Non-aqueous formulations for ram and screen extrusion-spheronisation.

The use of non-aqueous cellulose-based formulations for extrusion-spheronisation (E-S) is investigated. A 10 wt% hydroxypropyl cellulose/isopropyl alcohol solution (HPC/IPA) was identified as a suitable sticky liquid binder for preparing non-aqueous pastes. Preliminary tests were performed on a series of pastes using a ram as well as a laboratory roller screen extruder, since the former is commonly used in batch testing and the latter replicates the shear range in a manufacturing screen extruder. Pellets with acceptable size and shape distributions were obtained with Avicel® HFE-102 NF/HPC/IPA for ram E-S, and with Avicel® RC-591/HPC/IPA for screen E-S. Further investigation was performed with calcium carbonate added as a model active pharmaceutical ingredient. Both formulations were able to generate pellets with acceptable size and shape characteristics at up to 50 wt% carbonate loading: further work is required to optimise yields.

Laws of physics help explain capillary non-perfusion in diabetic retinopathy.

The purpose is to use laws of physics to elucidate the mechanisms behind capillary non-perfusion in diabetic retinopathy. In diabetic retinopathy, loss of pericytes weakens capillary walls and the vessel dilates. A dilated capillary has reduced resistance to flow, therefore increased flow in that vessel and decreased in adjoining capillaries. A preferential shunt vessel is thus formed from the dilated capillary and the adjacent capillaries become non-perfused. We apply the laws of Laplace and Hagen-Poiseuille to better understand the phenomena that lead to capillary non-perfusion. These laws of physics can give a foundation for physical or mathematical models to further elucidate this field of study. The law of Laplace predicts that a weaker vessel wall will dilate, assuming constant transmural pressure. The Hagen-Poiseuille equation for flow and the Ostwald-de Waele relationship for viscosity predict that a dilated vessel will receive a higher portion of the fluid flow than the adjoining capillaries. Viscosity will decrease in the dilated vessel, furthering the imbalance and resulting in a patch of non-perfused capillaries next to the dilated 'preferential' shunt vessel. Physical principles support or inspire novel hypotheses to explain poorly understood phenomena in ophthalmology. This thesis of pericyte death and capillary remodelling, which was first proposed by Cogan and Kuwabara, already agrees with histological and angiographical observations in diabetic retinopathy. We have shown that it is also supported by classical laws of physics.

What is rheology?

The topic of the 2017 Cambridge Ophthalmology Symposium was 'Go with the flow: rheology, fluid flow and the eye'. This paper is based on the opening presentation that was aimed to introduce key concepts within the topic of rheology to ophthalmologists and other scientists unfamiliar with the field. Examples are drawn from applications related to the eye.

Effects of culture conditions on the size, morphology and wet density of spores of Bacillus cereus 569 and Bacillus megaterium QM B1551.

The influence of variable culture conditions on the size and wet density of spores of Bacillus cereus and Bacillus megaterium were examined in this work. Culture temperature and initial pH was shown to have a significant impact on the size of both species, with increasingly alkaline culture media and elevated culture temperatures resulting in spores that were, on average, up to 25% reduced in volume. Increasing concentrations of inorganic salts in sporulation media exerted differing effects on each species; whereas a fivefold increase in the concentration of all salts resulted in only minor differences to the dimensions of B. cereus spores, B. megaterium spores became more elongated, displaying an average increase in volume of almost 30%. Similarly, as the spore elongated to yield aspect ratios larger than 1·4, their shape changed from typical prolate spheroids to cylinders with hemispherical ends. In contrast with previous studies, culture conditions employed in this study exerted no discernible impact on the wet density of B. cereus or B. megaterium spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial spores of the genera Bacillus and Clostridium represent nature's most durable cells in terms of their extreme resistance to a variety of deleterious environments. As a result, they are of concern in the food processing, healthcare and other sectors, and are of increasing biotechnological interest. Improved understanding of variance in spore size, morphology and density may aid the development of certain spore-associated applications (e.g. spore surface display) while contributing to active areas of research such as spore adhesion and resistance to heat.

Critical review: Injectability of calcium phosphate pastes and cements.

