[Perampanel as a therapy option in patients with epilepsy]. / Perampanel als Therapieoption bei Epilepsiepatienten.

BACKGROUND: This article describes a monocentric retrospective analysis of clinical experience with the latest antiepileptic drug perampanel with non-competitive modulation of postsynaptic AMPA receptors. MATERIAL AND METHODS: Evaluation of electronic medical charts of patients newly treated with perampanel between 2012 and 2014 at the epilepsy center of the University Hospital Freiburg regarding effectiveness and tolerability. RESULTS: A total of 85 patients (45 male, mean age 37.4 years, range 14-80 years) with therapy resistance to an average of 6 antiepileptic medications were newly treated with add-on perampanel. Of the patients 35 % experienced a relevant reduction in seizures. The most commonly reported side effects were tiredness (32.5 %), dizziness (24.5 %) and irritability (10.5 %). The dosages resulting in a significant reduction in seizures which varied between patients from 4 to 12 mg/day. Even multidrug-resistant patients who had not benefited from vagus nerve and deep brain stimulation, profited from add-on treatment with perampanel. CONCLUSION: In this cohort, even epilepsy patients who did not respond to multiple previous antiepileptic treatment profited from add-on therapy with the new mode of action of perampanel.

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

Developmental differences in methylation of human Alu repeats.

Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.

Apoptosis in sulfur mustard treated A549 cell cultures.

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

The TNM 8 M1b and M1c classification for non-small cell lung cancer in a cohort of patients with brain metastases.

BACKGROUND AND PURPOSE: According to the recent TNM 8 classification, patients with metastatic non-small cell lung cancer (NSCLC) and single extrathoracic metastasis should be classified as stage M1b, while those with 2 or more metastases comprise stage M1c. The purpose of this study was to analyze the impact of this classification in patients with brain metastases. MATERIALS AND METHODS: This retrospective study included 172 patients treated with individualized approaches. Actuarial survival was calculated. Uni- and multivariate analyses were performed. RESULTS: Thirty patients (17%) were staged as M1b. Those with squamous cell cancer were more likely to harbor M1b disease (29%, adenocarcinoma 14%, other histology 17%, p = 0.16). Median survival was 5.4 months (8.0 months in case of M1b disease and 4.5 months in case of M1c disease, p = 0.001). Multivariate analysis confirmed the role of M1b stage. M1b patients managed with upfront surgery or radiosurgery had significantly longer median survival than those who received whole-brain irradiation (21.0 vs. 3.5 months, p = 0.0001) and the potential to survive beyond 5 years. CONCLUSIONS: We found the M1b classification to provide clinically relevant information. The multivariate analysis suggested that patients with M1b disease, better performance status and younger age have better survival.

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

Colorectal cancer metastatic to the brain: analysis of prognostic factors and impact of KRAS mutations on presentation and outcome.

BACKGROUND: Treatment concepts for metastatic colorectal cancer continue to evolve. While the presence of RAS mutations impacts systemic therapy, little is known about the influence of such mutations in patients with brain metastases. PATIENTS AND METHODS: Pooled retrospective analysis was conducted of 57 patients with brain metastases from colorectal cancer treated in two different institutions (2005-2013). RESULTS: The only mutations analyzed in a relatively large subgroup were KRAS mutations (14 wild type, 12 mutated). Mutation status was not associated with baseline characteristics such as number or location of metastases, and did not impact prognosis. Three factors were significantly associated with survival in multivariate analysis: Karnofsky Performance Status (KPS), management strategy, and systemic treatment. Median survival was 0.6 months with best supportive care, 3.0 months with initial whole-brain radiotherapy (WBRT), and 12.7 months if initial treatment included surgery or stereotactic radiosurgery (SRS), p = 0.0001. The survival difference between the WBRT and surgery/SRS groups was largest in patients with KPS 80-100. CONCLUSION: Effective local treatment was a prerequisite for improved survival. The only significant prognostic baseline factor was KPS, which forms the basis of the diagnosis-specific graded prognostic assessment (DS-GPA) score. Thus, our results validate the DS-GPA in this patient population. So far, neither this nor other studies suggest a clinically important impact of KRAS mutations beyond their previously reported association with development of brain metastases. Studies focusing on patients who develop brain metastases early during the course of metastatic disease might be warranted, because the influence of different systemic therapies might be larger in this subgroup.

LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

Pharmacokinetics of orally administered acyclovir in patients with herpes progenitalis.

The pharmacokinetics of acyclovir administrated orally in a dose of 200 mg every four hours, five times a day to adults with herpes progenitalis was determined. Peak plasma acyclovir levels are found 1.5 to 1.75 hours after oral administration; peak levels range from 1.4 to 4.0 microM with a mean of 2.5 microM. Acyclovir levels in saliva are well correlated with simultaneous plasma levels, saliva levels being approximately 13 percent of plasma levels. Simultaneous plasma and vaginal secretion acyclovir levels are poorly correlated; peak levels in vaginal secretions range from 0.5 to 3.6 microM.

Treatment of cytomegalovirus pneumonia with high-dose acyclovir.

Cytomegalovirus pneumonia is a serious complication of marrow transplantation, with a 90 percent fatality rate. Acyclovir, a new antiviral agent with variable in vitro activity against cytomegalovirus, was administered to eight marrow transplant patients with biopsy-proven cytomegalovirus pneumonia; one patient survived. Doses were between 400 and 1200 mg/m2 and peak plasma levels between 47 and 316 microM were attained. Possible marrow toxicity occurred in three patients, and mild neurotoxicity occurred in one. High-dose acyclovir had mild toxicity but was not effective as treatment for cytomegalovirus pneumonia after marrow transplantation.

Treatment of herpes virus infections in immunocompromised patients with acyclovir by continuous intravenous infusion.

Sixteen immunocompromised patients with herpes virus infections were treated for three to five days with continuously administered intravenous acyclovir. Patients received initial acyclovir infusions over 5 minutes in dosages ranging from 1.5 to 5.0 mg/kg followed by continuously infused acyclovir at 7.2, 14.4, 21.6, 28.8, 36.0, or 43.2 mg/kg per day. The mean serum plateau levels of acyclovir determined by radioimmunoassay ranged from 4.1 microM for the 7.2 mg/kg per day dosage to 36.6 microM for the 43.2 mg/kg per day dose. A mean of 75 percent of acyclovir administered was recovered in the urine of patients treated. Eleven of 13 patients with varicella-zoster virus (VZV) infections had no new vesicle formation after three days of acyclovir treatment and all patients ceased to have new vesicles after five days of therapy. For the nine patients from whom complete viral cultures were available, six ceased to shed virus at three days, and viral shedding ceased by five days in all patients treated with acyclovir. No clinical or laboratory adverse reactions were associated with acyclovir therapy. These data suggest that acyclovir given by continuous intravenous infusion may be useful in the treatment of herpes virus infections in immunocompromised patients.

Neonatal acyclovir pharmacokinetics in patients with herpes virus infections.

A preliminary analysis is presented of the pharmacokinetics of acyclovir in neonatal patients with herpes simplex virus infections. Mean peak acyclovir levels (microM +/- SD) at 5, 10, and 15 mg/kg per dose were 30.0 +/- 9.9, 61.2 +/- 18.3, and 86.1 +/- 23.5, with corresponding mean trough levels (microM +/- SD) of 5.3 +/- 3.4, 10.1 +/- 8.4, and 13.8 +/- 11.1, respectively. The mean half-life (t 1/2 beta) of acyclovir was 3.78 +/- 1.21 hours. The mean percent urinary recovery of acyclovir (+/- SD) at each dosage level was similar, with an overall mean recovery of 65 percent. The mean acyclovir concentration in urine did not exceed the solubility of acyclovir in bladder urine (1,300 micrograms/ml). Generally, neonatal acyclovir pharmacokinetics was consistent with previous reports from studies of adults.

Double-blind controlled trial of topical acyclovir in genital herpes simplex virus infections.

