Collaborative study for the establishment of Etanercept Biological Reference Preparation Batch 1.

Three preparations of the human tumour necrosis factor (TNF) receptor II Fc fusion protein (TNFR II-Fc) Etanercept were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Etanercept. Seven laboratories tested the preparations using an in vitro cell-based bioassay (TNF-α neutralisation) prescribed by the Ph. Eur. monograph on Etanercept (2895). The results of this study indicated that the candidate preparation, coded 13/204, established as the first IS for Etanercept with an assigned potency for TNF neutralisation activity of 10 000 IU per ampoule was also suitable to serve as Ph. Eur. BRP batch 1. The results were compared to those obtained with different cell-based neutralisation assays that were used by further laboratories in the context of establishing the 1st WHO IS for Etanercept. Based on these analyses, preparation 13/204 was adopted by the Ph. Eur. Commission as Etanercept BRP batch 1 with an assigned potency of 10 000 IU per ampoule.

Chikungunya in a kidney transplant recipient: a case report / Chikungunya em um receptor de transplante renal: relato de caso

ABSTRACT In 2004, a global spread of Chikungunya fever affected most tropical and subtropical regions of the world. In 2016, an outbreak occurred in Northeast Brazil with hundreds of cases documented. Solid organ transplant recipients have a modified immune response to infection and the clinical course is usually different from immunocompetent patients. The diagnosis can be challenging in this population. Most reports describe patients residing in endemic areas, although we must emphasize the importance of differential diagnosis in kidney transplanted travelers who visit endemic regions, such as Northeast Brazil. Here, we reported a case of a kidney transplant recipient that acquired Chikungunya fever after a trip to an endemic region at Northeast Brazil during the outbreak in 2016, with a good clinical evolution. We also present warning recommendations for travelers to endemic areas as additional measures to prevent disease outbreaks.

RESUMO Em 2004, um surto global de Chikungunya afetou a maioria das regiões tropicais e subtropicais do mundo. Em 2016, um surto ocorreu no Nordeste do Brasil com centenas de casos documentados. Receptores de transplantes de órgãos sólidos têm uma resposta imune modificada à infecção, e o curso clínico é geralmente diferente daquele em pacientes imunocompetentes. O diagnóstico pode ser desafiador nessa população. A maioria dos relatos descreve pacientes residentes em áreas endêmicas, embora devamos enfatizar a importância do diagnóstico diferencial em viajantes transplantados renais que visitam regiões endêmicas, como o Nordeste do Brasil. Aqui, nós relatamos o caso de um receptor de transplante renal que adquiriu febre Chikungunya após uma viagem a uma região endêmica no Nordeste do Brasil durante o surto de 2016, com uma boa evolução clínica. Também apresentamos recomendações de alerta para viajantes em áreas endêmicas, como medidas adicionais para prevenir surtos de doenças.

Chikungunya in a kidney transplant recipient: a case report.

In 2004, a global spread of Chikungunya fever affected most tropical and subtropical regions of the world. In 2016, an outbreak occurred in Northeast Brazil with hundreds of cases documented. Solid organ transplant recipients have a modified immune response to infection and the clinical course is usually different from immunocompetent patients. The diagnosis can be challenging in this population. Most reports describe patients residing in endemic areas, although we must emphasize the importance of differential diagnosis in kidney transplanted travelers who visit endemic regions, such as Northeast Brazil. Here, we reported a case of a kidney transplant recipient that acquired Chikungunya fever after a trip to an endemic region at Northeast Brazil during the outbreak in 2016, with a good clinical evolution. We also present warning recommendations for travelers to endemic areas as additional measures to prevent disease outbreaks.

Measurement uncertainty and metrological traceability of whole blood cyclosporin A mass concentration results obtained by UHPLC-MS/MS.