Calcium phosphate cements (CPC) have seen clinical success in many dental and orthopaedic applications in recent years. The properties of CPC essential for clinical success are reviewed in this article, which includes properties of the set cement (e.g. bioresorbability, biocompatibility, porosity and mechanical properties) and unset cement (e.g. setting time, cohesion, flow properties and ease of delivery to the surgical site). Emphasis is on the delivery of calcium phosphate (CaP) pastes and CPC, in particular the occurrence of separation of the liquid and solid components of the pastes and cements during injection; and established methods to reduce this phase separation. In addition a review of phase separation mechanisms observed during the extrusion of other biphasic paste systems and the theoretical models used to describe these mechanisms are discussed. STATEMENT OF SIGNIFICANCE: Occurrence of phase separation of calcium phosphate pastes and cements during injection limits their full exploitation as a bone substitute in minimally invasive surgical applications. Due to lack of theoretical understanding of the phase separation mechanism(s), optimisation of an injectable CPC that satisfies clinical requirements has proven difficult. However, phase separation of pastes during delivery has been the focus across several research fields. Therefore in addition to a review of methods to reduce phase separation of CPC and the associated constraints, a review of phase separation mechanisms observed during extrusion of other pastes and the theoretical models used to describe these mechanisms is presented. It is anticipated this review will benefit future attempts to develop injectable calcium phosphate based systems.

Virtual milk for modelling and simulation of dairy processes.

The modeling of dairy processing using a generic process simulator suffers from shortcomings, given that many simulators do not contain milk components in their component libraries. Recently, pseudo-milk components for a commercial process simulator were proposed for simulation and the current work extends this pseudo-milk concept by studying the effect of both total milk solids and temperature on key physical properties such as thermal conductivity, density, viscosity, and heat capacity. This paper also uses expanded fluid and power law models to predict milk viscosity over the temperature range from 4 to 75°C and develops a succinct regressed model for heat capacity as a function of temperature and fat composition. The pseudo-milk was validated by comparing the simulated and actual values of the physical properties of milk. The milk thermal conductivity, density, viscosity, and heat capacity showed differences of less than 2, 4, 3, and 1.5%, respectively, between the simulated results and actual values. This work extends the capabilities of the previously proposed pseudo-milk and of a process simulator to model dairy processes, processing different types of milk (e.g., whole milk, skim milk, and concentrated milk) with different intrinsic compositions, and to predict correct material and energy balances for dairy processes.

Probing the epigenetic regulation of HIF-1α transcription in developing tissue.

HIF-1 is the master regulator of cellular hypoxia response; the oxygen sensitive HIF-1α subunit transactivates its own expression in hypoxia via a hypoxia response element (HRE) in the promoter of the HIF-1α gene. This transactivation loop significantly contributes to the build up of HIF-1α at the onset of hypoxia, with the binding of HIF-1 to the HIF-1α promoter being dependent on the epigenetic status of a CpG dinucleotide in the upstream HRE. Given the central role played by HIF-1 in tissue development, we sought to probe the epigenetic status of the HIF-1α HRE and that of its downstream target EPO in embryonic tissue. Our data shows that the CpG dinucleotide in HIF-1α HRE is unmethylated in several embryonic tissue samples, suggesting that transactivation of HIF-1α plays a significant role in HIF-1 mediated hypoxia response during development.

The effect of mixing on the extrusion-spheronisation of a micro-crystalline cellulose paste.

The effect of mixer shear strain rate on the performance of a model micro-crystalline cellulose pharmaceutical paste undergoing extrusion-spheronisation was studied using a laboratory scale planetary mixer and a screw-based mixer. The maximum shear strain rate in each mixer was estimated. Five pastes were prepared, one each at 97 and 265 s(-1) in the planetary mixer, and one each at 304, 988 and 2220 s(-1) in the screw mixer. The rheology of the pastes was quantified by Benbow-Bridgwater characterisation based on ram extrusion. Each paste was spheronised and pellet size and shape distributions obtained by automated size analysis. Mixer type (rather than shear strain rate) was found to have the strongest influence on the paste properties, with the screw-mixed material having a higher yield strength and forming smaller pellets with a narrower size distribution when spheronised under identical conditions.

A comparison of screen and ram extrusion-spheronisation of simple pharmaceutical pastes based on microcrystalline cellulose.

