize readers with the variety of theoretical models relevant to craving and the definitions of craving generated by them that are discussed in the supplement. These include phenomenological models, classical and operant conditioning models, the incentive-sensitization theory, a tonic-phasic model of dopamine system regulation, cognitive social learning theory and the cognitive processing theory of craving. The second objective is to provide a brief summary of the methodological articles which focus as a whole more on what can be done than on what has been done in alcohol craving research. The final objective is to emphasize the potential importance of transdisciplinary research--research that integrates components of different theoretical models--for delineating the role of alcohol and drug craving in the complex biobehavioral process known as addiction. It is the hope of the guest editors (the authors of this overview) that the Addiction supplement and this introduction to it will contribute to development of a framework for future transdisciplinary research on alcohol craving.

Craving research: future directions.

Many prospective clinical studies have concluded that craving does not reliably predict relapse and that the concept is of little or no clinical utility. Contrary to earlier more simplistic clinical models of addiction, more recent models do not require that craving be present for relapse to occur. New approaches to study human craving may enhance its predictive validity and yield more knowledge of its nature, course, behavioural sequelae and regulatory function in alcohol/drug consumption. These approaches include empirical research that focuses on: (1) the elucidation of the domains of craving (i.e. subjective experience, physiological responses, behavioural sequelae and their inter-relationships); (2) the temporal dynamics of craving (i.e. its course over minutes or days, as well as its natural history over the course of a drinking career); (3) the factors that may mediate/moderate/determine the development and resolution of craving; (4) studies of the predictive validity of craving measures; and (5) the development of valid methods of measuring the different domains of craving. The conclusions are that future craving research should: (1) incorporate more sophisticated general theories of behaviour (conditioning, cognitive social learning, neurobiological, and genetic); (2) apply more sophisticated and standardized measurement methods and experimental paradigms, including studies in which alcohol is made available to human subjects; and (3) effective development of new pharmacological and behavioural therapies for relapse prevention depend on greater understanding of the nature and measurement of craving.

A controlled trial of cue exposure treatment in alcohol dependence.

The clinical effectiveness of cue exposure (CE) treatment in alcohol dependence was evaluated in a controlled trial. Thirty-five men who were detoxified and severely alcohol dependent received either CE or relaxation control (RC) treatment. CE Ss had 400 min exposure to the sight and smell of preferred drinks over 10 days in a laboratory setting. RC Ss spent identical time in the laboratory but had relaxation therapy and only 20 min exposure to alcohol cues. During 6-month follow-up, personal interview was achieved with 91% of Ss. CE Ss had a more favorable outcome than the RC Ss in terms of latency (length of time) to relapse of heavy drinking (p < .01) and total alcohol consumption (p < .05). Significant predictors of latency to heavy drinking and dependence included skin conductance level (p < .001) and experimental condition (p < .01). Results point to the potential importance of cue exposure as a treatment for addictive behavior.

How should opiate withdrawal be measured?

Comparison is made between subjective and objective measurements of opiate withdrawal severity in a group of 24 regular opiate takers undergoing inpatient detoxification. A lack of correlation is found between patients' subjective ratings, objective nurse ratings and physiological parameters of withdrawal severity. This indicates that in future research the subjective and objective dimensions of withdrawal should be considered and measured separately.

The Alcohol Problems Questionnaire: reliability and validity.

The Alcohol Problems Questionnaire (APQ) was designed as a clinical instrument for measuring alcohol-related problems. It has been used to study the relationship between alcohol-related problems and dependence within the bi-axial model. The reliability of the APQ was, however, unknown. In the present study 101 subjects participated in a test-retest reliability study. Dependence and consumption scores were obtained along with socio-demographic information in order to study the construct validity of the APQ. The APQ was found to be highly reliable and the previous finding that dependence is a mediating factor in the relationship between consumption and problems was replicated. These findings add further weight to the view that alcohol-related problems represent a phenomenon which is conceptually, as well as statistically, distinct from dependence. Further, the APQ is reliable, simple to administer and is likely to be useful in the assessment and study of problem drinkers in the clinical and research settings.

