[Generic drugs--medical progress at a moderate price].
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Austria↗
Biomedical subjects
Markus Müller
Publications and source records attributed to Markus Müller.
Strategies for the determination of cefazolin in plasma and microdialysis samples by short-end capillary zone electrophoresis.
Capillary zone electrophoresis was employed to determine cefazolin, a first-generation cephalosporin antibiotic, in plasma and microdialysis samples from patients. To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min. Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions. For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape. For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape. Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution. The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible. A self-sealing film was used to seal each individual sample vial to suppress evaporation during long-term sequences. The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2-500 and 2.5-100 microg/mL, respectively. Limits of detection were 1.0 and 2.0 microg/mL in plasma and microdialysis samples, respectively. The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery.
Acetonitriles↗
Machine learning approaches to lung cancer prediction from mass spectra.
We addressed the problem of discriminating between 24 diseased and 17 healthy specimens on the basis of protein mass spectra. To prepare the data, we performed mass to charge ratio (m/z) normalization, baseline elimination, and conversion of absolute peak height measures to height ratios. After preprocessing, the major difficulty encountered was the extremely large number of variables (1676 m/z values) versus the number of examples (41). Dimensionality reduction was treated as an integral part of the classification process; variable selection was coupled with model construction in a single ten-fold cross-validation loop. We explored different experimental setups involving two peak height representations, two variable selection methods, and six induction algorithms, all on both the original 1676-mass data set and on a prescreened 124-mass data set. Highest predictive accuracies (1-2 off-sample misclassifications) were achieved by a multilayer perceptron and Naïve Bayes, with the latter displaying more consistent performance (hence greater reliability) over varying experimental conditions. We attempted to identify the most discriminant peaks (proteins) on the basis of scores assigned by the two variable selection methods and by neural network based sensitivity analysis. These three scoring schemes consistently ranked four peaks as the most relevant discriminators: 11683, 1403, 17350 and 66107.
Algorithms↗
Pharmacodynamics of piperacillin in severely ill patients evaluated by using a PK/PD model.
Penetration of antiinfective drugs into soft tissues is essential for antimicrobial killing at the target site, but is substantially lower in severely ill patients compared with healthy subjects. The present study was conducted to assess the antimicrobial effect of piperacillin in severely ill patients. Strains of Staphylococcus aureus and Pseudomonas aeruginosa were exposed in vitro to concentrations of piperacillin, simulating the pharmacokinetic profiles measured in soft tissue of patients and healthy subjects. The simulation for patients resulted in effective killing, whereas bacterial regrowth was detected for healthy subjects. Our in vitro simulation showed that bacterial killing may be effective in severely ill patients despite relatively low concentrations of piperacillin at the target site. This finding is due to impaired renal function and subsequently prolonged tissue and plasma half-lives of piperacillin in intensive care patients.
Colony Count, Microbial↗
Real-time quantitative reverse transcriptase-polymerase chain reaction for luteinizing hormone-releasing hormone receptor gene mRNA expression in human prostate cancer.
OBJECTIVES: To determine quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection of luteinizing hormone-releasing hormone (LHRH) receptor mRNA expressing prostate cancer (CaP) and benign prostatic hyperplasia (BPH) tissue by sequence-specific detection with hybridization probes using the LightCycler. Various in vitro studies have indicated that LHRH agonists and antagonists may have a direct inhibitory effect on CaP mediated by specific LHRH receptors. The incidence of LHRH receptor mRNA expression was demonstrated in 50% to 100% of clinical CaP and BPH tissue by current RT-PCR methods. However, qualitative RT-PCR can only demonstrate the existence of a gene; an exact quantification of LHRH-receptor mRNA expression has not yet been done. METHODS: Quantitative real-time RT-PCR for LHRH receptor mRNA was performed using the LightCycler system and the RNA Amplification Kit hybridization probes in 35 patients with CaP and 38 patients with BPH. RESULTS: Thirty-one (88.6%) of the 35 patients with CaP and 36 (94.7%) of the 38 patients with BPH had positive RT-PCR for LHRH receptor mRNA. The number of positive amplifications per 1 microg total RNA was not significantly lower in CaP at 695,428 +/- 350,860 than in BPH at 1,617,654 +/- 787,874. Patients with CaP evidenced a significant negative correlation between the amplification rate for LHRH receptor mRNA and Gleason score (r = -0.476; P = 0.004). CONCLUSIONS: In the past, the potential mechanisms acting on LHRH receptors in CaP have been identified as possible antitumor strategies for this type of cancer. Our study is the first to show that CaP does not have a higher expression of LHRH receptor mRNA expression than BPH when using quantitative sequence-specific oligonucleotide hybridization probes that only detect certain PCR products.
