Manifestation of acute appendicitis as known but paradox visceral side effect of ulcerative colitis anti-inflammatory therapy with januskinase-inhibitor Tofacitinib (Xeljanz™).

BACKGROUND: The etiopathogenesis of accompanying inflammatory phenomena and consequences of immunomodulation constitute a challenging and innovative field in the medical treatment of patients with autoimmune diseases. AIM: Based on i) clinical management experience gained from this challenging clinical case and ii) selective references of reports published in the scientific medical literature, we present an unusual counterfactual scientific case report. A patient diagnosed with ulcerative colitis undergoing januskinase (JAK)-inhibitor therapy developed acuteappendicitis as an unusual complication or as a visceral side effect of immunosuppressive/anti-inflammatory therapy. METHOD: Scientific case report. RESULTS: (case description): Medical history: A 52-year-old male presented with spasmodic pain in the right lower abdomen lasting for two days (no fever, no bowel movement changes (no stool irregularities), no vomiting). MEDICATION USED TO DATE: Steroid-resistant ulcerative colitis treated with immunosuppressive therapy (Adalimumab administered for 10 months [next generation anti-TNFα mAb], Vendolizumab for 9 months [α4ß7 integrin antagonist], Tofacitinib for 6 months); fructose intolerance, no previous abdominal surgery; medication: XeljanzTM (Tofacitinib, 5 mg 2x1; JAK-inhibitor; PFIZER PHARMA GmbH, Berlin,Germany); MutaflorTM (1x1; Ardeypharm GmbH, Herdecke, Germany). CLINICAL FINDINGS: Pressure pain in the right lower abdomen with local muscular defense (Mc-Burney's/Lanz's point positive), no peritonism, Psoas-muscle sign positive. DIAGNOSTIC MEASURES: Laboratory parameters: standard value of white blood cell count, CrP: 25 mg/l.-Transabdominal ultrasound revealed hypertrophic 'appendix vermiformis' with detectable target-phenomenon and surrounding fluid. DECISION-MAKING: Indication for laparoscopic exploration. THERAPY: Under perioperative single-shot antibiotic administration with UnacidTM, the patient underwent emergency laparoscopic appendectomy due to confirmed acute appendicitis with additional lavage and placement of local drainage. CLINICAL COURSE: The postoperative phase was uneventful (sufficient analgetic therapy, removal of local drainage on the 2nd postoperative day). The patient was discharged four days after surgery. Histopathology confirmed ulcero-phlegmonous, acute purulent appendicitis with fibrinous purulent mesenteriolitis. FURTHER MEASURES: Immunosuppressive therapy was continued. CONCLUSION: Based on the paradoxon of an acute inflammatory disease (acute appendicitis) seen in the case of a patient undergoing immunosuppressive/anti-inflammatory treatment using a JAK-Inhibitor for ulcerative colitis, we consider this case worthy of publication although this side effect has previously been described in patients with rheumatoid arthritis. This might be the manifestation of i) an immunomodulatory effect that reduced or at least altered mucosal defense, including an increased risk of opportunistic infections, presenting as a specific visceral 'side effect' of the JAK-Inhibitor and/or as a consequence; ii) an induced alternative inflammatory mechanism/proinflammatory signal transduction and - theoretically - an intestinal drainage defect in the segment of right colic artery with consecutive collection of necrotic cells and activation of inflammatory mediators.

Enzyme replacement therapy with alglucosidase alfa in 44 patients with late-onset glycogen storage disease type 2: 12-month results of an observational clinical trial.

Late-onset glycogen storage disease type 2 (GSD2)/Pompe disease is a progressive multi-system disease evoked by a deficiency of lysosomal acid alpha-glucosidase (GAA) activity. GSD2 is characterized by respiratory and skeletal muscle weakness and atrophy, resulting in functional disability and reduced life span. Since 2006 alglucosidase alfa has been licensed as a treatment in all types of GSD2/Pompe disease. We here present an open-label, investigator-initiated observational study of alglucosidase alfa enzyme replacement therapy (ERT) in 44 late-onset GSD2 patients with various stages of disease severity. Alglucosidase alfa was given i.v. at the standard dose of 20 mg/kg every other week. Assessments included serial arm function tests (AFT), Walton Gardner Medwin scale (WGMS), timed 10-m walk tests, four-stair climb tests, modified Gowers' maneuvers, 6-min walk tests, MRC sum score, forced vital capacities (FVC), creatine kinase (CK) levels and SF-36 self-reporting questionnaires. All tests were performed at baseline and every 3 months for 12 months of ERT. We found significant changes from baseline in the modified Gowers' test, the CK levels and the 6-min walk test (341 +/- 149.49 m, median 342.25 m at baseline; 393 +/- 156.98 m; median 411.50 m at endpoint; p = 0.026), while all other tests were unchanged. ERT over 12 months revealed minor allergic reactions in 10% of the patients. No serious adverse events occurred. None of the patients died or required de novo ventilation. Our clinical outcome data imply stabilization of neuromuscular deficits over 1 year with mild functional improvement.

