Optimization of critical quality attributes in continuous twin-screw wet granulation via design space validated with pilot scale experimental data.

In this study, the influence of key process variables (screw speed, throughput and liquid to solid (L/S) ratio) of a continuous twin screw wet granulation (TSWG) was investigated using a central composite face-centered (CCF) experimental design method. Regression models were developed to predict the process responses (motor torque, granule residence time), granule properties (size distribution, volume average diameter, yield, relative width, flowability) and tablet properties (tensile strength). The effects of the three key process variables were analyzed via contour and interaction plots. The experimental results have demonstrated that all the process responses, granule properties and tablet properties are influenced by changing the screw speed, throughput and L/S ratio. The TSWG process was optimized to produce granules with specific volume average diameter of 150µm and the yield of 95% based on the developed regression models. A design space (DS) was built based on volume average granule diameter between 90 and 200µm and the granule yield larger than 75% with a failure probability analysis using Monte Carlo simulations. Validation experiments successfully validated the robustness and accuracy of the DS generated using the CCF experimental design in optimizing a continuous TSWG process.

Evaluation of a temperate climate mosquito, Ochlerotatus detritus (=Aedes detritus), as a potential vector of Japanese encephalitis virus.

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21 days post-infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus.

Model-driven experimental evaluation of struvite nucleation, growth and aggregation kinetics.

Nutrient stewardship is emerging as an issue of global importance, which will drive the development of nutrient recovery in the near to medium future. This will impact wastewater treatment practices, environmental protection, sustainable agriculture and global food security. A modelling framework for precipitation-based nutrient recovery systems has been developed, incorporating non-ideal solution thermodynamics, a dynamic mass balance and a dynamic population balance to track the development of the precipitating particles. The mechanisms of crystal nucleation and growth and, importantly, aggregation are considered. A novel approach to the population balance embeds the nucleation rate into the model, enabling direct regression of its kinetic parameters. The case study chosen for the modelling framework is that of struvite precipitation, given its wide interest and commercial promise as one possible nutrient recovery pathway. Power law kinetic parameters for nucleation, crystal growth and particle aggregation rates were regressed from an ensemble data set generated from 14 laboratory seeded batch experiments using synthetic solutions. These experiments were highly repeatable, giving confidence to the regressed parameter values. The model successfully describes the dynamic responses of solution pH, the evolving particle size distribution subject to nucleation, growth and aggregation effects and the aqueous magnesium concentration in the liquid phase. The proposed modelling framework could well be extended to other, more complex systems, leading to an improved understanding and commensurately greater confidence in the design, operation and optimisation of large-scale nutrient recovery processes from complex effluents.

Risk factors for morphoea disease severity: a retrospective review of 114 paediatric patients.

BACKGROUND: Morphoea is a rare fibrosing disease of the skin and subcutaneous tissue with an unpredictable disease course, running the spectrum from mild skin involvement to severe disfigurement or extracutaneous complications. OBJECTIVES: Our objective was to describe the natural history of paediatric morphoea and determine patient variables that were associated with severe disease. PATIENTS AND METHODS: We conducted a retrospective chart review of patients with morphoea seen in one paediatric hospital system. Information about demographics, clinical characteristics, disease course and treatment were collected. Statistical analysis was performed using appropriate univariate tests and a multivariable model. RESULTS: One hundred and fourteen patients met study inclusion criteria. The female : male ratio was 2·6 : 1, and the median age of onset was 7 years old. There were 55 patients (48%) with linear morphoea, 38 patients (33%) with circumscribed morphoea, 12 patients (11%) with generalized morphoea, and nine patients (8%) with mixed morphoea. Neurological symptoms and joint involvement were present in 27 subjects (24%). Extracutaneous manifestations occurred in 38% of subjects with linear morphoea, compared with 15% with generalized morphoea and 3% with circumscribed morphoea (P = 0·0001). Thirty-six per cent of children with disease onset prior to 10 years of age and 5% of children with disease onset after 10 years of age had extracutaneous manifestations (P = 0·0002). Both linear morphoea and early-onset disease were significantly associated with extracutaneous involvement in a multivariable model. CONCLUSIONS: Children with linear morphoea and disease onset before 10 years of age should be monitored closely for extracutaneous manifestations and need early treatment with systemic medications to prevent disease complications.

