Intracranial hemorrhage with direct oral anticoagulants in patients with brain tumors.

Essentials Intracranial hemorrhage (ICH) is common in patients with brain tumors. We compared rates of ICH with DOACs and low molecular weight heparin. DOACs were associated with a lower incidence of ICH in primary brain tumors. DOACs appear safe to administer to patients with brain tumors. SUMMARY: Background Direct oral anticoagulants (DOACs) are efficacious in the treatment of cancer-associated thrombosis but are associated with an increased risk of hemorrhage compared with low-molecular-weight heparin in certain malignancies. Whether the DOACs increase the incidence of intracranial hemorrhage (ICH) in patients with brain tumors is not established. Objectives To determine the cumulative incidence of ICH in DOACs compared with Low-molecular-weight heparin (LMWH) in patients with brain tumors and venous thromboembolism. Patients and methods A retrospective comparative cohort study was performed. Radiographic images for all ICH events were reviewed and the primary endpoint was cumulative incidence of ICH at 12 months following initiation of anticoagulation. Results and conclusions A total of 172 patients with brain tumors were evaluated (42 DOAC and 131 LMWH). In the primary brain tumor cohort (n = 67), the cumulative incidence of any ICH was 0% in patients receiving DOACs vs. 36.8% (95% confidence interval [CI], 22.3-51.3%) in those treated with LMWH, with a major ICH incidence of 18.2% (95% CI, 8.4-31.0). In the brain metastases cohort (n = 105), DOACs did not increase the risk of any ICH relative to enoxaparin, with an incidence of 27.8% (95% CI, 5.5-56.7%) compared with 52.9% (95% CI, 37.4-66.2%). Similarly, DOAC did not increase the incidence of major ICH in brain metastases, with a cumulative incidence 11.1% (95% CI, 0.5-40.6%) vs. 17.8% (95% CI, 10.2-27.2%). We conclude that DOACs are not associated with an increased incidence of ICH relative to LMWH in patients with brain metastases or primary brain tumors.

Acute hepatic encephalopathy: diffusion-weighted and fluid-attenuated inversion recovery findings, and correlation with plasma ammonia level and clinical outcome.

BACKGROUND AND PURPOSE: In acute hepatic encephalopathy, MR imaging abnormalities have been described in the PVWM, thalami, and corticospinal tracts. We sought to determine characteristic regions of involvement on FLAIR and DWI, to evaluate their reversibility, and to correlate MR imaging extent with clinical severity. MATERIALS AND METHODS: Twenty patients who presented clinically with acute hepatic encephalopathy and MR imaging <21 days after symptom onset were reviewed retrospectively. Two neuroradiologists recorded involved regions on FLAIR and DWI in each, measured ADC values in affected regions and NAWM, and scored the MR imaging severity/extent. The initial severity (West Haven grade), follow-up clinical severity (degree of improvement), and maximal PAL within ±8 days of MR imaging were recorded and correlated with the MR imaging severity. RESULTS: On FLAIR and DWI respectively, there were abnormalities in the thalami (85%, 70%), PLIC (75%, 80%), PVWM (80%, 85%), and DBS (70%, 35%) and diffuse cortical involvement (30%, 25%). There were relatively strong significant (P < .005) correlations of FLAIR (r = 0.680, P = .001) and DWI severity (r = 0.690, P = .001) with PAL, and of PAL with the clinical outcome (r = 0.691, P = .001). Both FLAIR (r = 0.592, P = .006) and DWI (r = 0.487, P = .029) severity correlated moderately with the clinical outcome but were not significant at the P < .005 level after Bonferroni correction. CONCLUSIONS: Patients with acute hepatic encephalopathy may exhibit characteristic regions of involvement on FLAIR with DWI findings that can be reversible. The MR imaging extent on FLAIR and DWI strongly correlates with the maximal PAL, and PAL correlates well with the clinical outcome. Diffuse cortical involvement has a higher potential for neurologic sequelae but can be reversible.

Quantification of oligodeoxynucleotides in human plasma with a novel hybridization assay offers greatly enhanced sensitivity over capillary gel electrophoresis.