[Polymyositis associated with thymoma]. / Thymomassoziierte Polymyositis.

Neuromuscular diseases accompanying thymoma include myasthenia gravis, polymyositis, dermatomyositis, and neuromyotonia. Usually 50% of patients with thymoma develop myasthenia gravis. However, only 5% show polymyositis as an accompanying paraneoplastic phenomenon. We report the case of a patient with thymoma showing myasthenia gravis as well as polymyositis. Due to the simultaneous occurrence of these paraneoplastic diseases, the criteria for exact diagnosis (serum creatine kinase, EMG, ocular involvement) overlap. This diagnostic dilemma can appreciably complicate the therapeutic approach.

Bronchial epithelial cells cultured from clinically stable lung allograft patients promote the development of macrophages from monocytes rather than dendritic cells.

BACKGROUND: It is understood that chronic allograft failure occurs as a result of alloimmune and non-alloimmune injury. Dendritic cells (DC) are thought to be crucial in regulating (allo)immune airway damage and interactions with epithelial cells are likely. Studies in human lung transplantation are limited, however, and the available literature on DC is inconsistent. This study focused on the ex vivo influence of primary bronchial epithelial cells derived from lung allografts on DC differentiation. METHODS: Epithelial cell conditioned media (ECCM) were added to monocytes differentiating into DC under the influence of interleukin-4 and granulocyte macrophage-colony stimulating factor. The resultant cells were compared with DC cultured without ECCM and with monocyte-derived macrophages. Expression of typical DC (eg, CD1a) and macrophage (eg, CD14) markers was assessed by flow cytometry. Phenotypical assessments were complemented by functional studies of mannose receptor-mediated phagocytosis (FITC-dextran uptake) and antigen-presenting capability (mixed lymphocyte reactions). RESULTS: Cells exposed to ECCM expressed significantly lower levels of CD1a than unexposed DC. CD14 expression and phagocytic function were increased. ECCM cultured cells also expressed lower levels of T cell co-stimulatory molecules, secreted an anti-inflammatory cytokine profile and had significantly reduced antigen-presenting capability. CONCLUSION: Using phenotypic and functional approaches, this study has shown that ECCM from lung allografts drives the production of macrophage-like cells from monocytes rather than DC. The data suggest that epithelial cells may restrain airway DC and potential alloimmunity. It is unclear whether the observed effect is specifically seen in lung transplant recipients or is a general property of bronchial epithelial cells. This may reflect a homeostatic inter-relationship between airway epithelial and DC populations relevant both to lung allografts and the lung more generally.

Distinct muscle imaging patterns in myofibrillar myopathies.

OBJECTIVE: To compare muscle imaging findings in different subtypes of myofibrillar myopathies (MFM) in order to identify characteristic patterns of muscle alterations that may be helpful to separate these genetic heterogeneous muscular disorders. METHODS: Muscle imaging and clinical findings of 46 patients with MFM were evaluated (19 desminopathy, 12 myotilinopathy, 11 filaminopathy, 1 alphaB-crystallinopathy, and 3 ZASPopathy). The data were collected retrospectively in 43 patients and prospectively in 3 patients. RESULTS: In patients with desminopathy, the semitendinosus was at least equally affected as the biceps femoris, and the peroneal muscles were never less involved than the tibialis anterior (sensitivity of these imaging criteria to detect desminopathy in our cohort 100%, specificity 95%). In most of the patients with myotilinopathy, the adductor magnus showed more alterations than the gracilis muscle, and the sartorius was at least equally affected as the semitendinosus (sensitivity 90%, specificity 93%). In filaminopathy, the biceps femoris and semitendinosus were at least equally affected as the sartorius muscle, and the medial gastrocnemius was more affected than the lateral gastrocnemius. The semimembranosus mostly showed more alterations than the adductor magnus (sensitivity 88%, specificity 96%). Early adult onset and cardiac involvement was most often associated with desminopathy. In patients with filaminopathy, muscle weakness typically beginning in the 5th decade of life was mostly pronounced proximally, while late adult onset (>50 years) with distal weakness was more often present in myotilinopathy. CONCLUSIONS: Muscle imaging in combination with clinical data may be helpful for separation of distinct myofibrillar myopathy subtypes and in scheduling of genetic analysis.

