Increased target specificity of anti-HER2 genospheres by modification of surface charge and degree of PEGylation.

Genospheres are cationic lipid-nucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70-110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pH-dependent modulation of the Genosphere surface charge using pH-titratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particle-cell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol %) and high (5 mol %) grafting densities, as well as the effects of HER2-receptor-directed targeting by an internalizable anti-HER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pH-titratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1-(3-(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1,2-distearoyl-sn-glycero-3-hemiglutarate) rendered the particles surface-charge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zeta-potential measurements. In HER2-targeted systems, transfection activity and target specificity with HER2-overexpressing SKBR-3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol % PEG-lipid had only minor effects on Genosphere-cell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed.

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.

Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.

We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospheres) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.

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.

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.

A multicentre, randomized, double-blind, placebo-controlled trial of naltrexone in the treatment of alcohol dependence or abuse.

The opioid antagonist, naltrexone, is reported, in single centre studies, to improve the clinical outcome of individuals with alcohol dependence participating in outpatient psychosocial programmes. This is the first multicentre controlled study to evaluate the efficacy and safety of naltrexone as adjunctive treatment for alcohol dependence or abuse. Patients who met criteria for alcohol dependence (n = 169) or alcohol abuse (n = 6) were randomly assigned to receive double-blind oral naltrexone 50 mg daily (n = 90) or placebo (n = 85) for 12 weeks as an adjunct to psychosocial treatment. The primary efficacy variable was time to first episode of heavy drinking; secondary efficacy assessments included time to first drink, alcohol consumption, craving, and changes in the serum biological markers gamma-glutamyl transferase (GGT), and aspartate and alanine aminotransferases. Compliance was assessed by tablet counts and, in the naltrexone-treated group, by measurement of urinary concentrations of 6-ss-naltrexol. Forty-nine (58%) patients randomized to placebo and 53 (59%) randomized to naltrexone did not complete the study. In intention-to-treat analyses, there was no difference between groups on measures of drinking. The median reduction from baseline of serum GGT (P: < 0.05) and the reductions in alcohol craving (Obsessive and Compulsive Drinking Scale: OCDS) were greater in the naltrexone group (P: < 0.05), from approximately half-way through the study. Of 70 patients (35 placebo; 35 naltrexone) who met an a priori definition of compliance (80% tablet consumption, attendance at all follow-up appointments), those allocated to naltrexone reported consuming half the amount of alcohol (P: < 0.05), had greater median reduction in serum GGT activity (P: < 0.05), and greater reduction in alcohol craving (OCDS total score: P: < 0.05; Obsessive subscale score: P: < 0.05), compared to patients in the placebo group. Use of naltrexone raised no safety concerns. Naltrexone is effective in treating alcohol dependence/abuse in conjunction with psychosocial therapy, in patients who comply with treatment.

Liposome targeting to tumors using vitamin and growth factor receptors.

Liposome-encapsulated anticancer drugs reveal their potential for increased therapeutic efficacy and decreased nonspecific toxicities due to their ability to enhance the delivery of chemotherapeutic agents to solid tumors. Advances in liposome technology have resulted in the development of ligand-targeted liposomes capable of selectively increasing the efficacy of carried agents against receptor-bearing tumor cells. Receptors for vitamins and growth factors have become attractive targets for ligand-directed liposomal therapies due to their high expression levels on various forms of cancer and their ability to internalize after binding to the liposomes conjugated to receptors' natural ligands (vitamins) or synthetic agonists (receptor-specific antibodies and synthetic peptides). This chapter summarizes various strategies and advances in targeting liposomes to vitamin and growth factor receptors in vitro and in vivo with special emphasis on two extensively studied liposome-targeting systems utilizing folate receptor and HER2/neu growth factor receptor.

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.

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.

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.

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.

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.

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.

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.

Sexual abuse in people with alcohol problems. A study of the prevalence of sexual abuse and its relationship to drinking behaviour.

BACKGROUND: There is evidence that people with a history of sexual abuse may have an increased risk of developing alcohol and drug problems. METHOD: A self-completion sexual abuse questionnaire was designed and administered to a sample of attenders at three London alcohol services. Drinking behaviour was assessed using the Severity of Alcohol Dependence Questionnaire and the Alcohol Problems Questionnaire, and additional data were derived from case notes. RESULTS: Fifty-four per cent of women and 24% of men identified themselves as victims of sexual abuse or assault For the majority this had started before the age of 16 and involved non-relatives. Subjects with a history of sexual abuse were younger, reached drinking milestones earlier, were more likely to have a family history of alcohol misuse and had more alcohol-related problems than non-abused subjects. Sexual abuse, age and alcohol dependence predicted level of problems in a regression analysis. CONCLUSIONS: The high rates of sexual abuse and its association with indications of increased morbidity suggest it is an important issue for the management of alcohol problems. More use could be made of self-completion questionnaires for the investigation of sexual abuse.

PIP: Several community-based studies have found significantly higher rates of alcohol and drug abuse among those with a history of child sexual abuse. This association was investigated further in a sample of 126 consecutive attendees (89 men and 37 women) at three alcohol treatment centers in London, England. 25 men (24%) and 21 women (54%) reported a sexual abuse history. 43 (69%) of the 62 perpetrators reported by respondents were not family members. The mean age at first occurrence of abuse was 12.6 years and 75% of victims were 15 years of age or younger at first incident. Victims of sexual abuse were younger at presentation for treatment and had significantly higher scores on the Severity of Dependence Questionnaire and the Alcohol Problem Questionnaire than their nonabused counterparts. They also developed drinking problems at an earlier age and were more likely to have other psychiatric problems. 53.4% of the variance in drinking was contributed by alcohol dependence, age, and sexual abuse. These findings suggest that sexual abuse is a common childhood experience among men and women who seek help for alcohol problems. However, the lack of a control group of non-drinkers limits assessment of a possible causal link between child sexual abuse and subsequent alcohol misuse.