The performance of two laboratory-scale extrusion apparatuses used to approximate the action of an industrial screen extruder, namely a multi-holed die ram extruder and a roller screen extruder, were compared. Both devices featured short dies (ram 2mm, screen 1mm) with die diameter 1mm and hole area fraction approaching 0.25. A series of water/microcrystalline cellulose (MCC) pastes with water contents varying from 45 to 60 wt% were extruded and pellets obtained from subsequent spheronisation of the extrudates characterised in terms of size and shape. Each device exhibited a different range of processing windows for acceptable spheronised products, with the ram apparatus being able to extrude a wider range of paste water contents than the screen device. The pellets obtained from extrusion-spheronisation (E-S) of the pastes via the screen device were in general smaller, with a wider size distribution, than those from ram E-S. These results are attributed to the different mechanical histories experienced by the pastes in the two types of extruder, which lead to different extrudate densities being achieved. MCC/water/calcium carbonate pastes were also tested, where the latter component represented a 'hard' (non-deformable) active pharmaceutical ingredient. Addition of calcium carbonate increased the stiffness of the paste, which could be countered by adjusting the water content of the deformable MCC/water matrix within the extrudability limits of the latter material.

A novel lab-scale screen extruder for studying extrusion-spheronisation.

A novel apparatus, the laboratory roller screen extruder (termed the LRS), was developed to replicate key aspects of the geometry and shear strain rates generated near the screen of industrial screen extruders. The configuration of the LRS is reported alongside a commissioning study employing a cohesive 45 wt% water/microcrystalline cellulose paste. The key operating parameters which controlled the extrudate mass flowrate, force on the screen and roller torque were (i) the size of the gap between the top of the roller blade and the screen, and (ii) the roller rotational speed. The data suggest that the apparent shear rate, based on the blade-screen clearance, provides a quantitative criterion for scale-up. The amount of screen flex showed good agreement with a simple bending deformation model. Spheronisation of the extrudates gave pellets with a narrow size distribution and acceptable sphericity which would be acceptable for capsule filling. Optimisation of the pellet shape was not performed. The results indicate that the LRS can be used to assess formulations for industrial screen extrusion-spheronisation.

Characterising the structure of photosynthetic biofilms using fluid dynamic gauging.

A new configuration of the fluid dynamic gauging technique for measuring soft layers on surfaces was used to monitor the growth of a cyanobacterium, Synechococcus sp. WH 5701, on stainless steel (SS), glass and an indium tin oxide (ITO) on a polyethylene terephthalate (PET) substratum. The biofilm thickness increased steadily over 4 weeks and exhibited noticeable changes in microstructure and strength. The biofilms all exhibited a two-layer structure, with a compact layer next to the substratum and a loose layer above. Biofilms on ITO or SS exhibited cohesive failure when removed by fluid shear whereas those on glass exhibited adhesive failure. The technique is able to elucidate various aspects of biofilm behaviour, as illustrated by the action of a biocide (NaOCl) on a mature biofilm.

Optimisation and scale-up of a highly-loaded 5-ASA multi-particulate dosage form using a factorial approach.

Pellets with high loading of 5-aminosalicylic acid (5-ASA, mesalamine) are desired to reduce the number of tablets required to deliver the daily dosing regimen. Recently, we reported an extrusion-spheronisation route for the development of a 90 wt% 5-ASA/microcrystalline cellulose formulation based on milled 5-ASA which gave good yields of pellets at the lab scale. In the present work, such formulation was optimised further by preliminary studies using a lab-scale ram extruder, and then scaled up to the pilot plant scale on a Nica screen extruder using a mixed fractional factorial approach. The final formulation featured 95 wt% 5-ASA and 5 wt% Avicel RC591 (all dry basis) and yielded spherical pellets suitable for use as the drug core of a multi-particulate DDS.

A comparison of ram extrusion by single-holed and multi-holed dies for extrusion-spheronisation of microcrystalline-based pastes.

