Pharmacy legislation:public protector or professional hindrance?

Pharmacy originates from a background of medication compounding and supply. More recently, this role has developed away from an absolute focus on the supply of pharmaceuticals with, for example, the advent of pharmacist prescribing. Nevertheless, for a majority of the profession, medication supply remains a core activity. Regulation of the pharmacy profession is now the responsibility of the General Pharmaceutical Council, although up until 27 September 2010, this role fell to the Royal Pharmaceutical Society of Great Britain (RPSGB). Before this change, in one of the most high-profile legal cases involving a pharmacist in a professional capacity, R. v Lee, a pharmacist was prosecuted firstly for gross negligence manslaughter, later revised to offences under the Medicines Act 1968, for a single error relating to medication supply, and was given a suspended custodial sentence. Offences against sections 64 or 85 of the Medicines Act are absolute offences and there is no due diligence defence. Prosecution of a pharmacist for the supply of incorrect medication may seem a measured course of action to protect the public from the wrongful supply of potent pharmacotherapeutic agents; however, further analysis of Lee indicates that this approach may be counterproductive. An appeal of the original conviction in the Lee case has resulted in a clarification of the interpretation of section 85(5); however currently, prosecutions under section 64 are still a possibility. Owing to the seriousness of a criminal conviction under section 64, this continuation will potentially stifle the profession's ability to learn from dispensing errors. © The Author [2013]. Published by Oxford University Press; all rights reserved.

Saccharomyces Cerevisiae as a biotechnological tool for ageing research:studies on translation and metabolism

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

Pharmaceutical process optimisation of bulk lyophilisates:implications of powder handling

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

Impact in contact lenses and the anterior eye:challenges prevailing in 2015

As we settle into a new year, this second issue of Contact Lens and Anterior Eye allows us to reflect on how new research in this field impacts our understanding, but more importantly, how we use this evidence basis to enhance our day to day practice, to educate the next generation of students and to construct the research studies to deepen our knowledge still further. The end of 2014 saw the publication of the UK governments Research Exercise Framework (REF) which ranks Universities in terms of their outputs (which includes their paper, publications and research income), environment (infrastructure and staff support) and for the first time impact (defined as “any effect on, change or benefit to the economy, society, culture, public policy or services, health, the environment or quality of life, beyond academia” [8]). The REF is a process of expert review, carried out in 36 subject-based units of assessment, of which our field is typically submitted to the Allied Health, Dentistry, Nursing and Pharmacy panel. Universities that offer Optometry did very well with Cardiff, Manchester and Aston in the top 10% out of the 94 Universities that submitted to this panel (Grade point Average ranked order). While the format of the new exercise (probably in 2010) to allocate the more than £2 billion of UK government research funds is yet to be determined, it is already rumoured that impact will contribute an even larger proportion to the weighting. Hence it is even more important to reflect on the impact of our research. In this issue, Elisseef and colleagues [5] examine the intriguing potential of modifying a lens surface to allow it to bind to known wetting agents (in this case hyaluronic acid) to enhance water retention. Such a technique has the capacity to reduced friction between the lens surface and the eyelids/ocular surface, presumably leading to higher comfort and less reason for patients to discontinue with lens wear. Several papers in this issue report on the validity of new high precision, fast scanning imaging and quantification equipment, utilising techniques such as Scheimpflug, partial coherence interferometry, aberrometry and video allowing detailed assessment of anterior chamber biometry, corneal topography, corneal biomechanics, peripheral refraction, ocular aberrations and lens fit. The challenge is how to use this advanced instrumentation which is becoming increasingly available to create real impact. Many challenges in contact lenses and the anterior eye still prevail in 2015 such as: -While contact lens and refractive surgery complications are relatively rare, they are still too often devastating to the individual and their quality of life (such as the impact and prognosis of patients with Acanthmoeba Keratitis reported by Jhanji and colleagues in this issue [7]). How can we detect those patients who are going to be affected and what modifications do we need to make to contact lenses and patient management prevent this occurring? -Drop out from contact lenses still occurs at a rapid rate and symptoms of dry eye seem to be the leading cause driving this discontinuation of wear [1] and [2]. What design, coating, material and lubricant release mechanism will make a step change in end of day comfort in particular? -Presbyopia is a major challenge to hassle free quality vision and is one of the first signs of ageing noticed by many people. As an emmetrope approaching presbyopia, I have a vested interest in new medical devices that will give me high quality vision at all distances when my arms won’t stretch any further. Perhaps a new definition of presbyopia could be when you start to orientate your smartphone in the landscape direction to gain the small increase in print size needed to read! Effective accommodating intraocular lenses that truly mimic the pre-presbyopic crystalline lenses are still a way off [3] and hence simultaneous images achieved through contact lenses, intraocular lenses or refractive surgery still have a secure future. However, splitting light reaching the retina and requiring the brain to supress blurred images will always be a compromise on contrast sensitivity and is liable to cause dysphotopsia; so how will new designs account for differences in a patient's task demands and own optical aberrations to allow focused patient selection, optimising satisfaction? -Drug delivery from contact lenses offers much in terms of compliance and quality of life for patients with chronic ocular conditions such as glaucoma, dry eye and perhaps in the future, dry age-related macular degeneration; but scientific proof-of-concept publications (see EIShaer et al. [6]) have not yet led to commercial products. Part of this is presumably the regulatory complexity of combining a medical device (the contact lens) and a pharmaceutical agent. Will 2015 be the year when this innovation finally becomes a reality for patients, bringing them an enhanced quality of life through their eye care practitioners and allowing researchers to further validate the use of pharmaceutical contact lenses and propose enhancements as the technology matures? -Last, but no means least is the field of myopia control, the topic of the first day of the BCLA's Conference in Liverpool, June 6–9th 2015. The epidemic of myopia is a blight, particularly in Asia, with significant concerns over sight threatening pathology resulting from the elongated eye. This is a field where real impact is already being realised through new soft contact lens optics, orthokeratology and low dose pharmaceuticals [4], but we still need to be able to better predict which technique will work best for an individual and to develop new techniques to retard myopia progression in those who don’t respond to current treatments, without increasing their risk of complications or the treatment impacting their quality of life So what will your New Year's resolution be to make 2015 a year of real impact, whether by advancing science or applying the findings published in journals such as Contact Lens and Anterior Eye to make a real difference to your patients’ lives?