Adenocarcinoma↗
Methylation-specific PCR for DNA-based detection of occult tumor cells in lymph nodes of prostate cancer patients.
OBJECTIVES: To assess the feasibility of methylation-specific PCR (MSP) for the detection of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1) to detect occult prostate cancer cells in lymph nodes (LNs). METHODS: Paraffin-embedded pelvic LNs from 20 patients with pT2pN0M0R0 prostate cancer who developed PSA relapse were assessed by MSP. In 18 of the patients, samples of the primary tumor were obtained for MSP. In 19 patients, bone marrow (BM) aspirates were analyzed preoperatively for disseminated tumor cells by immunocytochemistry (mAb A45-B/B3). In 16 patients, biopsies of the anastomotic region were performed following PSA relapse. As a negative control GSTP1 methylation status was also assessed in LNs from 9 patients for whom an autopsy was performed for non-cancer-related causes. RESULTS: All primary tumors displayed GSTP1 hypermethylation (HM). Preoperative BM assessment showed disseminated tumor cells in 8/20 cases (40%). In 4 patients, biopsies of the vesico-urethral anastomosis showed local tumor recurrence. The LNs in the cancer patients showed GSTP1 HM in 18/20 cases (90%) versus 1/9 patients (11.1%) in the non-cancer cohort (p<0.0001). CONCLUSION: GSTP1 HM appears to be well suited for molecular staging of prostate cancer and accurately detects disseminated tumor cells in LNs, which was seen in 90% of the patients with PSA relapse. However, the limited number of patients and the finding of benign and malignant prostatic tissue at the vesico-urethral anastomosis as a putative local source of PSA recurrence does not allow us to draw conclusions on the prognostic significance of our findings, yet.
Aged↗
Synthesis of fluorine-18-labeled ciprofloxacin for PET studies in humans.
Ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylic acid), a widely-prescribed antibiotic, was labeled with fluorine-18 with the aim to perform positron emission tomography studies in humans for pharmacokinetic measurements. Due to a lack of chemical activation of ciprofloxacin for a direct nucleophilic exchange reaction a novel two-step synthetic approach, which employed an activated 6-fluoro-7-chloro substituted precursor molecule, was developed. The radiosynthesis yielded, starting from 52.5 +/- 11.3 GBq of [(18)F]fluoride, 1.3 +/- 0.6 GBq (n = 13) [(18)F]ciprofloxacin ready for intravenous administration in about 130 min synthesis time. A series of analytical tests was performed in order to prove the identity of the radiolabeled compound and its suitability for human applications.
Anti-Infective Agents↗
[Clinico-pharmacologic explanation models of cerivastatin associated rhabdomyolysis].
Because of fatal cases of rhabdomyolysis the HMG-CoA-reductase inhibitor cerivastatin had to be withdrawn from the global market in 2001. The high frequency and severity of cerivastatin-associated rhabdomyolysis caused concerns about the safety of the entire class of HMG-CoA-reductase inhibitors (statins). Still, the frequency of deadly incidents of rhabdomyolysis with cerivastatin was 16 to 80 times higher than with other statins. This seems to be due to a combination of several pharmacokinetic and pharmacodynamic characteristics of cerivastatin. Cerivastatin shows the highest oral bioavailability within its class. Interactions with other drugs like gemfibrocil may cause further elevation of cerivastatin plasma levels, thereby leading to higher frequencies of side effects in peripheral organs. With approximately 1 pM cerivastatin shows the lowest IC50 for inhibition of HMG-CoA-reductase of all statins. The combination of high systemic drug levels and high intrinsic activity potentially leads to apoptosis and energy-depletion of skeletal-muscle cells. Therefore cerivastatin-associated fatal rhabdomyolysis seems to be based on specific pharmacokinetic and pharmacodynamic properties of cerivastatin, and is not a general characteristic of all members of this drug-class. The experiences with cerivastatin support the importance of clinical studies even about well established drugs, and underline the relevance of precise reporting of adverse events by each physician.
Animals↗
Small interfering RNA targeting bcl-2 sensitizes malignant melanoma.