Chlordiazepoxide vs. methadone in opiate withdrawal: a preliminary double blind trial.

A double blind trial of chlordiazepoxide vs. methadone in the management of the opiate withdrawal syndrome was conducted in a group of 24 regular heroin takers. Subjective and objective measures including physiological parameters were recorded to compare the severity of opiate withdrawal between the two groups. No significant difference was found in terms of subjective withdrawal distress between the two treatment conditions, although there was a tendency to a return of withdrawal symptoms in the methadone group towards the end of treatment. A nurse rating scale demonstrated a significantly higher level of withdrawal signs in the chlordiazepoxide group on day 3. Physiological measures suggested that neither group experienced a severe withdrawal illness. A similar number in each group (37%) became completely drug free.

Synthesis and characterization of N-acylated, pH-sensitive 'caged' aminophospholipids.

A series of acid-labile 'caged' phosphatidylserine (PS) and phosphatidylethanolamine (PE) molecules have been synthesized by N-acylation of the aminophospholipid with maleic, citraconic, dimethylmaleic, phthalic, or 3,4,5,6-tetrahydrophthalic anhydride. N-citraconyl-dioleoylphosphatidylethanolamine (C-DOPE) and N-citraconyl-dioleoyl-phosphatidylserine (C-DOPS) exhibited the highest degree of sensitivity to acidic pH; incubation at pH 5.5 and 6.5, respectively, resulted in 50% cleavage to the native aminophospholipid within 60 min. Significant cleavage of the phthalyl- and maleyl-PE derivatives did not occur at physiologically relevant pH values (pH 5.5-8), while tetrahydrophthalyl-PE and dimethylmaleyl-PE could not be isolated, reflecting their inherent instability. At pH 5.5, the half time for cleavage of C-DOPE and C-DOPS was 110 min and 85 min, respectively. The utility of these 'caged' lipids was demonstrated in human erythrocytes. When mixed with cells, C-DOPS, or the short chain analog, N-citraconyl-dilauroylphosphatidylserine (C-DLPS), transferred from liposome membranes into erythrocytes and remained in the cell outer monolayer. Low pH treatment released the citraconyl group and the free PS was transported to the inner monolayer by the aminophospholipid transporter. These novel 'caged' phospholipids may be useful for the study of transmembrane aminophospholipid transport, protein-lipid interactions and membrane fusion.

Impact of increased physician supply on use of health services: a longitudinal analysis in rural Minnesota.

Prevalence studies of the use of ambulatory health care services have consistently reported relatively lower demand for services in rural areas. Such studies have implied that low use rates may be fixed characteristics of rural populations and may be resistant to the influence of manipulable variables such as supply of physicians. This longitudinal study suggests that use rates are in fact significantly changed after improvement of manpower resources, but that the effects are limited to the vicinity of new practice locations.

Substance abuse problems in Scotland.

In the wake of a recent epidemic of substance abuse in Scotland the governmental and medical response has involved a departure from the longstanding policies in Britain. There has been a shift from the medical model to a more community based, minimal intervention model. There have also been a number of changes in legislation relating to substance abuse. It may take a number of years for the effects of these policy changes to be felt. However, the way has been made clear for new initiatives and Scotland is encouraging its inhabitants to take more responsibility for their own lives. A detailed cost benefit analysis of the policy changes is now indicated.

Alcohol dependence and cue reactivity.

A group of 35 severely dependent male alcoholic patients undergoing cue exposure treatment were studied. Thirty-one subjects had complete data and their physiological and subjective responses to drink cues on the first day of the cue exposure program were subjected to a principal components analysis. The principal component loadings were then used to construct a single measure of responsivity and this responsivity measure was found to correlate significantly with Severity of Alcohol Dependence Questionnaire (SADQ) scores. Further analysis of the relationship between responsivity and the subscales of the SADQ showed that the experience of affective withdrawal symptoms, craving for alcohol and drinking to relieve withdrawal symptoms were the most strongly correlated with responsivity.