Freeze/thaw of IGG solutions.

In this communication, the effect of mannitol and trehalose crystallization on the unfolding of IgG1, a monoclonal antibody, in the frozen state with repeat freeze/thaw under different pH conditions was explored. Formulations were annealed at -20 °C for 20 h five times (interrupted by freeze/thaw). This was done to induce excipient crystallization. Characterization of the frozen-thawed samples was performed by circular dichroism, particle analysis, and size exclusion chromatography. At a pH of 3, formation of insoluble and soluble aggregates was observed however, these could be reduced by the use of a surfactant. Cryoprotectant free formulations showed higher monomer content after freeze/thaw. At pH5, a single freeze/thaw cycle did not result in a significant increase in particle numbers. At pH range of 4-7 however, aggregate formation in the size range of 1-25 µm was observed after 5freeze/thaw cycles.

Crystallizing amino acids as bulking agents in freeze-drying.

Bulking agents as mannitol (Man) and glycine (Gly) require high bulking agent to stabilizer ratios to ensure their crystallization during the freeze-drying process. The aim of this study was to investigate several amino acids (AA) as potential alternative bulking agents in low AA to sucrose (Suc) ratios. A fast freeze-drying process was performed above the collapse temperature (Tc) of the amorphous phase challenging the crystalline AA scaffold. Lyophilizates and liquid formulations were characterized by differential scanning calorimetry. Karl-Fischer titration and X-ray powder diffraction as well as macroscopic cake appearance and reconstitution times were evaluated. Stability of a monoclonal antibody was investigated by UV-Vis spectroscopy, light obscuration and size exclusion chromatography. Phenylalanine (Phe), leucine (Leu) and isoleucine (Ile) crystallized upon freeze-drying at 5/45 and 10/40 AA/Suc ratios. 2.5/47.5 AA/Suc ratio showed less pronounced crystallization. Crystallization peaks were not suppressed by 2 or 50 mg/mL antibody in Leu and Ile lyophilizates. Reconstitution times could be improved by addition of surfactant. No signs of protein aggregation were detected after freeze-drying. Man and Gly yielded inacceptable cake appearance when dried with the fast freeze-drying cycle in such low bulking agent to Suc ratios. Leu, Ile and Phe can be used as alternate bulking agents at lower bulking agent to Suc ratios compared to Man or Gly. The selected AAs provided promising cake characteristics and good protein process stability.

Impact of fast and conservative freeze-drying on product quality of protein-mannitol-sucrose-glycerol lyophilizates.

PURPOSE: Mannitol/sucrose formulations are employed to generate lyophilizates for biopharmaceuticals with an elegant cake appearance. The aim of this study was to dry protein/mannitol/sucrose formulations as fast as possible without loss of cake appearance and protein stability. Glycerol was included as potential additional protein stabilizer. Three proteins (lysozyme and two monoclonal antibodies) at low and high concentration were analyzed comparing fast with conservative freeze-drying. METHODS: Freeze-drying cycle development was carried out with mannitol/sucrose formulations. A product temperature (Tp) close to the Te of mannitol and clearly above the Tg' of sucrose was targeted. Protein formulations were exposed to the final fast lyophilisation process and to a conservative freeze-drying cycle. Lyophilizates were characterized by differential scanning calorimetry, Karl-Fischer titration and X-ray diffractometry. Additionally, macroscopic cake appearance and reconstitution times were evaluated. Protein stability was characterized by UV/Vis spectroscopy, light obscuration and size exclusion chromatography. RESULTS: The fast freeze-drying cycle resulted in a primary drying time of 7 h (Tp: -10 °C) and a secondary drying time of 2 h in contrast to 47 h (Tp: -39 °C) and 12 h for the conservative cycle. Lyophilizates showed Tg values above 60 °C, a residual moisture level of 1%, reconstitution times of less than 35 s, δ-mannitol and elegant cake appearance. Mannitol/sucrose ratios below 4/1 did not lead to complete mannitol crystallization and were therefore not suitable for the selected process conditions. Characterisation of protein stability rendered low aggregation and particle levels for both, fast and conservative freeze-drying conditions. CONCLUSIONS: It was shown that fast freeze-drying of mannitol/sucrose formulations above Tg' at a Tp of -10 °C resulted in good protein process stability and appropriate cake characteristics at maximum time reduction.

Detection of Collapse and Crystallization of Saccharide, Protein, and Mannitol Formulations by Optical Fibers in Lyophilization.