Molecular diagnosis of German patients with late-onset glycogen storage disease type II.

UNLABELLED: In patients with late-onset glycogen storage disease type II, one mutation, c.-32-13T>G, in the α-glucosidase (GAA) gene is identified frequently in European populations from different regions along with many rarer mutations. We have performed molecular genetic investigations in 18 German index patients with late-onset disease. The c.-32-13T>G, c.525delT (p.Glu176fsX45), and c.2481+102_2646+31del mutations were detected by PCR/restriction enzyme digest. Other mutations were detected by sequencing. All patients were compound heterozygous and 17 patients harboured the c.-32-13T>G mutation. Seven other previously described mutations (including the c.-32-13T>G) were identified, of which the p.C103G (c.307T>G) and the c.2481+102_2646+31del mutations were present each in three unrelated patients. Sequencing revealed five novel mutations. CONCLUSIONS: Genetic testing was able to identify the genetic defects in all patients and screening of the c.-32-13T>G mutation identified 94% of the cases. This is important for quick and reliable diagnosis, especially in view of enzyme replacement. Among the rarer mutations, c.2481+102_2646+31del and p.C103G are rather frequent in Germany.

[Neuromyelitis optica: a separate disease entity associated with aquaporin-4-antibodies]. / Neuromyelitis optica: eine mit Aquaporin-4-Antikörpern assoziierte nosologische Entität.

Neuromyelitis optica (NMO; Devic's Syndrome) is an idiopathic, often relapsing, severe inflammatory disorder preferentially affecting optic nerves and spinal cord. The distinction of NMO from multiple sclerosis (MS) as a separate disease entity has been controversally discussed for a long time. Though both diseases show demyelinisation, they differ in typical clinical, imaging and immunopathological findings. The recent identification of serum aquaporin (AQP)-4 antibody clearly separates NMO from MS. In addition, the identification of AQP-4 antibodies supports peripheral humoral autoimmune pathogenesis in NMO and permits early initiation of effective therapy for prevention of attack-related disability.

A simple HPLC-UV method for the determination of dimenhydrinate and related substances--dentification of an unknown impurity.

During the revision of the dimenhydrinate monograph of the European Pharmacopoeia a HPLC-UV method was developed. The procedure described allows a qualitative and quantitative determination of both dimenhydrinate compounds and of thirteen related substances. Furthermore a hitherto unknown impurity was identified and integrated into the purity check. Also 18 samples of dimenhydrinate have been tested. Thereby the relevant impurities of dimenhydrinate could be nominated and quantified.

High dose vitamin E therapy in amyotrophic lateral sclerosis as add-on therapy to riluzole: results of a placebo-controlled double-blind study.

UNLABELLED: Increasing evidence has suggested that oxidative stress may be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). The antioxidant vitamin E (alpha-tocopherol) has been shown to slow down the onset and progression of the paralysis in transgenic mice expressing a mutation in the superoxide dismutase gene found in certain forms of familial ALS. The current study, a double blind, placebo-controlled, randomised, stratified, parallel-group clinical trial, was designed to determine whether vitamin E (5000 mg per day) may be efficacious in slowing down disease progression when added to riluzole. METHODS: 160 patients in 6 German centres with either probable or definite ALS (according to the El Escorial Criteria) and a disease duration of less than 5 years, treated with riluzole, were included in this study and were randomly assigned to receive either alpha-tocopherol (5000 mg per day) or placebo for 18 months. The Primary outcome measure was survival, calculating time to death, tracheostomy or permanent assisted ventilation, according to the WFN-Criteria of clinical trials. Secondary outcome measures were the rate of deterioration of function assessed by the modified Norris limb and bulbar scales, manual muscle testing (BMRC), spasticity scale, ventilatory function and the Sickness Impact Profile (SIP ALS/19). Patients were assessed at entry and every 4 months thereafter during the study period until month 16 and at a final visit at month 18. Vitamin E samples were taken for compliance check and Quality Control of the trial. For Safety, a physical examination was performed at baseline and then every visit until the treatment discontinuation at month 18. Height and weight were recorded at baseline and weight alone at the follow-up visits. A neurological examination as well as vital signs (heart rate and blood pressure), an ECG and VEP's were recorded at each visit. Furthermore, spontaneously reported adverse experiences and serious adverse events were documented and standard laboratory tests including liver function tests performed. For Statistical Analysis, the population to be considered for the primary outcome measure was an "intent-to-treat" (ITT) population which included all randomised patients who had received at least one treatment dose (n = 160 patients). For the secondary outcome measures, a two way analysis of variance was performed on a patient population that included all randomised patients who had at least one assessment after inclusion. RESULTS: Concerning the primary endpoint, no significant difference between placebo and treatment group could be detected either with the stratified Logrank or the Wilcoxon test. The functional assessments showed a marginal trend in favour of vitamin E, without reaching significance. CONCLUSION: Neither the primary nor the secondary outcome measures could determine whether a megadose of vitamin E is efficacious in slowing disease progression in ALS as an add-on therapy to riluzol. Larger or longer studies might be needed. However, administration of this megadose does not seem to have any significant side effects in this patient population.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