Capillary gel electrophoresis using UV detection (CGE-UV) has been used to quantify oligodeoxynucleotides (ODN) in human plasma. Although the sensitivity of this method is adequate to detect antisense ODN, which are administered in daily doses up to 10 mg/kg, CGE-UV is not sensitive enough to detect the much lower quantities of ODN administered for other purposes, such as immune stimulation by CpG ODN. We have developed a very sensitive colorimetric hybridization assay that increases the sensitivity of detection by more than four logs compared with CGE-UV. The hybridization assay uses sequence-specific capture and detection ODN probes complementary to portions of the ODN sequence. Herein we provide a prototype for assay development and validation using a 24- mer immunostimulatory phosphorothioate ODN. Probes were locked nucleic acids (LNA), resulting in increased sensitivity and specificity. The linear range of the assay is 7.8-1000 pg/ml, with a 7.8 pg/ml lower limit of quantification (LLOQ) and a detection limit of 2.8 pg/ml. This translates to detection of 40 attamoles. Intraassay and interassay precision were < or =5.0% CV and < or =12.9% CV, respectively, for quality control samples. The assay is suitable for a variety of matrices, including monkey and rat plasma, allowing application to toxicokinetic samples. The methodology is highly specific, with the ability to distinguish almost all single-base mismatched ODN. The assay detects 100% of the parent as well as some metabolites up to N-4, which are known to be the primary metabolites forming in the first hours after in vivo administration and are physiologically active with in vitro assays.

Oligonucleotide technologies: synthesis, production, regulations and applications. 29-30th November 2000, Hamburg, Germany.

The two-day meeting 'Oligonucleotide technologies', organised by IBC Global Conferences Limited and chaired by M Gait (Medical Research Council, UK), was the first event of this type in Europe. It covered a wide range of oligonucleotide-based approaches useful for the identification and validation of new therapeutic targets as well as for the creation of novel oligonucleotide-based drugs. Recent progress in the preclinical and clinical development of new oligonucleotide-based therapeutics as well as new platforms and systems for DNA microarrays, allowing massively parallel gene expression studies, were discussed. Furthermore, new approaches for overcoming challenges in the large scale synthesis and functionalisation of oligonucleotides were revealed. The following meeting highlights are focussed on contributions devoted to the development of novel oligonucleotide-based drugs, such as antisense oligonucleotides, immunostimulatory CpG oligonucleotides and spiegelmers. Drug candidates discussed here are related to various indications, including cancer, infectious diseases and asthma. Besides highlighting of specific clinical candidates, emphasis is put on the basic principles of oligonucleotide-based drugs and approaches towards the improvement of their properties.

Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.

Neurotrophins and their receptors modulate cerebral cortical development. Tubers in the tuberous sclerosis complex (TSC) are characterized histologically by disorganized cortical cytoarchitecture and thus, we hypothesized that expression of neurotrophin mRNAs and proteins might be altered in tubers. Using in situ transcription and mRNA amplification to probe cDNA arrays, we found that neurotrophin-3 (NT3) and trkB mRNA expression were reduced whereas neurotrophin-4 (NT4) and trkC mRNA expression were increased in whole tuber sections. Alterations in mRNA abundance were defined in single microdissected dysplastic neurons (DNs) and giant cells (GCs). NT3 mRNA expression was reduced in GCs and trkB mRNA expression was reduced in DNs. NT4 mRNA expression was increased in DNs and trkC mRNA expression was increased in both DNs and GCs. In three patients, TSC2 locus mutations were confirmed and the mean tuberin mRNA expression levels was reduced across all nine cases. Consistent with these observations, NT3 mRNA expression was reduced but trkC mRNA expression was increased in vitro in human NTera2 neurons (NT2N) transfected with a tuberin antisense construct that reduced tuberin expression. Western analysis of tuber homogenates and computer-assisted densitometry of immunolabeled sections confirmed the neurotrophin mRNA expression data in whole sections and single neurotrophin immunoreactive cells. We conclude that alterations in NT4/trkB and NT3/trkC expression may contribute to tuber formation during brain development as downstream effects of the hamartin and tuberin pathway in TSC.

Prestorage universal WBC reduction of RBC units does not affect the incidence of transfusion reactions.