The collapse temperature (Tc) and the glass transition temperature of freeze-concentrated solutions (Tg') as well as the crystallization behavior of excipients are important physicochemical characteristics which guide the cycle development in freeze-drying. The most frequently used methods to determine these values are differential scanning calorimetry (DSC) and freeze-drying microscopy (FDM). The objective of this study was to evaluate the optical fiber system (OFS) unit as alternative tool for the analysis of Tc, Tg' and crystallization events. The OFS unit was also tested as a potential online monitoring tool during freeze-drying. Freeze/thawing and freeze-drying experiments of sucrose, trehalose, stachyose, mannitol, and highly concentrated IgG1 and lysozyme solutions were carried out and monitored by the OFS. Comparative analyses were performed by DSC and FDM. OFS and FDM results correlated well. The crystallization behavior of mannitol could be monitored by the OFS during freeze/thawing as it can be done by DSC. Online monitoring of freeze-drying runs detected collapse of amorphous saccharide matrices. The OFS unit enabled the analysis of both Tc and crystallization processes, which is usually carried out by FDM and DSC. The OFS can hence be used as novel measuring device. Additionally, detection of these events during lyophilization facilitates online-monitoring. Thus the OFS is a new beneficial tool for the development and monitoring of freeze-drying processes.

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability.

PURPOSE: The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported. METHODS: The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively. RESULTS: Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D2O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing. CONCLUSIONS: The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.

Synaptic mechanisms of adenosine A2A receptor-mediated hyperexcitability in the hippocampus.

Adenosine inhibits excitatory neurons widely in the brain through adenosine A1 receptor, but activation of adenosine A2A receptor (A2A R) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A2A R expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole-cell recording from identified postsynaptic neurons we show that A2A R facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A2A R enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A2A R has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A2A R activation alters the synaptic excitation - inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A2A R promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high-frequency electrical stimulation. Our results revealed selective synapse- and cell type specific adenosine A2A R effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A2A R's facilitatory effect on cortical network activity.

Genetic dissection of TrkB activated signalling pathways required for specific aspects of the taste system.

BACKGROUND: Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking. RESULTS: Here, we demonstrate, in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLCγ docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered. CONCLUSIONS: We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment.

Ablation of TrkB signalling in CCK neurons results in hypercortisolism and obesity.

Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity leads to debilitating neuroendocrine or metabolic disorders such as Cushing's syndrome (CS). Glucocorticoids control HPA axis activity through negative feedback to the pituitary gland and the central nervous system (CNS). However, the cellular mechanisms involved are poorly understood, particularly in the CNS. Here we show that, in mice, selective loss of TrkB signalling in cholecystokinin (CCK)-GABAergic neurons induces glucocorticoid resistance, resulting in increased corticotrophin-releasing hormone expression, chronic hypercortisolism, adrenocortical hyperplasia, glucose intolerance and mature-onset obesity, reminiscent of the human CS phenotype. Interestingly, obesity is not due to hyperphagia or decreased energy expenditure, but is associated with increased de novo lipogenesis in the liver. Our study therefore identifies CCK neurons as a novel and critical cellular component of the HPA axis, and demonstrates the requirement of TrkB for the transmission of glucocorticoid signalling.

Loss of NGF-TrkA signaling from the CNS is not sufficient to induce cognitive impairments in young adult or intermediate-aged mice.

Many molecules expressed in the CNS contribute to cognitive functions either by modulating neuronal activity or by mediating neuronal trophic support and/or connectivity. An ongoing discussion is whether signaling of nerve growth factor (NGF) through its high-affinity receptor TrkA contributes to attention behavior and/or learning and memory, based on its expression in relevant regions of the CNS such as the hippocampus, cerebral cortex, amygdala and basal forebrain. Previous animal models carrying either a null allele or transgenic manipulation of Ngf or Trka have proved difficult in addressing this question. To overcome this problem, we conditionally deleted Ngf or Trka from the CNS. Our findings confirm that NGF-TrkA signaling supports survival of only a small proportion of cholinergic neurons during development; however, this signaling is not required for trophic support or connectivity of the remaining basal forebrain cholinergic neurons. Moreover, comprehensive behavioral analysis of young adult and intermediate-aged mice lacking NGF-TrkA signaling demonstrates that this signaling is dispensable for both attention behavior and various aspects of learning and memory.

Clients' perceptions of opioid substitution treatment: an input to improving the quality of treatment.

