Preparation of medicines for children - A hierarchy of classification

There is some confusion about the types of paediatric pharmaceutical preparation (in a regulatory and pharmaceutical development context) that are acceptable for approval by medicines regulators. Some of the confusion relates to terminology which may mean different things to different stakeholders. It may not always be possible to provide authorised, commercially manufactured, age appropriate, ready-to-administer preparations. In terms of assurance of quality and bioavailability there is a continuum from this ideal through intermediate products through authorised compounding and manipulation of commercial dosage forms to ad hoc compounding using only the skills and experience of the individual pharmacist. Additionally, it is widely known that caregivers may manipulate medicines at home, for example by segmenting tablets and by addition to foods or liquids. The first intent of the manufacturer should be to provide for children an age appropriate, ready-to-administer preparation which is commercially manufactured and approved by the competent authorities. However, there will still be a place for providing other age appropriate preparations such as approved products that are 'intermediates' requiring reconstitution before use, or instructions for compounding or manipulation of a dosage form. If compounding or manipulation is likely to be required it is preferable that data are generated by Industry, approved by the competent authorities and provided in the Summary of Product Characteristics (SmPC). It is acknowledged however, that ad hoc compounding or manipulation may also take place in certain circumstances such as logistical difficulties or to satisfy the needs of the child who does not find the authorised product to be 'age appropriate'. This paper explores compounding and manipulation of medicines in relation to approval by medicines regulators and non-approved preparation to fulfil the needs of the individual patient. Definitions are proposed to provide a hierarchical classification based on assurances of quality and bioavailability.     

Development and validation of a streamlined method designed to detect residues of 62 veterinary drugs in bovine kidney using ultra-high performance liquid chromatography--tandem mass spectrometry

In the USA, the US Department of Agriculture's Food Safety and Inspection Service (FSIS) conducts the National Residue Program designed to monitor veterinary drug and other chemical residues in beef and other slaughtered food animals. Currently, FSIS uses a 7-plate bioassay in the laboratory to screen for antimicrobial drugs in bovine kidneys from those animals tested positive by inspectors in the slaughter establishments. The microbial inhibition bioassay has several limitations in terms of monitoring scope, sensitivity, selectivity, and analysis time. Ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) has many advantages over the bioassay for this application, and this study was designed to develop, evaluate, and validate a fast UHPLC-MS/MS method for antibiotics and other high-priority veterinary drugs in bovine kidney. Five existing multi-class, multi-residue methods from the literature were tested and compared, and each performed similarly. Experiments with incurred samples demonstrated that a 5-min shake of 2 g homogenized kidney with 10 ml of 4/1 (v/v) acetonitrile/water followed by simultaneous clean-up of the initial extract with 0.5 g C18 and 10 ml hexane gave a fast, simple, and effective sample preparation method for the <10 min UHPLC-MS/MS analysis. An extensive 5-day validation process demonstrated that the final method could be used to acceptably screen for 54 of the 62 drugs tested, and 50 of those met qualitative MS identification criteria. Quantification was not needed in the application, but the method gave ≥ 70% recoveries and ≤ 25% reproducibilities for 30 of the drugs.     

Ablation of TNF-RI/RII expression in Alzheimer's disease mice leads to an unexpected enhancement of pathology: implications for chronic pan-TNF-α suppressive therapeutic strategies in the brain

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-β phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-β peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.     

Age-dependent alterations in nerve growth factor (NGF)-related proteins, sortilin, and learning and memory in rats

The objective of this study was to evaluate the effects of aging on the performance of specific memory-related tasks in rats as well as to determine the levels of several nerve growth factor (NGF)-related proteins in relevant brain regions. The results indicated age-related impairments in spatial learning in a water maze task as well as deficits in recognition memory in a Spontaneous Novel Object Recognition task. In the prefrontal cortex and hippocampus, aged rats (compared to young controls) had elevated levels of the proneurotrophin, proNGF (+ 1.8-1.9 fold), p75^NTR receptors (+ 1.6-1.8 fold) and sortilin (+ 1.8-2.1 fold), and decreased levels of mature NGF (−36 to 44%), and phospho-TrkA receptors (−45 to 49%). The results of this study support the argument that NGF signaling is altered in the aging brain, and that such alterations may contribute to an age-related decline in cognitive function. These results may also help to identify specific components of the NGF-signaling pathway that could serve as targets for novel drug discovery and development for age-related disorders of cognition (e.g., Alzheimer's disease).     

Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis.Pneumonic tularemia is a highly debilitating disease caused by the Gram-negative coccobacillus Francisella tularensis. Strains classified under subspecies tularensis (type A) are the most virulent and pose the biggest challenge from a clinical perspective (28), with a mortality rate estimated to exceed 30% in untreated patients (11). Prophylactic vaccination is the best countermeasure, and there is good historical evidence that pneumonic tularemia can be prevented by vaccination with the attenuated F. tularensis live vaccine strain (LVS) (37). However, LVS is unlikely to be licensed for mass vaccination because the mechanism of attenuation has not been defined. Due to the potential of a major public health threat, there is an urgent need to understand the protective mechanisms associated with an effective immune response so that novel vaccines can be developed.Protective immunity against F. tularensis infection is usually attributed to an effective T cell response. However, F. tularensis has a significant extracellular phase, which makes it accessible to humoral immune responses (18). Indeed, there is ample evidence that B cells and antibodies are necessary for mice to develop their natural resistance to primary and secondary LVS infections. Purified lipopolysaccharide (LPS) from LVS induced a population of B1-a cells within 2 to 3 days of administration that protected mice against intraperitoneal (i.p.) LVS challenge (6, 7, 14). Consistent with these results, μMT mice lacking mature B cells exhibited increased susceptibility to primary intradermal (i.d.) LVS infection and delayed bacterial clearance (15, 40). μMT mice were also more susceptible to secondary i.p. LVS infection, and this defect was corrected by reconstitution with LVS-primed B cells (15). The contribution of antibodies has been addressed repeatedly in passive immunization experiments, which showed that immune serum from humans and mice vaccinated with live or inactivated LVS protected naïve mice against challenges with LVS or other low virulence strains given by a variety of routes (13, 19, 26, 29, 33, 36, 40). The dominant antibody response was directed at LPS, but antibodies against protein antigens have also been found (17, 23, 31, 41, 43). Monoclonal antibodies specific for LPS or the outer membrane protein FopA provided significant protection against LVS challenge when given either prophylactically (38) or therapeutically (30, 38). Together, these results suggest that antibodies contribute toward effective control of attenuated or low-virulence F. tularensis strains.It has been much more difficult to demonstrate antibody-mediated protection against type A strains in mice (1, 20, 21, 38), even though they express many antigens recognized by LVS immune serum (13, 30). This is not surprising given the historical difficulties in generating protective immunity against type A strains in this animal model (5). However, Ray et al. recently showed that oral LVS vaccination protected mice against a pulmonary SCHU S4 challenge in an antibody-dependent manner (35). Klimpel et al. also reported a similar finding using immune serum from mice cured of a lethal intranasal (i.n.) SCHU S4 infection with levofloxacin in a passive immunization model (27). Thus, the protective effects of antibodies appear not to be restricted only to low-virulence strains but may also contribute to the protection against highly virulent type A strains.To further characterize the mechanism of antibody-mediated protection, we utilized the recently characterized Fischer 344 (F344) rat model (45). Since F344 rats developed much stronger resistance to respiratory SCHU S4 challenge after LVS vaccination than previously observed in mice, we speculated that antibodies may provide better protection in this model and allow us to define their protective mechanism more thoroughly. We now show in a passive immunization model that serum antibodies from LVS-vaccinated rats conferred protection against a lethal intratracheal (i.t.) SCHU S4 challenge. Protection correlated with reduced systemic bacterial growth and less severe histopathology during the early phase of infection and bacterial clearance by a T cell-dependent mechanism. Thus, antibodies contribute to but are not sufficient for the effective control of respiratory infections by fully virulent type A strains. Our studies provide valuable insights into the protective mechanisms of antibodies that will guide future development of tularemia vaccine candidates.     

Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods

An important tool in cell biology is the combination of immunogold labelling and transmission electron microscopy (TEM) by which target molecules (e.g. antigens) are bound specifically to affinity markers (primary antibodies) and then detected and localised with visualisation probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron-dense, punctate and available in different sizes whilst TEM provides high-resolution images of particles and cell compartments. By virtue of these properties, the combination can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labelling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labelling between compartments or groups of cells). The following approaches are possible: (i) A target molecule can be tested for preferential labelling by mapping the localisation of gold particles across a set of compartments. (ii) Data from wild-type and knockdown/knockout control cells can be used to correct raw gold particle counts, estimate specific labelling densities and then test for preferential labeling. (iii) The same antigen can be mapped in two or more groups of cells to test whether there are experimental shifts in compartment labelling patterns. (iv) A variant of this approach uses more than one size of gold particle to test whether or not different antigens colocalise in one or more compartments. (v) In studies involving antigen translocation, absolute numbers of gold particles can be mapped over compartments at specific positions within polarised, oriented or dividing cells. Here, the current state of the art is reviewed and approaches are illustrated with virtual datasets.     

Western diet consumption and cognitive impairment: links to hippocampal dysfunction and obesity

Intake of saturated fats and simple carbohydrates, two of the primary components of a modern Western diet, is linked with the development of obesity and Alzheimer's Disease. The present paper summarizes research showing that Western diet intake is associated with cognitive impairment, with a specific emphasis on learning and memory functions that are dependent on the integrity of the hippocampus. The paper then considers evidence that saturated fat and simple carbohydrate intake is correlated with neurobiological changes in the hippocampus that may be related to the ability of these dietary components to impair cognitive function. Finally, a model is described proposing that Western diet consumption contributes to the development of excessive food intake and obesity, in part, by interfering with a type of hippocampal-dependent memory inhibition that is critical in the ability of animals to refrain from responding to environmental cues associated with food, and ultimately from consuming energy intake in excess of that driven solely by caloric need.