Clinical presentation of terbinafine-induced severe liver injury and the value of laboratory monitoring: a Critically Appraised Topic.

CONTEXT: Many physicians monitor liver function tests during terbinafine therapy. OBJECTIVES: To evaluate the symptoms of published cases of terbinafine-associated severe drug-induced liver injury (DILI) to assess the utility of laboratory monitoring. DATA SOURCES: We based our search on the LiverTox database of the National Institutes of Health, but we also searched both PubMed and Embase. In addition, we hand searched the references of the papers we found. STUDY SELECTION: All reports of patients with DILI on terbinafine and with reported clinical symptoms, or absence thereof, were evaluated. DATA EXTRACTION: Two independent reviewers (J.A. and O.N.K.) assessed articles for eligibility of inclusion, and collected and evaluated the data. DATA SYNTHESIS: Thirty-eight papers fulfilled the inclusion criteria, with reports of 69 symptomatic patients. The mean duration of terbinafine treatment until onset of symptoms was 30·2 days (range 5-84). Symptoms in order of frequency were jaundice, flu-like symptoms, dark urine and pruritus. Patients experienced symptoms for a mean and median of 14·8 and 16 days, respectively (range 0-42) until seeking medical attention. CONCLUSIONS: Patients who had DILI were symptomatic, usually with jaundice, abdominal pain and general malaise, but also with severe pruritus. No asymptomatic patient was identified through laboratory screening. The timeline of DILI onset varies significantly, but most cases occur between 4 and 6 weeks. There was no time point at which monitoring was meaningful, and we do not recommend monitoring of liver function tests on terbinafine; however, patients should be advised to discontinue treatment and look for medical care when symptoms of DILI occur.

Not every patient needs a triglyceride check, but all can get pancreatitis: a systematic review and clinical characterization of isotretinoin-associated pancreatitis.

Monitoring of triglycerides for patients on isotretinoin is practised primarily to avoid hypertriglyceridaemia-associated pancreatitis. The aim of this study was to describe clinically the published cases of hypertriglyceride-associated pancreatitis. A comprehensive search strategy using MEDLINE, Embase and grey literature was conducted (1960 to January 2016) to identify all case reports of isotretinoin-associated pancreatitis and all relevant studies of isotretinoin and triglycerides for any indication (≥ 20 patients). Terms related to isotretinoin, triglycerides and pancreatitis were searched with all available synonyms. Any studies that used isotretinoin and mentioned triglycerides or pancreatitis were searched in full text, where available, for cases of pancreatitis. Studies from all countries and published in any language were included, but Korean and Turkish studies could not be analysed. Two authors independently reviewed the publications to determine eligibility, and for data extraction. In total, 125 papers fulfilled the inclusion criteria and were searched for cases of pancreatitis. Eleven papers with 25 cases of pancreatitis associated with isotretinoin were identified; four of these cases were likely due to hypertriglyceridaemia. Three patients had elevated baseline triglycerides, but no monitoring. Pancreatitis occurred 6 and 7 weeks, and 6 months after initiation of therapy. For the fourth patient who was treated for glioblastoma and died, no detailed clinical information was available. Idiosyncratic pancreatitis associated with isotretinoin is the most frequent pancreatitis on isotretinoin, and patients should be warned about it. Hypertriglyceride-associated pancreatitis is an exceedingly rare adverse event of isotretinoin therapy. Our data cannot give a frequency or risk for either adverse event. Based on the clinical information of the patients available, we conclude that for patients without elevated baseline triglycerides, or risk thereof, monitoring of triglycerides during therapy is of little value.

Antagonism between granulocytic maturation and deacetylase inhibitor-induced apoptosis in acute promyelocytic leukaemia cells.

BACKGROUND: Transcriptional repression is a key mechanism driving leukaemogenesis. In acute promyelocytic leukaemia (APL), the fusion protein promyelocytic leukaemia-retinoic acid receptor-α fusion (PML-RARα) recruits transcriptional repressors to myeloid differentiation genes. All-trans-retinoic acid (ATRA) induces the proteasomal degradation of PML-RARα and granulocytic differentiation. Histone deacetylases (HDACs) fall into four classes (I-IV) and contribute to the transcription block caused by PML-RARα. METHODS: Immunoblot, flow cytometry, and May-Grünwald-Giemsa staining were used to analyze differentiation and induction of apoptosis. RESULTS: A PML-RARα- and ATRA-dependent differentiation programme induces granulocytic maturation associated with an accumulation of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP)ɛ and of the surface protein CD11b. While this process protects APL cells from inhibitors of class I HDAC activity, inhibition of all Zinc-dependent HDACs (classes I, II, and IV) with the pan-HDACi (histone deacetylase inhibitor(s)) LBH589 induces apoptosis of immature and differentiated APL cells. LBH589 can eliminate C/EBPɛ and the mitochondrial apoptosis regulator B-cell lymphoma (BCL)-xL in immature and differentiated NB4 cells. Thus, BCL-xL and C/EBPɛ are newly identified molecular markers for the efficacy of HDACi against APL cells. CONCLUSIONS: Our results could explain the therapeutic limitations occurring with ATRA and class I HDACi combinations. Pro-apoptotic effects caused by pan-HDAC inhibition are not blunted by ATRA-induced differentiation and may provide a clinically interesting alternative.