Demographic and clinical characteristics of cutaneous lupus erythematosus at a paediatric dermatology referral centre.

BACKGROUND: Paediatric cutaneous lupus erythematosus (CLE) is uncommon and inadequately described in the literature. Similar to adults, children with CLE develop LE-specific and/or LE-nonspecific skin findings. Similarities and differences in demographics and clinical course between paediatric and adult CLE have not been sufficiently described. OBJECTIVES: To detail the demographic and clinical features of paediatric CLE and compare these findings with those reported in the adult literature. METHODS: A retrospective chart review was performed of 53 children seen in a paediatric dermatology clinic with cutaneous manifestations of LE. RESULTS: Patients presented with all five major subtypes of CLE, with some notable differences from adult CLE and previously published reports of paediatric CLE. Progression from discoid LE to systemic LE (SLE) did not occur in our cohort. Patients with subacute CLE were more likely than adults to have lesions below the waist as well as concomitant SLE. Sex distribution for CLE in our study was equal prior to puberty and female predominant in post-pubertal patients. CONCLUSIONS: Children with CLE have variable clinical presentations and progression to SLE that may be different from adult disease. Specifically, children with acute and subacute CLE may be more likely than adults to have systemic disease; therefore, patients with these subtypes should be monitored closely for evidence of SLE. Study limitations included small patient numbers that may limit the ability to generalize these data and relatively short follow-up intervals.

Correlates and characteristics of hepatitis C virus-specific T-cell immunity in exposed uninfected high-risk prison inmates.

Some hepatitis C (HCV)-uninfected, high-risk individuals have HCV-specific cellular immunity without viraemia or seroconversion. The characteristics of these responses and the risk behavioural associations were studied in 94 subjects in a prospective cohort of recently seronegative prisoners reporting injecting drug use (IDU). Detailed behavioural data were collected. HCV antibody and PCR testing were performed. ELISpot assays for HCV-induced interferon (IFN)-γ and interleukin (IL)-2 production by T lymphocytes, as well as multiplex in vitro cytokine production assays, were performed. Seventy-eight subjects remained antibody and PCR negative and 16 seroconverted. Of the seronegative group, 22 (28%) had IFN-γ ELISpot responses in comparison with 13 of the 16 seroconverters (82%). This seronegative immune status was associated positively with injecting anabolic steroids and negatively with a recent break from IDU. The IFN-γ ELISpot responses involved both CD4 and CD8 T lymphocytes and were comparable in magnitude, but narrower in specificity, in uninfected subjects than in seroconverters. A subset of seronegative subjects had HCV-induced cytokine production patterns comparable with the seroconverters with increased production of IFN-γ, IL-2 and tumour necrosis factor (TNF)-α and reduced IL-10 in response to nonstructural peptides. In conclusion, comparable patterns of HCV-specific cellular immunity are found in recently infected subjects and in a minority of high-risk, uninfected subjects. Further characterization of these responses and their protective efficacy will inform HCV vaccine development.

The continued emergence of hantaviruses: isolation of a Seoul virus implicated in human disease, United Kingdom, October 2012.

Following a suspected case of hantavirus in a patientsuffering from acute kidney injury, rodents fromthe patient's property in Yorkshire and the Humber,United Kingdom (UK) were screened for hantaviruses.Hantavirus RNA was detected via RT-PCR in two Rattusnorvegicus. Complete sequencing and phylogeneticanalysis established the virus as a Seoul hantavirus,which we have provisionally designated as strainHumber. This is the first hantavirus isolated from wildrodents in the UK and confirms the presence of a pathogenicSeoul virus in Europe.