BACKGROUND: Traceable and accurate results of cyclosporine A (CsA) mass concentrations in whole blood are required to ensure the monitoring of immunosuppressive therapy in transplant recipients. Metrological traceability and measurement uncertainty can allow ensuring reliability and comparability of these results over time and space. In this study, we provide a practical and detailed example of how the traceability and uncertainty of mass concentration of CsA results, obtained using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure, can be described and estimated. METHODS: Traceability was described mainly according to ISO 17511 and information obtained from certificates facilitated with the manufacturer's calibrators. Uncertainty estimation was performed using the bottom-up and top-down approaches. For the bottom-up approach, the most relevant sources of uncertainty were identified and later used to estimate the standard, combined and expanded uncertainties. For the top-down approach, expanded uncertainty was estimated directly using intralab quality control data mainly. RESULTS: Mass concentration of CsA results was traceable to the manufacturer's product calibrators used to calibrate the UHPLC-MS/MS procedure. The expanded uncertainties estimated by the bottom-up and top-down approaches were 7.4% and 7.2%, respectively. CONCLUSIONS: After performing the bottom-up and top-down approaches, we observed that their results were quite similar. This fact would confirm that the top-down approach could be sufficient for estimating uncertainty of CsA mass concentrations in whole blood results in clinical laboratories. Finally, we hope that this study can help and motivate clinical laboratories to describe metrological traceability and to perform measurement uncertainty studies based on the simpler top-down approach.

Generic immunosuppressants.

Immunosuppressive drugs for solid organ transplantation are critical dose drugs with a narrow therapeutic index. Many of the most commonly used innovator drugs are off patent and have been replicated by generic counterparts, often at substantial cost-savings to the patient. However, serious adverse events caused by the transition from innovator to generic medications, specifically in pediatric solid organ transplant recipients, have questioned these autosubstitutions. The purpose of this review is to summarize the criteria set forth by the regulatory bodies, and to examine how major immunosuppressive drugs conform to these recommendations. Regulatory bodies have established inconsistent criteria to demonstrate bioequivalence between innovator and generic medications, causing approved generic variations to have varying levels of equivalence with the innovator drugs. In order to minimize the risk for under-immunosuppression, the following recommendations have been concluded. Brand prescribing of cyclosporine and tacrolimus are recommended due to evidence of adverse events after conversion to generic formulations and differences in dissolution parameters. Mycophenolate mofetil (MMF) shows better bioequivalence between innovator and generic formulations, however caution should be advised when switching between formulations. The institution of 'innovator only' policies may be appropriate at this time in order to minimize the risk of under-immunosuppressing patients until the evidence of more stringent bioequivalence has been established.

Clinical outcomes in adult patients with aplastic anemia: A single institution experience.

Newer treatment modalities are being investigated to improve upon historical outcomes with standard immunosuppressive therapy (IST) in aplastic anemia (AA). We analyzed outcomes of adult patients with AA treated with various combinatorial anti-thymoglobulin-based IST regimens in frontline and relapsed/refractory (R/R) settings. Pretreatment and on-treatment clinical characteristics were analyzed for relationships to response and outcome. Among 126 patients reviewed, 95 were treatment-naïve (TN) and 63, R/R (including 32 from the TN cohort); median ages were 49 and 50 years, respectively. Overall survival (OS) was superior in IST responders (P < .001). Partial response to IST was associated with shorter relapse-free survival (RFS), as compared with complete response (P = .03). By multivariate analysis, baseline platelet and lymphocyte count predicted for IST response at 3 and 6 months, respectively. While additional growth factor interventions led to faster count recovery, there were no statistically significant differences in RFS or OS across the various frontline IST regimens (i.e., with/without G-CSF or eltrombopag). While marrow cellularity did not correlate with peripheral-blood counts at 3 months, cytomorphological assessment revealed dyspoietic changes in all nonresponders with hypercellular-marrow indices. Covert dysplasia, identified through early bone marrow assessment, has implications on future therapy choices after IST failure. Salvage IST response depended upon prior response to ATG: prior responders (46%) vs. primary refractory (0%) (P < .01). In the R/R setting, there was no survival difference between IST and allogeneic stem cell transplant groups, with a trend toward superior OS in the former. Transplant benefits in the R/R setting may be underrealized due to transplant-related mortality.