Sixty-nine patients with first episodes and 111 with recurrent episodes of genital herpes simplex virus (HSV) infection were enrolled in a double-blind trial comparing a 5 percent topical acyclovir ointment versus placebo, polyethylene glycol (PEG). Among acyclovir recipients with first episodes of genital herpes, the mean duration of viral shedding from genital lesions, 2.0 days, mean duration of local pain or itching, 3.6 days, and mean time to healing of lesions, 11.2 days, were less than in placebo recipients 4.6, 6.7, and 15.8 days, respectively (p less than 0.05 for each comparison). Among patients with recurrent genital herpes, the mean duration of viral shedding from genital lesions was 0.8 days in acyclovir recipients compared with 1.7 days in placebo recipients (p less than 0.001). Among men with recurrent genital herpes, the mean time to crusting and healing of lesions was 3.5 and 7.5 days in acyclovir recipients compared with 5.0 and 9.7 days in placebo recipients, p = 0.03 and 0.07, respectively. No significant differences in the duration of symptoms or healing times were noted between acyclovir- and placebo-treated women with recurrent genital herpes. Acyclovir therapy was not associated with a decrease in frequency of clinical recurrences or an increase in the time of the next recurrence in patients with either first or recurrent genital herpes. Topical acyclovir appears effective in shortening some of the clinical manifestations of genital HSV infections.

Brugia spp. and Litomosoides carinii: identification of a covalently cross-linked microfilarial sheath matrix protein (shp2).

A microfilarial sheath protein gene (shp2) coding for the major constituent of the insoluble, cross-linked sheath remnant (SR) from Brugia malayi, Brugia pahangi and Litomosoides carinii has been cloned and sequenced, based on peptide partial amino-acid sequences. All three closely related single-copy shp2 genes in the two genera carry a single intron in identical position; shp2 mRNAs are post-transcriptionally modified by both cis-splicing and trans-splicing. In accordance with their extracellular destinations the encoded proteins include signal peptide sequences; molecular masses of approx. 23 kDa are hence predicted for the mature secreted polypeptides. In their structures sheath matrix proteins shp2 may be regarded as extreme cases of a modular constitution, since these proteins largely consist of two different segments of multiple sequence repetitions, PAA and QYPQAP (or QYPQ), separated by elements of unique sequence. Extreme insolubility and cross-linking are likely to originate from these repetitive sequences within shp2, and to constitute the basic properties of a microfilarial matrix largely consisting of an shp2 network.

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

alpha-Melanotropin: the minimal active sequence in the frog skin bioassay.

The minimal sequence required for biological activity of alpha-MSH (alpha-melanotropin, alpha-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10(-4) M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-alpha-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-alpha-MSH11-13-NH2). We prepared a series of fragment analogues of alpha-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-alpha-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-alpha-MSH6-10-NH2 (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-alpha-MSH5-10-NH2, which was only slightly more potent than Ac-alpha-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of alpha-MSH in this assay. Addition of methionine to the N-terminus of Ac-alpha-MSH5-10-NH2 resulted in the heptapeptide, Ac-alpha-MSH4-10-NH2, which was only about 4-fold more potent than Ac-alpha-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-alpha-MSH4-10-NH2 sequence yielded the peptide, Ac-alpha-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-alpha-MSH4-10-NH2. This peptide was only about 6-fold less potent than alpha-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-alpha-MSH4-10-NH2 was about 4 times more potent than Ac-alpha-MSH4-10-NH2. Ac-[Nle4]-alpha-MSH4-11-NH2 also was about 4 times more potent than Ac-alpha-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of alpha-MSH on the frog skin melanocyte receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

C-terminal deletion analogs of a crustacean pigment-dispersing hormone.

This study deals with the effect of deamidation and C-terminal truncation on the potency of an octadecapeptide pigment-dispersing hormone (PDH: Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala- NH2), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus borealis. Bioassay of synthetic analogs for melanophore pigment dispersion in destalked fiddler crabs (Uca pugilator) showed that deamidation causes a 300-fold decrease in potency. The analogs 1-17 NH2 and 1-16 NH2 were about 3 times more potent than 1-18-OH. Further truncation led to decreases in potency, with the peptide 1-9-NH2 being the smallest C-terminal deletion analog to display activity (0.001% potency). Smaller analogs (1-8-NH2, 1-6-NH2 and 1-4-NH2) were inactive when tested in doses as high as 500 nmoles/crab. On the basis of our earlier work on N-terminal deletion analogs and the present findings the residues 6 to 9 seem to be important for PDH action.

Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa.

Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli. The purified P. aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method. A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P. aeruginosa DXP synthase. Fluoropyruvate is described as a first inhibitor of DXP synthase.

Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease.

A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.