Circular economy in drinking water treatment: reuse of ground pellets as seeding material in the pellet softening process.

Calcium carbonate pellets are produced as a by-product in the pellet softening process. In the Netherlands, these pellets are applied as a raw material in several industrial and agricultural processes. The sand grain inside the pellet hinders the application in some high-potential market segments such as paper and glass. Substitution of the sand grain with a calcite grain (100% calcium carbonate) is in principle possible, and could significantly improve the pellet quality. In this study, the grinding and sieving of pellets, and the subsequent reuse as seeding material in pellet softening were tested with two pilot reactors in parallel. In one reactor, garnet sand was used as seeding material, in the other ground calcite. Garnet sand and ground calcite performed equally well. An economic comparison and a life-cycle assessment were made as well. The results show that the reuse of ground calcite as seeding material in pellet softening is technologically possible, reduces the operational costs by €38,000 (1%) and reduces the environmental impact by 5%. Therefore, at the drinking water facility, Weesperkarspel of Waternet, the transition from garnet sand to ground calcite will be made at full scale, based on this pilot plant research.

p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors.

BACKGROUND: Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status. METHODS: Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling. RESULTS: Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53. CONCLUSION: These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.

Effects of high passage cultivation on CHO cells: a global analysis.

Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.

Predicting ligand interactions with ABC transporters in ADME.

ABC-type drug efflux pumps, e.g., ABCB1 (=P-glycoprotein, =MDR1), ABCC1 (=MRP1), and ABCG2 (=MXR, =BCRP), confer a multi-drug resistance (MDR) phenotype to cancer cells. Furthermore, the important contribution of ABC transporters for bioavailability, distribution, elimination, and blood-brain barrier permeation of drug candidates is increasingly recognized. This review presents an overview on the different computational methods and models pursued to predict ABC transporter substrate properties of drug-like compounds. They encompass ligand-based approaches ranging from 'simple rule'-based efforts to sophisticated machine learning methods. Many of these models show excellent performance for the data sets used. However, due to the complex nature of the applied methods, useful interpretation of the models that can be directly translated into chemical structures by the medicinal chemist is rather difficult. Additionally, very recent and promising attempts in the field of structure-based modeling of ABC transporters, which embody homology modeling as well as recently published X-ray structures of murine ABCB1, will be discussed.

Model-based operational constraints for fluidised bed crystallisation.

Fluidised bed reactors are used for water softening in water treatment plants. Recent research shows that under current operation of reactors the crystallisation of calcium carbonate can be hampered. Until now the operational constraints on the fluidised bed have not been exactly known. Experiments were carried out to investigate the fluidisation behaviour of calcium carbonate pellets in water. The results of the fluidisation experiments are compared to two commonly used modelling approaches of Ergun and Richardon-Zaki. Using the experimental data the models are calibrated. The calibrated Richardson-Zaki model is used to determine operational constraints on pellet size at the bottom of the reactor and water flow through the reactor. The model-based constraints are compared to operational data of the Weesperkarspel full-scale treatment plant of Waternet (The Netherlands). It can be concluded that the current operation of the treatment plant violates the calculated constraints with consequences for effluent quality and corrective maintenance. By using models for determining the operation of the fluidised bed, the softening process can thus be improved.

In silico prediction of substrate properties for ABC-multidrug transporters.

Overexpression of ABC (ATP-binding cassette)-type drug efflux pumps, such as ABCB1, ABCC1 and ABCG2 in cancer cells confers multi-drug resistance (MDR) and represents a major cause of treatment failures in cancer therapy. Furthermore, there is increasing evidence for the important contribution of ABC-transporters to bioavailability, distribution, elimination and blood-brain barrier permeation of drug candidates. This review presents an overview on the different computational methods and models pursued to predict ABC-transporter substrate properties of drug-like compounds. They range from linear discriminant analysis to pharmacophore modelling and machine learning algorithms. Many of these models show a satisfying performance within the study-specific, defined chemical space but general applicability for the whole drug-like chemical space still needs to be proven. First attempts aiming towards selectivity profiling for ligands of the two polyspecific transporters ABCB1 and ABCG2 is also discussed. This might pave the way for a pharmacological profiling of compound series with special focus on their ADMET (absorption, distribution, metabolism, excretion and toxicity) properties.

O-GlcNAcylation of STAT5 controls tyrosine phosphorylation and oncogenic transcription in STAT5-dependent malignancies.

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.

HDAC1 and HDAC2 integrate the expression of p53 mutants in pancreatic cancer.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Lymphangioma-like Kaposi Sarcoma in Transplant-Associated Iatrogenic Immunosuppression: A Case Report.