Apoptosis by 6-O-palmitoyl-L-ascorbic acid coincides with JNK-phosphorylation and inhibition of Mg2+-dependent phosphatase activity.

6-O-Palmitoyl ascorbic acid (PAA) has recently been used as a substitute for ascorbic acid because of its greater potency as an antioxidant. In detailed concentration response studies distinct cytotoxic effects of PAA at concentrations exceeding 100 microM were reported. Here we examined and further characterized this cytotoxicity. While ascorbic acid was tolerated well up to millimolar concentrations, PAA revealed an LC50 between 125 and 150 microM in rat GH3 tumor cells. Morphological and biochemical observations suggested the induction of apoptosis at concentrations exceeding 125 microM with a prominent activation of caspase 3 at 250 microM after 4 hr. A subsequent pronounced fragmentation of DNA (DNA-ladder) was detected after 6 hr and was further enhanced after 12 hr. The activation of caspases and the cleavage of its substrate PARP was preceded by a distinct increase in the phosphorylation of stress activated JNK-kinases. This observation suggested that the agent affected signal transduction mechanisms regulating protein phosphorylation at serine/threonine residues in the cell. No effect of PAA on protein phosphatase 2A (PP2A)-like activity was observed while magnesium-dependent protein phosphatase activity, presumably PP2C, was inhibited concentration-dependently up to 75% at the respective concentrations. Thus, the cytotoxic, pro-apoptotic effect of PAA might be related to the inhibition of PP2C and the activation of JNK.

Effects of medicinal compounds on the differentiation of the eukaryotic microorganism dictyostelium discoideum: can this model be used as a screening test for reproductive toxicity in humans?

Dictyostelium discoideum is a single-cell, eukaryotic microorganism that can undergo multicellular development in order to produce dormant spores. We investigated the capacity of D. discoideum to be used as a rapid screening system for potential developmental toxicity of compounds under development as pharmaceuticals. We used a set of four transgenic D. discoideum strains that expressed a reporter gene under the control of promoters that are active at certain time periods and in distinct cell types during D. discoideum development. We found that teratogens such as valproic acid, tretinoin, or thalidomide interfered to various extents with D. discoideum development, and had different effects on prestalk and prespore cell-specific reporter gene expression. Phenytoin was inactive in this assay, which may point to limitations in metabolization of the compound in Dictyostelium required to exert developmental toxicity. D. discoideum cell culture is cheap and easy to handle compared to mammalian cell cultures or animal teratogenicity models. Although the Dictyostelium-based assay described in this report may not securely predict the teratogenic potential of these drugs in humans, this organism may be qualified for rapid large-scale screenings of synthetic compounds under development as new pharmaceuticals for their potential to interfere with developmental processes and thus help to reduce the amount of teratogenicity tests in animal models.

[Facioscapulohumeral muscular dystrophy. The spectrum of clinical manifestations and molecular genetic changes]. / Fazioskapulohumerale Muskeldystrophie. Spektrum der klinischen Manifestationen und molekulargenetischen Veränderungen.

Although the gene for facioscapulohumeral muscular dystrophy (FSHD) has not been identified so far, 4q35 deletion represents a diagnostic marker of the disease. In the present study, 46 consecutive symptomatic patients with 4q35 FSHD deletions or typical FSHD clinical features were evaluated. The patients were divided into three groups: 33 patients (72%) with typical FSHD phenotype and 4q35 FSHD deletion, eight (17%) with atypical (non-Landouzy-Dejerine) FSHD phenotype but with 4q35 FSHD deletion, and five patients (11%) with the typical FSHD phenotype but without FSHD 4q35 deletion. Apparently, the 4q35 deletion is associated not only with Landouzy-Dejerine FSHD but also with a variety of "atypical" FSHD forms. On the other hand, the Landouzy-Dejerine FSHD phenotype is possibly a polyetiological syndrome caused in some patients by other genetic effects than 4q35 deletion.

Profiling indomethacin impurities using high-performance liquid chromatography and nuclear magnetic resonance.