The use of multi-holed dies as an alternative to single-holed dies for generating extrudates for spheronisation was investigated both in terms of extrusion and spheronisation performance. A model 45wt% microcrystalline cellulose (MCC)/water paste was employed in ram extrusion tests with square-ended dies with 1, 6, 33 and 137 holes, all of diameter 1mm and length 2mm. The extrudates generated using the multi-holed dies yielded pellets with comparable sphericity to those using the single-holed die. Multi-holed dies could also be operated with lower paste flow rates before encountering liquid phase migration (LPM). The characteristic processing velocity for the onset of LPM was determined for each die configuration and supported the hypothesis that LPM was caused by suction effects. A simple model of the flow pattern in a lab-scale Fuji-Paudal frontal screen extruder is presented which yields estimates of velocities and shear rates involved in these devices. The pressure required to extrude the paste through multi-holed dies was compared with the model proposed by Benbow and co-workers. The paste rheology was characterised using the Benbow-Bridgwater approach, employing 1, 2 and 3mm diameter dies of various lengths. The Benbow et al. model under-predicted the observed extrusion pressure, which was attributed to its failure to account for the redundant work contribution in these complex flows.

Developmental plasticity of human foetal femur-derived cells in pellet culture: self assembly of an osteoid shell around a cartilaginous core.

This study has examined the osteogenic and chondrogenic differentiation of human foetal femur-derived cells in 3-dimensional pellet cultures. After culture for 21-28 days in osteogenic media, the pellets acquired a unique configuration that consisted of an outer fibrous layer, an osteoid-like shell surrounding a cellular and cartilaginous region. This configuration is typical to the cross section of the foetal femurs at the same age and was not observed in pellets derived from adult human bone marrow stromal cells. Time course study showed that after 7-14 days, the cells of the inner cellular region were viable, proliferated rapidly, and were immuno-positive for c-myc, as well as for bone sialoprotein and type I collagen. After 21-28 days, the cells accumulated at the inner edge of the osteoid shell. The direction of osteoid formation thus differed from that of periosteal bone formation. Following micro-dissection of the human foetal femurs into epiphyses, bone cylinder and hypertrophic cartilage, epiphyseal chondrocytes and osteoblasts both gave rise to osteoid-shell forming cells. These studies demonstrate the developmental plasticity of human foetal skeletal and epiphyseal chondrocytes and suggest that the microenvironment modulates lineage commitment and matrix formation. Furthermore, this ex vivo model offers a new approach to delineate human bone development as well as a model with potential application for evaluation of therapeutic compounds for bone formation.

Extrusion-spheronisation of highly loaded 5-ASA multiparticulate dosage forms.

The aim of the current work was to develop an extrusion-spheronisation (E-S) route to manufacture pellets with a high loading (≥90wt%) of 5-aminosalicylic acid (5-ASA). Ram extrusion studies, supported by centrifuge testing, were employed to investigate the effect of the chemical (acidity) and physical (particle size and shape) characteristics of 5-ASA on the ability of microcrystalline cellulose (MCC)-based pastes to retain water when subjected to pressure. Liquid phase migration (LPM) within the paste during the extrusion, and hence variation in water content of extrudates and reproducibility of the final E-S product, was generally observed. The extent of LPM was found to be related to both the drug loading and its physical properties, most notably the particle shape (needle-like). A reduction in particle size, combined with a change in the shape of the 5-ASA particles, allowed LPM to be reduced considerably or eliminated. The performance of colloidal grades of MCC (Avicel RC591 and CL611) as alternative extrusion aids to the standard Avicel PH101 was also investigated: these proved to be superior aids for the highly loaded 5-ASA pastes as their greater water retention capacity mitigated LPM. Combining these results yielded a route for manufacturing pellets with 5-ASA loading ≥90wt%.

Extrusion-spheronisation of microcrystalline cellulose pastes using a non-aqueous liquid binder.

An experimental investigation of extrusion-spheronisation (E-S) of a novel microcrystalline cellulose (MCC) formulation, comprising dimethyl sulfoxide (DMSO) as the granulating liquid, is reported. The extrusion-spheronisation performance of DMSO/MCC pastes is compared to that of similar water/MCC formulations and their rheological behaviours similarly assessed using lubricated squeeze flow. A case study involving water, DMSO and anhydrous ethanol yields important information regarding the physico-chemical properties necessary for solvents to be suitable for the extrusion of MCC.