Malignant melanoma is a prime example of a treatment-resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low, and the duration of responses is short. More than 90% of all melanomas express the antiapoptotic protein Bcl-2, shown to contribute to a chemoresistant phenotype in melanoma. We previously demonstrated that antisense-mediated inhibition of Bcl-2 sensitizes malignant melanoma to apoptosis-inducing treatment modalities. In the present study, we evaluated synthetic small interfering RNA (siRNA) compounds targeting Bcl-2 as a novel approach to downregulate Bcl-2 expression in melanoma cells. siRNA treatment led up to a 19-fold reduction of bcl-2 mRNA levels and only barely detectable Bcl-2 protein expression at low nanomolar concentrations. Silencing of Bcl-2 in melanoma cells by specific siRNA led to a moderate increase in apoptotic cell death and inhibition of cell growth. However, if siRNA compounds targeting Bcl-2 were combined with the apoptosis-inducing chemotherapeutic agent cisplatin, a massive increase in apoptotic cell death compared with controls was observed. Notably, the combination of Bcl2 siRNA and low-dose cisplatin resulted in a supra-additive effect, with nearly complete suppression of cell growth, whereas cell growth in cisplatin-only-treated cells was only moderately affected (96% vs. 25%, p < 0.001). These findings underline a key role for Bcl-2 in conferring chemoresistance to melanoma and highlight Bcl-2 siRNA strategies as novel and highly effective tools, with the potential for future targeted therapy of malignant melanoma.
Apoptosis↗
Penetration of moxifloxacin into healthy and inflamed subcutaneous adipose tissues in humans.
The present study addressed the ability of moxifloxacin to penetrate into healthy and inflamed subcutaneous adipose tissues in 12 patients with soft tissue infections (STIs). Penetration of moxifloxacin into the interstitial space fluid of healthy and inflamed subcutaneous adipose tissues was measured by use of in vivo microdialysis following administration of a single intravenous dosage of 400 mg in six diabetic and six nondiabetic patients with STIs. For the entire study population, the mean time-concentration profile of free moxifloxacin in plasma was identical to the time-concentration profile of free moxifloxacin in tissue (P was not significant). For healthy and inflamed adipose tissues for the diabetic subgroup, the mean moxifloxacin areas under the concentration-time curves (AUCs) from 0 to 8 h (AUC(0-8)s) were 8.1 +/- 7.1 and 3.7 +/- 1.9 mg.h/liter, respectively (P was not significant). The ratios of the mean AUC(0-8) for inflamed tissue/AUC(0-8) for free moxifloxacin in plasma were 0.5 +/- 0.4 for diabetic patients and 1.2 +/- 0.8 for nondiabetic patients (P was not significant). The ratios of the AUCs from 0 to 24 h for free moxifloxacin in plasma/MIC at which 90% of isolates are inhibited were >58 and 121 h for Streptococcus species and methicillin-sensitive Staphylococcus aureus, respectively. Concentrations of moxifloxacin effective against clinically relevant bacterial strains are reached in plasma and in inflamed and healthy adipose tissues. Thus, the pharmacokinetics of moxifloxacin in tissue and plasma support its use for the treatment of STIs in diabetic and nondiabetic patients.
Adipose Tissue↗
Treatment options in vertebrobasilar dolichoectasia--case report and review of the literature.
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Aged↗
Quantitative real-time rt-PCR for detection of circulating prostate-specific antigen mRNA using sequence-specific oligonucleotide hybridization probes in prostate cancer patients.
OBJECTIVE: A great number of studies have failed thus far to demonstrate that the presence of PSA-expressing tumor cells in the blood of prostate cancer (PC) patients is a highly sensitive prognostic marker, particularly after radical prostatectomy (RPX). These studies have only relied on qualitative or semiquantitative detection techniques, however. We report our initial experience in testing real-time RT-PCR for the detection of PSA mRNA using a quantitative online PCR system, the LightCycler(TM), and sequence-specific oligonucleotide hybridization probes. METHODS: Blood samples were obtained before and after surgery from 129 patients undergoing RPX for localized PC and from 19 patients undergoing transurethral resection of the prostate for benign prostatic hyperplasia (BPH). Quantitative RT-PCR for the detection PSA mRNA was performed using the LightCycler system with RNA Amplification Kit Hybridization Probes and sequence-specific oligonucleotide hybridization probes. RESULTS: PSA mRNA was detected by the LightCycler in 28 patients (39%) with pT2 tumors, in 22 patients (38%) with >pT2 tumors, but in only 3 patients (16%) with BPH. The mean values of the LNCaP cell equivalents were higher in >pT2 patients than in pT2 patients (37 x 10(3) and 104 x 10(3)) or BPH patients (7.1 x 10(3) and 4.8 x 10(3)) both preoperatively (333 x 10(3)/ml blood) and postoperatively (545 x 10(3)). CONCLUSION: Real-time RT-PCR with the LightCycler appears to be a promising method for the preoperative detection of circulating LNCaP tumor cells in PC as reflected by PSA mRNA. Considering the low detection rates in BPH patients, the method may also be suitable for patient monitoring after RPX and could thus play an important role in deciding on early radiotherapy or even hormone ablation therapy. Additional long-term follow-up will have to show whether patients with high expression of PSA mRNA actually have an increased risk or recurrence and whether the method is suitable as well to detect progression.