Detection of herpes viruses in the cerebrospinal fluid of adults with suspected viral meningitis in Malawi.

PURPOSE: We looked for herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA in Malawian adults with clinically suspected meningitis. METHODS: We collected cerebrospinal fluid (CSF) from consecutive adults admitted with clinically suspected meningitis to Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, for a period of 3 months. Those with proven bacterial or fungal meningitis were excluded. Real-time polymerase chain reaction (PCR) was performed on the CSF for HSV-1 and HSV-2, VZV, EBV and CMV DNA. RESULTS: A total of 183 patients presented with clinically suspected meningitis. Of these, 59 (32 %) had proven meningitis (bacterial, tuberculous or cryptococcal), 39 (21 %) had normal CSF and 14 (8 %) had aseptic meningitis. For the latter group, a herpes virus was detected in 9 (64 %): 7 (50 %) had EBV and 2 (14 %) had CMV, all were human immunodeficiency virus (HIV)-positive. HSV-2 and VZV were not detected. Amongst those with a normal CSF, 8 (21 %) had a detectable herpes virus, of which 7 (88 %) were HIV-positive. CONCLUSIONS: The spectrum of causes of herpes viral meningitis in this African population is different to that in Western industrialised settings, with EBV being frequently detected in the CSF. The significance of this needs further investigation.

Phase I dose-escalation study to determine the safety, pharmacokinetics and pharmacodynamics of brivanib alaninate in combination with full-dose cetuximab in patients with advanced gastrointestinal malignancies who have failed prior therapy.

BACKGROUND: The objectives of this phase I study were to determine the safety, pharmacokinetics (PK), pharmacodynamics and efficacy of brivanib combined with full-dose cetuximab in patients with advanced gastrointestinal malignancies. METHODS: Patients with advanced gastrointestinal malignancies who had failed prior therapies received brivanib (320, 600 or 800 mg daily) plus cetuximab (400 mg m(-2) loading dose then 250 mg m(-2) weekly). Assessments included adverse events, PK, tumour response, 2[18F]fluoro-2-deoxyglucose positron-emitting tomography and K-Ras mutation analyses. RESULTS: Toxicities observed were manageable; the most common treatment-related toxicities (>10% of patients) were fatigue, diarrhoea, anorexia, increase in aspartate aminotransferase and alanine aminotransferase, acneiform dermatitis, headache, mucosal inflammation, nausea, dry skin, vomiting, hypertension, pruritus, proteinuria and weight loss. Of 62 patients, 6 (9.7%) had objective radiographic partial responses, with an overall response rate of 10%. Median duration of response was 9.2 months; median progression-free survival was 3.9 months. CONCLUSIONS: The acceptable toxicity profile and efficacy of brivanib observed in this study were promising. These findings are being further evaluated in a phase III study of brivanib plus cetuximab vs cetuximab alone in patients previously treated with combination chemotherapy for K-Ras wild-type advanced metastatic colorectal cancer.

A phase I study to determine the safety, pharmacokinetics and pharmacodynamics of a dual VEGFR and FGFR inhibitor, brivanib, in patients with advanced or metastatic solid tumors.

BACKGROUND: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD. RESULTS: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180-800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly ≤1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ≥600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts. CONCLUSION: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses ≤800 mg orally q.d., the recommended phase II study dose.

Effect of intranasal administration of Semliki Forest virus recombinant particles expressing interferon-ß on the progression of experimental autoimmune encephalomyelitis.