[Guidelines for vaccination of immunocompromised individuals]. / Impfungen bei Immundefekten/Immunsuppression - Expertenstatement und Empfehlungen.

Immunosuppression of various origins is associated with an increased risk of infection; therefore the prevention of infectious diseases by vaccination is especially important in immunocompromised patients. However, the response to vaccinations is often reduced in these risk groups and the application of live vaccines is contraindicated during immunosuppression.In the following expert statement, recommendations for vaccination were created on the basis of current evidence and theoretical/immunological considerations. A first, general part elaborates on efficacy and safety of vaccinations during immunosuppression, modes of action of immunosuppressive medications and recommended time intervals between immunosuppressive treatments and vaccinations. A core piece of this part is a graduation of immunosuppression into three stages, i. e. no relevant immunosuppression, mild to moderate and severe immunosuppression and the assignment of various medications (including biologicals) to one of those stages; this is followed by an overview of possible and necessary vaccinations in each of those stages.The second part gives detailed vaccination guidelines for common diseases and therapies associated with immunosuppression. Primary immune deficiencies, chronic kidney disease, diabetes mellitus, solid and hematological tumors, hematopoetic stem cell transplantation, transplantation of solid organs, aspenia, rheumatological-, gastroenterologic-, dermatologic-, neurologic diseases, biologicals during pregnancy and HIV infection are dealt with.These vaccination guidelines, compiled for the first time in Austria, aim to be of practical help for physicians to facilitate and improve vaccination coverage in immunocompromised patients and their household members and contact persons.

Fingolimod: a review of its mode of action in the context of its efficacy and safety profile in relapsing forms of multiple sclerosis.

Fingolimod is an orally administered, first-in-class therapy for the treatment of relapsing forms of multiple sclerosis. Data from pivotal clinical trials show that fingolimod has a robust, significant effect on annualized relapse rates and MRI outcomes. Fingolimod has a novel, well-characterized mechanism of action. It acts through a specific set of receptors, sphingosine 1-phosphate receptors, present on the surface of a wide range of human cells and tissues, including neural cells, neurons and lymphocytes. Here we review the current literature to describe the mechanism of action of fingolimod in the context of its well-established clinical efficacy and safety profile. Understanding of the mechanisms behind any non-therapeutic effects of fingolimod facilitates their prediction and management in the clinical setting.

Compounded Formulations of Dimethyl Fumarate Show Significant Variability in Product Characteristics.

BACKGROUND: Pharmacy compounded products are not regulated to the same standards as commercially available, approved products, increasing potential safety and efficacy risks to patients. This report describes an in vitro study examining consistency of content and release profile of compounded dimethyl fumarates. METHODS: Samples of compounded dimethyl fumarate (cDMF-A, cDMF-B, cFumaderm) from separate Austrian compounding pharmacies were analyzed for drug content, uniformity of dosage, impurity, and in vitro release. RESULTS: The average dimethyl fumarate (DMF) content ranged from 102.5 to 96.7% of the 120 mg content listed on the product labels. While the DMF capsule-to-capsule content of cDMF-A was somewhat uniform (~20% difference), there was greater variability amongst cDMF-B and cFumaderm capsules (> 30% difference). Impurity testing revealed 2 and 4 unknown components within cDMF-A and cDMF-B, respectively, with levels ranging from 0.05 to 0.81% of total drug content. In in vitro testing of cDMF-A,>10% (12 mg) of DMF was released after 120 min in simulated gastric fluid and 17% (20 mg) released in simulated intestinal fluid after 60 min. While minimal amount of DMF was released from cDMF-B in simulated gastric fluid, 50% (60 mg) of DMF was released after 60 min in simulated intestinal fluid. Similarly for cFumaderm, a fraction (< 20 mg) of the 120 mg target dose was released after several hours in simulated intestinal fluid. The uniformity of release rates across capsules varied significantly. CONCLUSION: These results demonstrate that compounded fumarates are not equivalent to licensed products and may present unknown safety, efficacy, or quality risks.