Shear rheology of molten crumb chocolate.

The shear rheology of fresh molten chocolate produced from crumb was studied over 5 decades of shear rate using controlled stress devices. The Carreau model was found to be a more accurate description than the traditional Casson model, especially at shear rates between 0.1 and 1 s(-1). At shear rates around 0.1 s(-1) (shear stress approximately 7 Pa) the material exhibited a transition to a solid regime, similar to the behavior reported by Coussot (2005) for other granular suspensions. The nature of the suspension was explored by investigating the effect of solids concentration (0.20 < phi < 0.75) and the nature of the particles. The rheology of the chocolate was then compared with the rheology of (1) a synthetic chocolate, which contained sunflower oil in place of cocoa butter, and (2) a suspension of sugar of a similar size distribution (volume mean 15 mum) in cocoa butter and emulsifier. The chocolate and synthetic chocolate showed very similar rheological profiles under both steady shear and oscillatory shear. The chocolate and the sugar suspension showed similar Krieger-Dougherty dependency on volume fraction, and a noticeable transition to a stiff state at solids volume fractions above approximately 0.5. Similar behavior has been reported by Citerne and others (2001) for a smooth peanut butter, which had a similar particle size distribution and solids loading to chocolate. The results indicate that the melt rheology of the chocolate is dominated by hydrodynamic interactions, although at high solids volume fractions the emulsifier may contribute to the departure of the apparent viscosity from the predicted trend.

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development.

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

Botulinum toxin injections for adults with overactive bladder syndrome.

BACKGROUND: Overactive bladder syndrome is a common condition with a significant negative impact on quality of life. Intravesical injection of botulinum toxin is increasingly used as an intervention for refractory overactive bladder, with a considerable body of case reports and series in the literature suggesting beneficial effects. OBJECTIVES: The objective was to compare intravesical botulinum toxin injection with other treatments for neurogenic and idiopathic overactive bladder in adults. The hypotheses addressed were whether intravesical injection of botulinum toxin was better: than placebo or no treatment, pharmacological and other non-pharmacological interventions, whether higher doses of botulinum toxin were better than lower doses, whether botulinum toxin in combination with other treatments was better than other treatments alone, whether one formulation of botulinum toxin is better than another, and whether one injection technique was better than another. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (searched 22 November 2005). The register contains trials identified from MEDLINE, CINAHL, the Cochrane Central Register of Controlled Trials (CENTRAL), and handsearching of journals and conference proceedings. Additionally, all reference lists of selected trials were searched. No limitations were placed on the searches. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of treatment for overactive bladder syndrome in adults in which at least one management arm involved intravesical injection of botulinum toxin were included. Participants had either neurogenic or idiopathic overactive bladder with or without stress incontinence. Comparison interventions could include no intervention; placebo; lifestyle modification; bladder retraining; pharmacological treatments; surgery; bladder instillation techniques; neuromodulation; and different types, doses, and injection techniques of botulinum toxin. DATA COLLECTION AND ANALYSIS: Binary outcomes were presented as relative risk and continuous outcomes by mean differences. No data could be synthesised across studies due to differing designs and outcome measures. Data were tabulated where possible with results taken from trial reports where this was not possible. Where multiple publications were found, the reports were treated as a single source of data. MAIN RESULTS: Eight studies met the inclusion criteria. Results varied between studies. For the most part, studies reported superiority of botulinum toxin A to placebo in such outcomes as incontinence episodes, bladder capacity, maximum detrusor pressure, and quality of life. Low doses of botulinum toxin (100U to150U) appeared to have beneficial effects, but higher doses (300U) may have been more effective. Botulinum toxin appeared to have beneficial effects in overactive bladder that quantitatively exceeded the effects of intravesical resiniferatoxin. AUTHORS' CONCLUSIONS: Intravesical botulinum toxin shows promise as a therapy for overactive bladder symptoms, but as yet too little controlled trial data exist on benefits and safety compared with other interventions, or with placebo. Practitioners should be aware that at present there is little more than anecdotal evidence, in the form of case reports to support the efficacy of intravesical botulinum toxin; there is not much in the way of substantial, robust safety data. Furthermore, the optimal dose of botulinum toxin for efficacy and safety has not yet been demonstrated.