Bioequivalence of macitentan and tadalafil given as fixed-dose combination or single-component tablets in healthy subjects.

AIMS: To demonstrate the bioequivalence of macitentan/tadalafil fixed-dose combination (FDC) tablets with single-component tablets of macitentan and tadalafil in healthy subjects. METHODS: Studies AC-077-101 and AC-077-103 were single-centre, open-label, single-dose, 2-period, randomized, crossover Phase 1 studies conducted in healthy subjects. Two FDCs were investigated: FDC-1 and FDC-2 in Study AC-077-101 and FDC-2 in Study AC-077-103. Both FDCs contained 10 mg/40 mg of macitentan/tadalafil and differed in excipients and coating materials used. In both studies, pharmacokinetic sampling over 216 hours was conducted, and pharmacokinetic parameters were derived using noncompartmental methods. RESULTS: Bioequivalence of macitentan, its active metabolite ACT-132577, and tadalafil was established for FDC-2 in both studies AC-077-101 and AC-077-103 in which tadalafil as a single component was sourced from the USA and EU, respectively, to fulfil regional regulatory requirements. The area under the plasma concentration-time curve and maximum plasma concentration with 90% confidence intervals of all components were entirely within the bioequivalence limits (0.8000-1.2500). No subject died and no serious adverse events were reported in either studies. CONCLUSION: The FDC-2 tablet containing 10 mg/40 mg of macitentan/tadalafil was bioequivalent to the free combination of 10 mg macitentan and 40 mg tadalafil (both US and EU sourced). Macitentan and tadalafil were well tolerated when administered as FDC or as a free combination.

A bosentan pharmacokinetic study to investigate dosing regimens in paediatric patients with pulmonary arterial hypertension: FUTURE-3.

AIM: The aim of the present study was to investigate whether increasing the bosentan dosing frequency from 2 mg kg-1 twice daily (b.i.d.) to 2 mg kg-1 three times daily (t.i.d.) in children with pulmonary arterial hypertension (PAH) (from ≥3 months to <12 years of age) would increase exposure. METHODS: An open-label, prospective, randomized, multicentre, multiple-dose, phase III study was conducted. Patients (n = 64) were randomized 1:1 to receive oral doses of bosentan of 2 mg kg-1 b.i.d. or t.i.d. The main pharmacokinetic endpoint was the daily exposure to bosentan over 24 h corrected to the 2 mg kg-1 dose (AUC0-24C ). The maximum plasma concentration corrected to the 2 mg kg-1 dose (CmaxC ), the time to reach the maximum plasma concentration (tmax ) and safety endpoints were also assessed. RESULTS: The geometric mean [95% confidence interval (CI)] for AUC0-24C was 8535 h.ng ml-1 (6936, 10 504) and 7275 h.ng ml-1 (5468, 9679) for 2 mg kg-1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.85 (0.61, 1.20)]. The geometric mean (95% CI) for CmaxC was 743 ng ml-1 (573, 963) and 528 ng ml-1 (386, 722) for 2 mg kg-1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.71 (0.48, 1.05)]. The median (range) for tmax was 3.0 h (0.0-7.5) and 3.0 h (1.0-8.0) for 2 mg kg-1 b.i.d. and t.i.d., respectively. The proportions of patients who experienced ≥1 adverse event were similar in the b.i.d. (66.7%) and t.i.d. (67.7%) groups. CONCLUSIONS: There appeared to be no clinically relevant difference in exposure to bosentan, or in safety, when increasing the frequency of bosentan dosing from b.i.d. to t.i.d. Therefore, the present study provides no indication that the dosing recommendation should be changed, and 2 mg kg-1 b.i.d. remains the recommended dosing regimen for bosentan in paediatric PAH patients.

Microarray screening for novel preeclampsia biomarker candidates.

INTRODUCTION: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies. MATERIAL AND METHODS: Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level. RESULTS: In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (ß-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and ß-hCG) were confirmed to be significantly different between preeclampsia cases and controls. DISCUSSION: We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

Echocardiographic Changes and Long-Term Clinical Outcomes in Pediatric Patients With Pulmonary Arterial Hypertension Treated With Bosentan for 72 Weeks: A Post-hoc Analysis From the FUTURE 3 Study.