The anti-inflammatory drug indomethacin was investigated regarding new related impurities. Therefore, related substances 2-9 were prepared by independent synthesis and physicochemically characterized. To determine indomethacin and its related substances, a new HPLC-UV method was developed and validated. Indomethacin and its impurities were eluted on a C(18) column with a mobile phase consisting of methanol and an aqueous solution of 0.2% phosphoric acid at a flow rate of 1.5 ml/min and were quantified by UV detection at 320 nm. Overall, the HPLC-UV method was simple and reliable for the detection of eight impurities in indomethacin. In addition to the HPLC-UV method, 1H nuclear magnetic resonance (NMR) was used to investigate indomethacin regarding impurities. For that purpose, related substances 2-9 were systematically added to indomethacin and investigated. The NMR method was found to be very useful for the identification of impurities in bulk substance without prior separation. Both HPLC-UV and NMR were used to analyze 38 batches of indomethacin available on the European market. The outcome was that 42% of the batches did not meet the compendial requirements although they met the specifications of current compendial methods. Some batches contained the previously undescribed impurity 8, while other batches contained by-products from two distinct synthetic routes. The methods presented herein are important contributions to the ongoing efforts to reduce impurities and therefore the risk of adverse side-effects in drugs that are no longer under patent protection.

Aza analogues of thalidomide: synthesis and evaluation as inhibitors of tumor necrosis factor-alpha production in vitro.

A synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported. These 5-phthalimidopyrimidines as well as phthalimido-2,4-difluorobenzenes were designed as analogues of thalidomide, a well known inhibitor of TNF-alpha production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-alpha production was performed. Among the compounds of the present series, 5-ethyl-1-phenyl-5-(tetrafluorophthalimido)barbituric acid and 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione were proved to be potent inhibitors. Both compounds showed inhibitory activity in the lower micromolar range on the LPS-induced TNF-alpha production in human monocytes.

Separation, analyses and syntheses of trimethoprim impurities.

The analysis of several impurities of the chemotherapeutic agent trimethoprim with various methods, including thin layer chromatography, gas chromatography, capillary electrophoresis as well as nuclear magnetic resonance is described. These methods were used to identify new impurities in trimethoprim batches. The main impurities were separated by column chromatography. To ensure the identity of the impurities, de novo syntheses were successfully carried out. With the methods described, it was possible to detect, separate and identify new impurities in trimethoprim batches.

Rapid and sensitive LC separation of new impurities in trimethoprim.

Trimethoprim is a chemotherapeutic often used in combination with sulfonamides. Herein, we report on the development and validation of a new HPLC assay of trimethoprim. The test allows the identification of new impurities that have not been detectable with any other known method including European Pharmacopoeia and USP. Trimethoprim and its impurities were eluted on a C18 column with a mobile phase consisting of methanol and a solution of sodium perchlorate at a flow rate of 1.3 ml/min and was quantified by UV detection at 280 nm. Overall, the method is simple, rapid and reliable for the detection of six impurities in trimethoprim batches.

Synthesis and immunological activity of water-soluble thalidomide prodrugs.

A series of new water-soluble thalidomide prodrugs was prepared. All compounds were derivatized on the nitrogen of the glutarimide ring. Esters of natural amino acids and succinic acid derivatives have been introduced by reaction with the hydroxymethyl thalidomide 2. Nicotinic acid derivatives were prepared from halomethyl derivatives. Additionally, a methoxymethyl derivative and a carboxymethyl derivative were prepared directly from thalidomide. Most compounds showed a very large increase in water solubility compared to thalidomide itself (0.012mg/mL). The amorphous hydrochlorides of the N-methylalanine ester 8, valine ester 9, and glycylglycine ester 10, respectively, were the most soluble compounds showing solubility greater than 300mg/mL, which equals an increase greater than 15,000-fold. The lipophilicity of the prodrugs has been determined by their HPLC capacity factors k'. The stability of selected compounds was determined. The hydrolysis rates follow pseudo-first order kinetics. In order to assess the immunological activity, the prodrugs were tested using tumor necrosis factor-alpha and interleukin-2 inhibition assays. Selected compounds were additionally investigated on their abililty to inhibit the local Shwartzman reaction, an assay to determine the vascular permeability. The prodrugs retained high effectiveness in the inhibition of TNF-alpha release. Our results indicated that the more stable prodrugs exhibited higher activity in the immunological assays. Some compounds showed higher activity than thalidomide itself, suggesting a high affine binding to the pharmacophore. In conclusion, the prodrugs exhibited high water solubility and high activity and might therefore be used in therapeutic applications.