Despite the emphasis on providing high quality mental health and addiction treatment, there has been relatively little consideration given to examining clients' perceptions of addiction treatment (consumer satisfaction) as a quality improvement strategy. The present article reports on a survey of a representative sample of 93 clients receiving opioid substitution treatment (OST). Employed participants reported higher treatment satisfaction and a pattern of positive associations was found between satisfaction and general health, mental health, social functioning, lower methadone doses, and participants' ratings of their treatment progress. Lower satisfaction was associated with higher frequency of benzodiazepine use, and, for women, longer treatment duration. Maori participants rated their treatment progress as lower than that of non-Maori. Results strongly endorse recording participants' comments to provide a deeper understanding of survey item ratings. The study findings highlight the need for a research focus on the roles of mental health and other registered nurses who work with people receiving OST in specialist service and primary care settings, and endorse a partnership approach to future research in this area. The pattern of findings arising from this study suggests key strategies for improving the flexibility and client responsiveness of OST.

PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity.

BACKGROUND: The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. RESULTS: Here we report that a point mutation at the phospholipase Cγ (PLCγ) docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2) specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF), is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system) on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. CONCLUSIONS: These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

Burnout in hospital-based medical consultants in the New Zealand public health system.

AIM: To assess the prevalence and severity of burnout in hospital-based medical consultants, and investigate associated demographic and professional characteristics. METHOD: Utilising standardised measures of burnout (Maslach Burnout Inventory) and job satisfaction (Job Satisfaction Scale) this cross-sectional study recruited 267 consultants working in a large tertiary hospital in Christchurch, New Zealand. RESULTS: Seventy-one percent of all eligible participants were recruited. The prevalence of burnout in each of the three dimensions was as follows: High Emotional Exhaustion=29.7%; High Depersonalisation=24.4%; Low Personal Accomplishment=31.2%. One in five consultants was assessed as having high overall burnout. Considered against the psychometric norms for medical workers, significantly more consultants than expected reported low Emotional Exhaustion (p<0.001) and low Depersonalisation (p<0.01). Working longer hours (p<0.01), lower job satisfaction (p<0.001), and shorter time in the current job (p<0.05) independently increased the risk of high Emotional Exhaustion. Working longer hours (p<0.05) and lower job satisfaction (p<.01) independently increased the risk of high Depersonalisation. Longer time in the same job increased the risk of low Personal Accomplishment (p<0.05). Longer hours worked (p<0.05), shorter vocational experience as a consultant (p<0.05), and lower job satisfaction (p<0.001) independently increased the risk of high overall burnout. CONCLUSION: An unexpected proportion of consultants experience robust emotional well-being and healthy work engagement. However, for those experiencing high burnout, by severity or dimension, working long hours and low job satisfaction appear to be particularly contributory factors. Whilst remedial interventions should target the minority who experience significant burnout, studies using robust research designs are required to assess the meaningful clinical utility of these. The challenge remains to determine the optimal organisational practices to minimise burnout in this workforce.

Timed feeding of mice modulates light-entrained circadian rhythms of reticulated platelet abundance and plasma thrombopoietin and affects gene expression in megakaryocytes.

Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light-dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light-dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light-dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.

Development of a brief treatment instrument for routine clinical use with methadone maintenance treatment clients: the methadone treatment index.

Routine measurement of treatment outcome between clinician and client in alcohol and drug user treatment services is an important quality improvement initiative. It is particularly important for clients receiving long-term treatment such as methadone maintenance treatment, as fluctuations in substance use, functioning, and health are to be expected. Although there are a number of standardized alcohol and drug user treatment outcome instruments available for research and clinical use, a key challenge is to develop clinical instruments that will actually be used routinely in busy practice settings by a range of staff. Such instruments need to be brief, acceptable to staff and clients, easy to use, provide immediate feedback, and meet adequate psychometric requirements. This report describes development work undertaken in three studies of the Methadone Treatment Index (MTI). The MTI is a brief instrument comprising measures of recent substance use, aspects of social and behavioral functioning, and physical and psychological health. The MTI was designed in consultation with clinicians and clients for use in monitoring treatment progress with clients receiving methadone maintenance treatment. Key findings were that the MTI was acceptable to clients, produced clinically relevant information, and has satisfactory psychometric properties, although it was not used to measure change in this study. Further evaluation of the MTI on a longitudinal basis is supported.

Specific and nonspecific comorbidity in anorexia nervosa.