[Current aspects of therapy conversion for multiple sclerosis]. / Aktuelles zur Therapieumstellung bei Multipler Sklerose.

In recent years the approval of new substances has led to a substantial increase in the number of course-modifying immunotherapies available for multiple sclerosis. Therapy conversion therefore represents an increasing challenge. The treatment options sometimes show complex adverse effect profiles and necessitate a long-term and comprehensive monitoring. This article presents an overview of therapy conversion of immunotherapies for multiple sclerosis in accordance with the recommendations of the Disease-Related Competence Network for Multiple Sclerosis and the German Multiple Sclerosis Society as well as the guidelines on diagnostics and therapy for multiple sclerosis of the German Society of Neurology and the latest research results. At the present point in time it should be noted that no studies have been carried out for most of the approaches for therapy conversion given here; however, the recommendations are based on theoretical considerations and therefore correspond to recommendations at the level of expert consensus, which is currently essential for the clinical daily routine.

[Pemphigoid diseases. Autoimmune diseases in the elderly]. / Pemphigoiderkrankungen. Autoimmunerkrankungen des Alters.

BACKGROUND: The incidence and thereby also the relevance of pemphigoid diseases have significantly increased in elderly patients. METHODS: The clinical features, diagnostic workup, and therapy of pemphigoid diseases based on the recently published German guidelines and expert opinions are described. RESULTS: The diagnosis is based on medical history, clinical manifestations, histopathology, and autoimmune serology. Treatment options depend on the diagnosis and disease severity. Both local and systemic immunosuppressive or immunomodulatory strategies have been proven to be effective. CONCLUSIONS: Chronic, itchy, inflammatory skin disorders in elderly patients are generally suspicious for pemphigoid diseases. The prognosis is usually good. For decisions about (local or systemic) immunosuppressive therapy, gerontological aspects should be taken into consideration.

Ultra-fast cyclosporin A quantitation in whole blood by Laser Diode Thermal Desorption-tandem mass spectrometry; comparison with High Performance Liquid Chromatography-tandem mass spectrometry.

In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization-tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM). A simple protein precipitation step was used prior to depositing 5 µL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS. The laser pattern was set to ramp from 0 to 45% laser power within 2 s. The APCI parameters were set to negative needle voltage (-2 µA), carrier gas temperature (30°C) and air flow rate (3 L min(-1)). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of -40 V. The analysis was achieved within 9 s from sample to sample. The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5 ng mL(-1) (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965 ng mL(-1) was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC-ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982-0.991). The Passing-Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.

Standardization of LC-MS for therapeutic drug monitoring of tacrolimus.

BACKGROUND: LC-MS is increasingly used for therapeutic drug monitoring of tacrolimus. A recent summary from an international proficiency-testing scheme demonstrated that the mass spectrometry respondents were the largest method group. However, these methods lack standardization, which may explain the relatively poor interlaboratory agreement for such methods. This study aimed to provide one path toward the standardization of tacrolimus quantification by use of LC-MS. METHODS: A 40-member whole blood tacrolimus proficiency panel was circulated to 7 laboratories, 4 in the US and 3 in Europe, offering routine LC-MS-based quantification of tacrolimus. All laboratories used a common LC-MS platform and followed the manufacturer's instructions that accompanied an LC-MS reagent kit intended for tacrolimus quantification in whole blood samples. Four patient pools were prepared that had sufficient volume to allow comparison with a tacrolimus reference measurement procedure. RESULTS: For the 40-member panel, the standardized MassTrak LC-MS assay demonstrated excellent agreement with a validated LC-MS method used by Analytical Services International (y = 1.02x - 0.02; r = 0.99). The CVs for the pooled patient samples ranged from 2.0% to 5.4%. The mean difference from the reference measurement procedure ranged from 0.4% to 4.4%. CONCLUSIONS: Tacrolimus assay standardization, which must include all facets of the analysis, is necessary to compare patient results between laboratories and to interpret consensus guidelines. LC-MS can provide accurate and precise measurement of tacrolimus between laboratories.