Freezing process optimisation of pharmaceutical formulations for freeze drying

The body of work presented in this thesis are in three main parts: [1] the effect of ultrasound on freezing events of ionic systems, [2] the importance of formulation osmolality in freeze drying, and [3] a novel system for increasing primary freeze drying rate. Chapter 4 briefly presents the work on method optimisation, which is still very much in its infancy. Aspects of freezing such as nucleation and ice crystal growth are strongly related with ice crystal morphology; however, the ice nucleation process typically occurs in a random, non-deterministic and spontaneous manner. In view of this, ultrasound, an emerging application in pharmaceutical sciences, has been applied to aid in the acceleration of nucleation and shorten the freezing process. The research presented in this thesis aimed to study the effect of sonication on nucleation events in ionic solutions, and more importantly how sonication impacts on the freezing process. This work confirmed that nucleation does occur in a random manner. It also showed that ultrasonication aids acceleration of the ice nucleation process and increases the freezing rate of a solution. Cryopreservation of animal sperm is an important aspect of breeding in animal science especially for endangered species. In order for sperm cryopreservation to be successful, cryoprotectants as well as semen extenders are used. One of the factors allowing semen preservation media to be optimum is the osmolality of the semen extenders used. Although preservation of animal sperm has no relation with freeze drying of pharmaceuticals, it was used in this thesis to make a case for considering the osmolality of a formulation (prepared for freeze drying) as a factor for conferring protein protection against the stresses of freeze drying. The osmolalities of some common solutes (mostly sugars) used in freeze drying were determined (molal concentration from 0.1m to 1.2m). Preliminary investigation on the osmolality and osmotic coefficients of common solutes were carried out. It was observed that the osmotic coefficient trend for the sugars analysed could be grouped based on the types of sugar they are. The trends observed show the need for further studies to be carried out with osmolality and to determine how it may be of importance to protein or API protection during freeze drying processes. Primary drying is usually the longest part of the freeze drying process, and primary drying times lasting days or even weeks are not uncommon; however, longer primary drying times lead to longer freeze drying cycles, and consequently increased production costs. Much work has been done previously by others using different processes (such as annealing) in order to improve primary drying times; however, these do not come without drawbacks. A novel system involving the formation of a frozen vial system which results in the creation of a void between the formulation and the inside wall of a vial has been devised to increase the primary freeze drying rate of formulations without product damage. Although the work is not nearly complete, it has been shown that it is possible to improve and increase the primary drying rate of formulations without making any modifications to existing formulations, changing storage vials, or increasing the surface area of freeze dryer shelves.