Aged↗
Salvage radiotherapy in patients with persisting prostate-specific antigen after radical prostatectomy for prostate cancer.
Salvage radiotherapy in patients with persisting prostate-specific antigen (PSA) after radical prostatectomy for prostate cancer offers an approach to reduce local recurrence rates and to improve the rate of biochemical freedom from relapse. 30-70% of these patients experience a decrease in their PSA to an undetectable range; in about 40-50% of these patients, PSA remains stable after 5 years. Therefore, radiation therapy offers these patients an ultimate chance of cure. The pre-irradiation PSA value is of particular importance. The PSA level should not exceed 2 ng/ml because otherwise the rate of distant metastases increases significantly. Serious side effects are apparently low, thus confirming the suitability of this therapeutic approach.
Humans↗
Single- and multiple-dose pharmacokinetics of oral creatine.
Supplementation with exogenous creatine (Cr) has shown physiological benefits in humans, but little is known about the pharmacokinetics of Cr in humans. Six healthy males completed an open-label study consisting of a full pharmacokinetic analysis following a single oral dose of Cr monohydrate (71 mg kg-1) and at steady-state after 6 days of Cr administration (71 mg kg-1 qid). After the single oral dose, the clearance (CL/F) was 0.20 +/- 0.066 L h-1 kg-1, tmax was 1.9 +/- 0.88 hours, and Cmax = 102.1 +/- 11.2 mg h L-1. At steady-state, CL/F decreased to 0.12 +/- 0.016 L h-1 kg-1, tmax did not change, and Cmax increased to 162.2 +/- 30.0 mg L-1. Penetration (AUCMUSCLE/AUCPLASMA) of Cr into the interstitial muscle space, as determined by microdialysis, was 0.47 +/- 0.09 and 0.37 +/- 0.27 for the single dose and at steady-state, respectively. Plasma and muscle data were simultaneously fitted with a model incorporating a saturable absorption and first-order elimination process. In conclusion, repeated dosing of Cr caused a reduction in clearance that could result from saturation of the skeletal muscle pool of Cr.
Administration, Oral↗
The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase.
BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT). METHODS: Telomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. RESULTS: High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression. CONCLUSIONS: These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status.
Catalytic Domain↗
Microdialysis: an in vivo approach for measuring drug delivery in oncology.
Microdialysis (MD) is a catheter-based sampling method that provides the opportunity to directly study tumor drug exposure and metabolism in a minimally invasive way. Tumor drug exposure, which is directly linked to clinical outcome, may be substantially reduced due to diffusion barriers in solid tumors. Therefore plasma drug profiles are frequently inappropriate for predicting outcome in oncology. This contribution focuses on the application of MD in preclinical and clinical oncological research and presents an overview of the current literature. It is concluded that MD, in combination with pharmacokinetic/pharmacodynamic modeling, has the potential to contribute to the design of optimal treatment schedules and to select appropriate drug candidates, doses, and dosing intervals for established and new anticancer drugs.
Animals↗
Science, medicine, and the future: Microdialysis.
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Blood Glucose↗
Telomerase: its clinical relevance in the diagnosis of bladder cancer.
More than 50 years ago, Papanicolaou recognized the importance of a non-invasive technique for the diagnosis and follow-up of patients with carcinoma of the urinary bladder. Cystoscopy, however, has remained the 'gold standard' since no currently available non-invasive method can compete with cystoscopy's sensitivity and specificity. The detection of the ribonucleoprotein telomerase or the telomerase subunits human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in urine samples offer new diagnostic perspectives. The present article presents a review of publications in the literature and evaluates their clinical relevance. The experimental studies reported to date are very promising and show that telomerase exactly fulfils the requirements for a good diagnostic marker for carcinoma of the urinary bladder. The diagnostic application remains in an experimental stage and telomerase is still several steps away for routine use as a clinical parameter. The remaining steps leading to its routine clinical application will be discussed.
Biomarkers, Tumor↗