OBJECTIVE: This article reports lifetime Axis I and II comorbidity in women with anorexia nervosa (AN), and ascertains specific and nonspecific comorbidity in AN compared to clinical samples of women with bulimia nervosa (BN) or major depression (DEP). METHOD: Outpatient AN (n = 56), BN (n = 132), and DEP (n = 100) samples were assessed using Structured Clinical Interviews I and II for DSM-III-R. Baseline data were compared using univariate statistics and logistic regression. RESULTS: In the AN sample as a whole, specific elevations were found for prevalences of obsessive compulsive disorder. The AN-binge eating purging subtype (AN-BP) and the BN sample had elevated prevalences of Cluster B personality disorders. Cluster C prevalences were elevated across samples. CONCLUSION: Evidence of AN-specific, eating disorder-specific, and nonspecific comorbidity illustrates the heterogeneity in AN. Further research is need to examine the relative impact of specific and nonspecific comorbidity in AN subtypes and AN as a whole.

Entrainment to feeding but not to light: circadian phenotype of VPAC2 receptor-null mice.

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2-/-) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2-/- mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2-/- mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

Nursing student attitudes to psychiatric nursing and psychiatric disorders in New Zealand.

Attitudes towards mental illness and psychiatric nursing can have far-reaching consequences for patients and the perceived desirability of this as a future career choice. Attitudes can be both brought into training and affected by training experiences, although the direction of these associations is unclear. Using a cross-section design, this study investigates the associations between attitudes, demographic variables, mental illness exposure, and career aspirations in 164 nursing students. Year of training and area of intended practice independently predicted attitudes. These findings have important implications for nursing training. They demonstrate the need for specifically focusing training to target the students with the most negative attitudes.

Health status of clients receiving methadone maintenance treatment using the SF-36 health survey questionnaire.

This study investigated the health status of a representative sample of clients (35 Maori, 72 non-Maori) receiving methadone maintenance treatment in New Zealand, using the SF-36 health survey. The publication of New Zealand norms in 1999 enabled comparisons of the health of the Methadone Treatment Programme study participants with that of the New Zealand population. Although over 50% of participants rated their health as good, very good or excellent, 44% rated their health as fair or poor and compared with population norms, the health of the study participants was significantly poorer on all eight SF-36 scales. Male and female participants rated their health similarly to male and female clients attending another New Zealand Methadone Treatment Programme. Results highlighted the impact of a chronic disorder and co-existing health-related problems on the health and well-being and day-to-day functioning of this client group. Higher frequency of benzodiazepine use was associated with poorer social functioning, mental health and role functioning and higher frequency of cannabis use was associated with poorer role functioning due to emotional problems. Findings support routine monitoring of health status with clients receiving methadone maintenance treatment as a guide to preventative and treatment interventions and health maintenance strategies.

Anxiety and psychoactive substance use disorder comorbidity in anorexia nervosa or depression.

OBJECTIVE: This study considered whether the prevalence and type of anxiety and psychoactive substance use disorder (PSUD) diagnoses differ between women with spectrum anorexia nervosa (AN) (N=40) and women with major depressive disorder (N = 58) participating in outpatient clinical trials. METHOD: Anxiety and PSUD diagnoses (according to criteria in the 3rd Rev. ed. of the Diagnostic and Statistical Manual of Mental Disorders) were assessed using structured clinical interviews. Comparisons were made between AN subtypes (restricting or binge eating/purging) and by history of depression within the AN sample. RESULTS: A high prevalence of obsessive-compulsive disorder (OCD) was found in women with AN. However, social phobia, simple phobia, and PSUD were significantly elevated in both women with depression and women with AN. Prevalences were similar for anxiety and PSUD diagnoses between AN subtypes. DISCUSSION: Women with anorexia or depression were comparable in all respects, except for the elevated OCD prevalence in AN, emphasizing the need to use clinical comparison groups to avoid inadvertently attributing elevated prevalences of comorbid conditions to specific disorders.

"Knowing one's self" anorexic: implications for therapeutic practice.

OBJECTIVE: Recent postmodernist studies of anorexia nervosa (AN) challenge current clinical understandings and therapies by illuminating not what AN is but how what it is known to be by clinicians helps construct the disorder and therapy for it. This study points to the equal if not greater importance of how patients know AN. METHODS: Using a deconstructive approach, the discourses of a group of women diagnosed with severe AN were analyzed to reveal radically different versions of "knowing one's self" anorexic. RESULTS: These versions of "self" have strategically different implications for, and meanings of, any therapeutic endeavour. DISCUSSION: Postmodernist approaches point to the need for social reconstruction of lay and community understandings of AN. They also have implications at the level of individual therapy, and could be deployed with patients to establish individual but authentic bases for therapy.