The effect of intranasal (IN) administration of Semliki Forest virus (SFV) recombinant particles expressing interferon-ß [IFN-ß, a partially effective treatment for multiple sclerosis (MS)] on the progression of experimental autoimmune encephalomyelitis (EAE, a murine model for MS) was investigated. The murine IFN-ß gene was cloned from SFV-infected mouse brain by RT-PCR into an SFV-enhanced expression vector, pSFV10-E, from which IFN-ß-expressing recombinant particles (rSFV10-E-IFN-ß) were prepared. Expression studies using immunohistochemistry and viral inhibition assay in BHK and murine L929 cells confirmed increased expression of IFN-ß. High level expression in the central nervous system (CNS) following IN inoculation was confirmed by the excision of olfactory bulbs, brain and spinal cord, and the detection of IFN-ß levels in homogenised tissue by ELISA. rSFV10-E-IFN-ß particles were administered IN to C57/Bl6 mice that had been induced for EAE using the encephalogenic peptide myelin oligodendrocyte glycoprotein (MOG) 35-55. The progression of EAE was measured by clinical score, weight loss and pathology. As previously shown, treatment with empty rSFV10-E particles moderately exacerbated EAE, as did continuous treatment with rSFV10-E-IFN-ß particles. Inhibition of disease with rSFV10-E-IFN-ß particles was dependent on the number and timing of treatments. Fewer treatments, administered before the effector stage, led to an improvement in clinical and pathology score. In conclusion, the timing and frequency of IN administration of rSFV10-E-IFN-ß particles are critical to disease outcome, with treatment prior to the effector stage being most effective.

Human mass balance study of the novel anticancer agent ixabepilone using accelerator mass spectrometry.

Ixabepilone (BMS-247550) is a semi-synthetic, microtubule stabilizing epothilone B analogue which is more potent than taxanes and has displayed activity in taxane-resistant patients. The human plasma pharmacokinetics of ixabepilone have been described. However, the excretory pathways and contribution of metabolism to ixabepilone elimination have not been determined. To investigate the elimination pathways of ixabepilone we initiated a mass balance study in cancer patients. Due to autoradiolysis, ixabepilone proved to be very unstable when labeled with conventional [14C]-levels (100 microCi in a typical human radio-tracer study). This necessitated the use of much lower levels of [14C]-labeling and an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS). Eight patients with advanced cancer (3 males, 5 females; median age 54.5 y; performance status 0-2) received an intravenous dose of 70 mg, 80 nCi of [14C]ixabepilone over 3 h. Plasma, urine and faeces were collected up to 7 days after administration and total radioactivity (TRA) was determined using AMS. Ixabepilone in plasma and urine was quantitated using a validated LC-MS/MS method. Mean recovery of ixabepilone-derived radioactivity was 77.3% of dose. Fecal excretion was 52.2% and urinary excretion was 25.1%. Only a minor part of TRA is accounted for by unchanged ixabepilone in both plasma and urine, which indicates that metabolism is a major elimination mechanism for this drug. Future studies should focus on structural elucidation of ixabepilone metabolites and characterization of their activities.

Inhibition of angiogenesis by a Semliki Forest virus vector expressing VEGFR-2 reduces tumour growth and metastasis in mice.

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.

Cutaneous Crohn's disease mimicking Melkersson-Rosenthal syndrome: treatment with methotrexate.

A woman with a 5-year history of unilateral orofacial granulomatosis required repeated evaluations (including sequential colonoscopies) to establish the diagnosis of cutaneous Crohn's disease, a condition that proved responsive to low doses of oral methotrexate administered weekly. To our knowledge this is the first report describing the use of methotrexate for treatment of orofacial granulomatosis caused by underlying Crohn's disease.

5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent: phase I clinical and pharmacokinetic study.