[Vaccination in adult patients with chronic inflammatory rheumatic diseases]. / Impfung bei erwachsenen Patienten mit chronisch entzündlichen rheumatischen Erkrankungen.

Patients with chronic inflammatory rheumatic diseases often have an intrinsic and therapy associated increased susceptibility to infections which substantially contributes to morbidity and mortality of the patients. A large proportion of these infections are preventable by vaccination. For this reason in 2005 the standing vaccination committee (STIKO) recommended for patients with immunosuppression vaccination against pneumococcus, influenza, Haemophilus influenza b and meningococcus in addition to standard vaccinations, independent of age. Every patient should therefore be informed about a possible increase in susceptibility to infections and the recommended prevention by vaccination before implementation of immunosuppressive therapy.

Fulminant myocarditis.

Myocarditis is most often caused by a viral infection. Less common causes include other infectious agents and autoimmune diseases. Fulminant myocarditis is an unusual complication with a rapidly progressive course resulting in severe heart failure and cardiogenic shock. Fulminant myocarditis should be treated with full supportive care, using aggressive pharmacologic therapy and mechanical circulatory support, because significant improvement in left ventricular function will often occur. Cardiac transplantation is required in a small minority of patients. Cardiac magnetic resonance imaging is becoming a frequently used modality to aid in the diagnosis of myocarditis.

Correlation of thermal analysis and pyrolysis coupled to GC-MS in the characterization of tacrolimus.

In recent years, thermal analysis has assumed major role in the pharmaceutical industry because it can be used to evaluate the stability both in the control of raw materials and the finished product, having employment potential in the development and characterization of new products and assessment processes. Tacrolimus (TCR) is a macrolide lactone with potent immunosuppressive activity. The purpose of this study was to characterize tacrolimus raw material using Thermal analysis and Pyrolysis coupled to Gas chromatography-Mass spectrometry (Pyr-GC-MS). It was analyzed four samples of tacrolimus named TCR A, B, C and D. Thermal analysis experiments was performed in Shimadzu equipment, under nitrogen and synthetic air atmosphere in different heating rate. Pyrolysis analysis was conducted in isothermal conditions of 300°C and 400°C coupled to GC-MS, in which the mass spectrometer was operated in scan mode to detect ions in the range of mass of m/z 25-900. The thermal studies by DSC, DTA and DSC-Photovisual showed desolvation process for all tacrolimus raw materials and TG-dynamical demonstrated two pseudo-polymorphic forms (monohydrate and sesquihydrate) of tacrolimus. It was observed good correlation between the stoichiometric mass losses of the TG-dynamical and identification of product ion in Pyr-GC/MS technique. It was possible to correlate the five pyrolytic product ions with the Ozawa kinetic analysis from the thermal decomposition of TG-dynamical. The thermal studies (DSC, DSC-Photovisual, DTA and TG-dynamical) were applied in the thermal characterization of the raw materials of tacrolimus which showed pseudo-polymorphic forms, which must be monitored by pharmaceutical industry, avoiding future problems in pharmaceutical process, chemical stability and bioavailability of the tacrolimus product.

Evaluation, synthesis and characterization of tacrolimus impurities.

Tacrolimus is an immunosuppressant macrolactam of fermentative origin. By means of HPLC, LC-MS and NMR analyses, coupled with the reference standard synthesis, the main impurities of tacrolimus bulk drug samples were identified and their chemical-physical properties reported. Known ascomycin and tautomers I and II were detected. The correct relative retention time HPLC value of 39,40-dihydro tacrolimus was established. The not described 23,24-anhydro derivative was detected and completely characterized. A full characterization of ascomycin and 39,40-dihydro tacrolimus was also reported.