EPCIS Event-Based traceability in pharmaceutical supply chains via automated generation of linked pedigrees

In this paper we show how event processing over semantically annotated streams of events can be exploited, for implementing tracing and tracking of products in supply chains through the automated generation of linked pedigrees. In our abstraction, events are encoded as spatially and temporally oriented named graphs, while linked pedigrees as RDF datasets are their specific compositions. We propose an algorithm that operates over streams of RDF annotated EPCIS events to generate linked pedigrees. We exemplify our approach using the pharmaceuticals supply chain and show how counterfeit detection is an implicit part of our pedigree generation. Our evaluation results show that for fast moving supply chains, smaller window sizes on event streams provide significantly higher efficiency in the generation of pedigrees as well as enable early counterfeit detection.

"Trade creep" and implications of the Transatlantic Trade and Investment Partnership Agreement for the United Kingdom National Health Service

The ambitious and comprehensive Transatlantic Trade and Investment Partnership Agreement (TTIP/TAFTA) agreement between the European Union and United States is now being negotiated and may have far-reaching consequences for health services. The agreement extends to government procurement, investment, and further regulatory cooperation. In this article, we focus on the United Kingdom National Health Service and how these negotiations can limit policy space to change policies and to regulate in relation to health services, pharmaceuticals, medical devices, and health industries. The negotiation of TTIP/TAFTA has the potential to "harmonize" more corporate-friendly regulation, resulting in higher costs and loss of policy space, an example of "trade creep" that potentially compromises health equity, public health, and safety concerns across the Atlantic.

OntoPedigree:modelling pedigrees for traceability in supply chains

The sharing of product and process information plays a central role in coordinating supply chains operations and is a key driver for their success. "Linked pedigrees" - linked datasets, that encapsulate event based traceability information of artifacts as they move along the supply chain, provide a scalable mechanism to record and facilitate the sharing of track and trace knowledge among supply chain partners. In this paper we present "OntoPedigree" a content ontology design pattern for the representation of linked pedigrees, that can be specialised and extended to define domain specific traceability ontologies. Events captured within the pedigrees are specified using EPCIS - a GS1 standard for the specification of traceability information within and across enterprises, while certification information is described using PROV - a vocabulary for modelling provenance of resources. We exemplify the utility of OntoPedigree in linked pedigrees generated for supply chains within the perishable goods and pharmaceuticals sectors.

Characterization of Grewia gum, a potential pharmaceutical excipient

Grewia gum was extracted from the inner stem bark of Grewia mollis and characterized by several techniques such as gas chromatography (GC), gel permeation chromatography (GPC), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis of the extracted sample. Spectroscopic techniques such as x-ray photoelectron spectroscopy (XPS), fourier-transformed infrared (FT-IR), solid-state nuclear magnetic resonance (NMR), and 1H and 13C NMR techniques were also used to characterize the gum. The results showed that grewia gum is a typically amorphous polysaccharide gum containing glucose, rhamnose, galactose, arabinose and xylose as neutral sugars. It has an average molecular weight of 5925 kDa expressed as the pullulan equivalent. The gum slowly hydrated in water, dispersing and swelling to form a highly viscous dispersion exhibiting pseudoplastic flow behaviour. The polysaccharide gum is thermally stable and may have application as stabilizer or suspending agent in foods, cosmetics and in pharmaceuticals. It may have application as a binder or sustained-release polymer matrix in tablets or granulations. © IPEC-Americas Inc.