The purpose of this phase I, dose-escalation study was to determine the toxicity, maximum tolerated dose, pharmacokinetics, and pharmacodynamic end points of 5,6-dimethylxanthenone acetic acid (DMXAA). In all, 46 patients received a total of 247 infusions of DMXAA over 15 dose levels ranging from 6 to 4900 mg x m(-2). The maximum tolerated dose was established at 3700 mg x m(-2); dose-limiting toxicities in the form of urinary incontinence, visual disturbance, and anxiety were observed at the highest dose level (4900 mg x m(-2)). The pharmacokinetics of DMXAA were dose dependent. Peak concentrations and area under the curve level increased from 4.8 microM and 3.2 microM h, respectively, at 6 mg x m(-2) to 1290 microM and 7600 microM h at 3700 mg x m(-2), while clearance declined from 7.4 to 1.7 l h(-1) x m(-2) over the same dose range. The terminal half-life was 8.1+/-4.3 h. More than 99% of the drug was protein bound at doses up to 320 mg x m(-2); at higher doses the percent free drug increased to a maximum of 6.9% at 4900 mg x m(-2). Dose-dependent increases in the serotonin metabolite 5-hydroxyindoleacetic acid were observed at dose levels of 650 mg x m(-2) and above. There was one unconfirmed partial response at 1300 mg x m(-2). In conclusion, DMXAA is a novel vascular targeting agent and is well tolerated.

Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumour vascular targeting activity in vivo.

BACKGROUND: Combretastatin A4 Phosphate (CA4P) is a tubulin binding agent which causes rapid tumour vascular shutdown. It has anti-proliferative and apoptotic effects on dividing endothelial cells after prolonged exposure, but these effects occur on a much longer time scale than the reduction in tumour blood flow. This study compared the time course of CA4P effects on endothelial cell shape and reduction in red cell velocity. METHODS: Endothelial cell area and form factor (1-4 pi x area x perimeter-2) were measured for proliferating and confluent HUVECs after CA4P treatment. Recovery of shape after CA4P and colchicine was compared. Window chamber studies of tumours were used to measure red cell velocity. Results 70% reduction in red cell velocity and 44% reduction in HUVEC form factor occurred by 10 minutes. Proliferating HUVECs underwent greater cell shape change after CA4P, which occurred at lower doses than for confluent cells. Cell shape recovered 24 hours after 30 minutes exposure to CA4P, but not after colchicine. CONCLUSIONS: The similar time course of cell shape change and red cell velocity reduction suggests endothelial cell shape change may be involved early in the in vivo events leading to vascular shutdown. Differences in the recovery from the shape changes induced by CA4P and colchicine could underlie the different toxicity profiles of these drugs.

Transient virus infection and multiple sclerosis.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the CNS in which autoimmunity to myelin plays a role in pathogenesis. The epidemiology of MS indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with MS have produced equivocal results. Many studies of the aetiology of MS have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in MS research. The most studied animal model of MS is chronic relapsing experimental autoimmune encephalomyelitis (CREAE), which is induced in susceptible animals following injection of myelin components. While CREAE cannot provide information on the initiating factor for MS, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. The disease process may comprise separate triggering and relapse phases. The triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. The relapse phase could be similar to CREAE, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the CNS, but which may involve the recrudescence of anti-myelin autoimmunity. Although current vaccines have a high degree of biosafety, it is suggested that the measles-mumps-rubella vaccine in particular could be modified to obviate any possibility of triggering anti-myelin autoimmunity.

Medico-legal issues surrounding medical countermeasures used in the Gulf War--part 1.

When the multinational force deployed to the Gulf after Iraq's invasion of Kuwait in 1990, military intelligence assessed the Iraqi's as possessing and being capable of using weapons of mass destruction. There was judged to be a real threat that chemical weapons, especially nerve agents, and certain biological weapons would be used. Coalition countries attempted to reduce the effects by the use of medical countermeasures. Since the Gulf conflict a series of medico legal problems from this policy have arisen; some of which have formed the basis of claims against MOD. In this paper I shall review how consent to treatment may have been modified in the military operational context and by the interface with military law. I shall look at the issues of clinical negligence and how duty of care may be affected. In the next article I shall look at relevant employment law; briefly review how medicines regulatory provision applies to medical countermeasures, whether they were properly licensed and whether in any case this applied to the Ministry of Defence in the context of deployment to the Gulf War.