BACKGROUND: Febrile nonhemolytic transfusion reaction (FNHTR) has been identified as a pivotal reason for prestorage universal WBC reduction. A regional blood center implemented universal prestorage WBC reduction for RBCs on January 1, 2000. Whether prestorage universal WBC reduction of RBC units will affect FNHTR is not known. STUDY DESIGN AND METHODS: All reports of RBC transfusion reactions at Barnes-Jewish Hospital submitted for evaluation to the blood bank, before and after the implementation of WBC reduction of RBCs, were retrospectively evaluated. RESULTS: For the 36,303 allogeneic RBC transfusions administered in 1999, 85 reactions (0.23%) were reported. These reactions were classified as FNHTR in 43 cases, allergic in 13, delayed hemolytic in 19, and miscellaneous in 10. For the 31,543 non-WBC-reduced RBC transfusions performed in 1999, 78 reactions (0.25%) were reported. These reactions were classified as FNHTR in 39 cases, allergic in 13, delayed hemolytic in 19, and miscellaneous in 7. In the first half of 2000, 32 reactions (0.20%) were reported for 16,093 prestorage WBC-reduced RBC transfusions (p = 0.41). There were 13 FNHTRs and 10 allergic, 7 delayed hemolytic, and 2 miscellaneous reactions. The use of prestorage WBC-reduced RBCs did not significantly affect the rate of reactions classified as allergic (0.04% in 1999; 0.06% in 2000; p = 0.43) or as FNHTR (0.12% in 1999; 0.08% in 2000; p = 0.33). For all patients, universal WBC reduction in 2000 did not reduce the rate of FNHTR from the rate seen with selective bedside WBC reduction, the practice used in 1999 (0.12% in 1999; 0.08% in 2000; p = 0.36). CONCLUSION: No significant difference was found in the incidence of transfusion reactions in patients receiving prestorage WBC-reduced RBCs and non-WBC-reduced RBCs. In addition, no difference was found in transfusion reaction rates when periods of prestorage universal WBC reduction were compared to those of selective WBC reduction.

The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Tumor suppressor gene regulation of cell growth: recent insights into neurofibromatosis 1 and 2 gene function.

The development of cancer involves a myriad of genetic changes that impact on multiple processes important for the orderly regulation of cell growth and differentiation. Genes whose protein products are disrupted during neoplastic transformation are termed "tumor suppressor genes" (TSGs). Many of these TSGs are associated with familial cancer predisposition syndromes, in which affected individuals have an increased risk of certain malignancies. Studies on the mechanism of action for known TSGs have revealed three intracellular loci of critical importance: environmental sensing and signal initiation, signal propagation and transduction, and cell cycle control. The neurofibromatosis 1 and neurofibromatosis 2 genes are discussed as illustrative examples of tumor suppressors that function at the levels of signal transduction and environmental sensing, respectively.

Bile acid-oligodeoxynucleotide conjugates: synthesis and liver excretion in rats.

The synthesis of bile acid oligodeoxynucleotide conjugates via the 3-OH group of the bile acids is described. When used in vivo in rats, covalent conjugation of an oligodeoxynucleotide via a linker to cholic acid resulted in an increased biliary excretion of bile acid-oligodeoxynucleotide conjugates compared to unconjugated oligodeoxynucleotides.

Inhibition of Ras p21 synthesis by antisense undecamers with uniform and specifically arranged phosphorothioate linkages.

The design of chimeric oligodeoxynucleotides (ODNs) in which certain phosphodiester linkages are replaced by phosphorothioate (PS) aims to decrease non-sequence-specific effects of uniform PS ODNs and to preserve the PS-provided protection against exo- and endonucleases. This study has, for the fist time, directly compared the differences in nuclease resistance, cellular uptake, antisense potency and sequence specificity of PS and end-capped, pyrimidine-protected (PPS) undecamer ODNs, that are complementary to the initiation codon region of human Ha-ras mRNA. At concentrations above 5 microM, both PS and PPS undecamers were moderately and equally stable for over 48 h in complete medium with RS485 cells overexpressing Ha-ras. They were completely stable at 0.4 microM when complexed with Lipofectin reagent that enhanced cellular uptake up to 9-fold. Both the antisense PPS and PS undecamers produced well-defined inhibition of Ras p21 synthesis in both cell-free and cell-based assays. However, non-sequence-specific effects of the uniform phosphorothioates were still significant. In contrast, the antisense PPS undecamer, when delivered to RS485 cells with Lipofectin reagent, inhibits human Ras p21 synthesis by more than 90% at a concentration of 3.2 microM, while the effect of controls with inverted, mismatched or scrambled sequence was minimal (5% or less) on p21 synthesis and RS485 cell growth.

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells.