Lymphangioma-like Kaposi sarcoma (LLKS) is a rare histologic presentation of Kaposi sarcoma (KS), with only 28 cases reported in the literature. LLKS has been described in acquired immunodeficiency syndrome and in endemic African-type as well as classic indolent KS. We present the 1st reported case of LLKS in a transplant-associated iatrogenic immunosuppressed patient.

Histone deacetylase as a therapeutic target.

The maintenance of health depends on the coordinated and tightly regulated expression of genetic information. Certain forms of leukemia have become paradigms for the pathogenic role of aberrant repression of differentiation genes. In these acute leukemias, fusion proteins generated by chromosomal translocations no longer function as transcriptional activators, but instead repress target genes by recruiting histone deacetylases (HDACs). The potential benefit of HDAC inhibition has been established by the use of enzyme inhibitors in vitro and in a single reported case of experimental therapy. Because recently identified HDAC inhibitors appear to overcome many drawbacks of early inhibitory compounds in clinical use, the stage is set to test the therapeutic value of HDAC inhibition in leukemias and in other diseases, including solid tumors and aberrant hormonal signaling. This review summarizes the range of diseases expected to respond to HDAC inhibition.

SIAH proteins: critical roles in leukemogenesis.

The delicate balance between the synthesis and the degradation of proteins ensures cellular homeostasis. Proteases act in an irreversible manner and therefore have to be strictly regulated. The ubiquitin-proteasome system (UPS) is a major pathway for the proteolytic degradation of cellular proteins. As dysregulation of the UPS is observed in most cancers including leukemia, the UPS is a valid target for therapeutic intervention strategies. Ubiquitin-ligases selectively bind substrates to target them for poly-ubiquitinylation and proteasomal degradation. Therefore, pharmacological modulation of these proteins could allow a specific level of control. Increasing evidence accumulates that ubiquitin-ligases termed mammalian seven in absentia homologs (SIAHs) are not only critical for the pathogenesis of solid tumors but also for leukemogenesis. However, the relevance and therapeutic potential of SIAH-dependent processes has not been fully elucidated. Here, we summarize functions of SIAH ubiquitin-ligases in leukemias, how they select leukemia-relevant substrates for proteasomal degradation, and how the expression and activity of SIAH1 and SIAH2 can be modulated in vivo. We also discuss that epigenetic drugs belonging to the group of histone deacetylase inhibitors induce SIAH-dependent proteasomal degradation to accelerate the turnover of leukemogenic proteins. In addition, our review highlights potential areas for future research on SIAH proteins.

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

Inhibitors of HDACs--effective drugs against cancer?

Alterations in genomic and non-genomic mechanisms can disturb homeostasis and cause severe human diseases. Histone deacetylases (HDACs) are epigenetic regulators which catalyze the removal of acetyl moieties from histones and non-histone proteins. Aberrant histone deacetylation, due to increased HDAC activity and expression, often correlates with pathological gene repression and neoplastic transformation. Therefore, intense efforts have been made to find small molecule inhibitors of HDACs (HDACIs). Such compounds indeed alter cellular signaling networks relevant for tumorigenesis, and several HDACIs are currently tested in clinical trials against different types of cancer. Although HDACs share a conserved deacetylase domain and an at least similar mechanism of catalysis, isoenzyme-specific HDACIs could be identified and certain HDACIs even evoke degradation of HDACs. Here, we summarize molecular actions of HDACs and of different classes of HDACIs. In addition, we review data obtained in clinical studies involving HDACIs and we discuss how such agents might be beneficial for the treatment of cancer.

Ubiquitin conjugase UBCH8 targets active FMS-like tyrosine kinase 3 for proteasomal degradation.

The class III receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) regulates normal hematopoiesis and immunological functions. Nonetheless, constitutively active mutant FLT3 (FLT3-ITD) causally contributes to transformation and is associated with poor prognosis of acute myeloid leukemia (AML) patients. Histone deacetylase inhibitors (HDACi) can counteract deregulated gene expression profiles and decrease oncoprotein stability, which renders them candidate drugs for AML treatment. However, these drugs have pleiotropic effects and it is often unclear how they correct oncogenic transcriptomes and proteomes. We report here that treatment of AML cells with the HDACi LBH589 induces the ubiquitin-conjugating enzyme UBCH8 and degradation of FLT3-ITD. Gain- and loss-of-function approaches show that UBCH8 and the ubiquitin-ligase SIAH1 physically interact with and target FLT3-ITD for proteasomal degradation. These ubiquitinylating enzymes though have a significantly lesser effect on wild-type FLT3. Furthermore, physiological and pharmacological stimulation of FLT3 phosphorylation, inhibition of FLT3-ITD autophosphorylation and analysis of kinase-inactive FLT3-ITD revealed that tyrosine phosphorylation determines degradation of FLT3 and FLT3-ITD by the proteasome. These results provide novel insights into antileukemic activities of HDACi and position UBCH8, which have been implicated primarily in processes in the nucleus, as a previously unrecognized important modulator of FLT3-ITD stability and leukemic cell survival.