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.

An antisense oligonucleotide targeting the alphaV integrin gene inhibits adhesion and induces apoptosis in breast cancer cells.

The aim of this study was to show the anti-adhesive potential of an antisense oligodeoxynucleotide (ODN) approach when designed to suppress the cellular function of the alphaV integrin subunit in breast cancer cells. The alphaV integrins play major roles in favouring breast cancer spreading. In this study, we inhibited alphaV subunit synthesis in the human breast carcinoma cell line, MDA-MB231, by a partially phosphorothioated antisense oligodeoxynucleotide (5543-ODN). The alphaV antisense 5543-ODN reduced alphaV, but not actin, mRNA transcription and protein expression by 55% and 65% respectively (1 microM, 72 h). Control sense and mismatch reagents were inactive. The antisense, but not the sense and mismatch, 5543-ODN induced dose- and time-dependent inhibition of MDA-MB231 adhesion to serum, vitronectin, fibrinogen and fibronectin substrates but was inactive on adhesion to laminin. Thus, the alphaV integrin was located in adhesion structures, which were disrupted by treatment with the alphaV antisense 5543-ODN. Antisense treated cells also showed evidence of programmed cell death with the appearance of apoptotic bodies. MDA-MB231 cells express a mutant form of the pro-apoptotic factor p53; however, no changes in the expression of p53 were observed by Western blotting. Immunofluorescence did reveal an increased nuclear translocation of p53 suggesting activation of the protein, but such a translocation did not lead to significant changes in either the expression of the cyclin dependent kinase inhibitor, p21(WAF1/CIP1) the cell survival factor Bcl-2 or the pro-apoptotic factor Bax.

Recent advances in the medicinal chemistry of antisense oligonucleotides.

The therapeutic index of an antisense oligonucleotide is determined by its nucleotide sequence and the type of chemical modification. In the last decade, a great deal of effort has been devoted to the development of a second generation of novel antisense oligonucleotide analogs with improved properties compared to the uniformly phosphorothioate-modified oligonucleotides of the first generation. In this review, the advances in the medicinal chemistry of antisense oligonucleotides of the last 2 years will be discussed with emphasis on modifications of the phosphate backbone, heterocyclic bases, carbohydrate moiety and pendent groups for improved delivery. Understanding the basic rules by which a certain modification influences the mechanism of antisense action, binding affinity, metabolic stability, cellular uptake, toxicity and bioavailability of an oligonucleotide, will have a profound impact on the further development of antisense therapeutics.

Oligodeoxynucleotide targeted to the alphav gene inhibits alphav integrin synthesis, impairs osteoclast function, and activates intracellular signals to apoptosis.

The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases.

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.

Peptide nucleic acids (PNA) and PNA-DNA chimeras: from high binding affinity towards biological function.

Oligonucleotide analogs are of major interest as tools in molecular biology, as diagnostics, and as potential pharmaceuticals which bind in a predictable way to certain nucleic acid target sequences, aiming at the inhibition of expression of disease-causing genes. One of the most promising nucleic acid mimetics are the peptide - or polyamide- nucleic acids (PNA) which bind with higher affinity to DNA and RNA than natural oligonucleotides. In these non-ionic PNAs, the entire sugar-phosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure. A unique property of PNA is its ability to displace one strand of a DNA double-helix. This strand displacement process, which is inefficient with DNA, is supported by the formation of an unusually stable internal (PNA)2 x DNA triple helix. The combination of PNA and DNA in one molecule results in PNA/DNA chimeras with new properties. They show improved aqueous solubility compared to pure PNAs due to their partially negatively charged structure. Furthermore, the cellular uptake of the chimeras is better than of pure PNAs. In contrast to PNA, the chimeras bind exclusively in the antiparallel orientation under physiological conditions. The binding affinity is generally stronger when the PNA/DNA chimeras are hybridized to RNAthan to DNA, whereby the strength of binding strongly depends on the PNA: DNA ratio. Most interestingly, PNA/DNA chimeras are recognized as substrates by various nucleic acid processing enzymes, and consequently can also assume biological functions, such as a primer function for DNA polymerases. Pure PNA cannot induce RNase H cleavage of target RNA, which is believed to support the biological efficacy of antisense agents. However, DNA-PNA chimeras are able to stimulate cleavage of the target RNA by RNase H upon formation of an RNA x chimera duplex.