FormUlation of bosenTan in pUlmonary arterial hypeRtEnsion (FUTURE) 3 was a 24-week open-label, prospective, and randomized phase 3 study that assessed the pharmacokinetics of bosentan 2 mg/kg b.i.d. or t.i.d. in children with pulmonary arterial hypertension (PAH). We report findings from a post-hoc analysis that explored the prognostic value of echocardiographic changes during FUTURE 3 in relation to clinical outcomes observed during the 24-week core study and 48-week extension. Patients aged ≥3 months to <12 years (n = 64) received oral doses of bosentan 2 mg/kg b.i.d. or t.i.d. (1:1) for 24 weeks, after which they were eligible to enter the extension with continued bosentan administration. Echocardiographic evaluations were performed at baseline, Week 12, and 24 of the core study via central reading, and analyzed post-hoc for correlation with clinical outcomes (time to PAH worsening, time to death, and vital status). Sixty-four patients were randomized in the core study [median (IQR) age 3.8 (1.7-7.8) years]; and 58 patients (90.6%) entered the 48-week extension. Most of the patients (68.8%) were receiving ≥1 PAH medication at baseline. Echocardiographic changes during the core study were small but with high variability. There were statistically significant associations at Week 24 between worsening of the parameters, systolic left ventricular eccentricity index (LVEIS) and E/A ratio mitral valve flow, and the outcomes of time to death and time to PAH worsening. Additional studies that utilize simple and reproducible echocardiographic assessments are needed to confirm these findings and subsequently identify potential treatment goals in pediatric PAH.

Potential markers of preeclampsia--a review.

Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today.

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry.

PURPOSE: To examine the potential high throughput capability and efficiency of an automated DNA extraction system in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status. METHODS: A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray mass spectrometric system. RESULTS: We determined that as little as 15 pg of RHD-positive genomic DNA could be detected in a background of 585 pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and specificity of 96.1 and 96.1%, respectively. CONCLUSION: Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using real-time PCR.

Size fractionation of cell-free DNA in maternal plasma improves the detection of a paternally inherited beta-thalassemia point mutation by MALDI-TOF mass spectrometry.

OBJECTIVES: The selective enrichment of cell-free fetal DNA in maternal plasma by size fractionation leads to the improved detection of paternally inherited fetal point mutations when using conventional, real-time PCR, or as has more recently been shown by MALDI-TOF mass spectrometry. We have now examined the use of size fractionation in conjunction with mass spectrometry for the detection of a paternally inherited codon 39 mutation of the beta-globin gene. METHODS: Maternal plasma was obtained from an early second trimester pregnancy at risk for beta-thalassemia, where the father carried the codon 39 mutation and the mother was a carrier for the IVSI-110 mutation of the beta-globin gene. Cell-free DNA was analyzed by mutation-specific PCR and MALDI-TOF mass spectrometry for the presence of the codon 39 mutation. A comparison was made between total cell-free DNA and that which had been enriched for a size of 100-300 bp. RESULTS: The paternally inherited codon 39 mutant allele was detectable in both cell-free DNA preparations, but the signal was much more pronounced and precise in the size-fractionated sample. CONCLUSIONS: Size fractionation of cell-free DNA may lead to the improved non-invasive detection of fetal point mutations for beta-thalassemia by MALDI-TOF mass spectrometry.

Prenatal RhD Testing: A Review of Studies Published from 2006 to 2008.

The availability of noninvasive prenatal diagnosis for the fetal RhD status (NIPD RhD) is an obvious benefit for alloimmunized pregnant women. This review gives information about the performance characteristics of current diagnostic technologies and recent promising proof-of-principle studies. Notably, during the past 3 years almost twice as much samples have been investigated with NIPD RhD compared with the studies from 1998 to 2005. Thus we have now a lot more information compared with the knowledge before 2006. There is no doubt that funding of the SAFE Network of Excellence (2004-2009) from the European Commission within the framework 6 program has massively increased the worldwide experience in NIPD RhD. In 2009 European funding has been stopped. Because of this large investment from public funding sources, it is now the duty of policy makers (scientific boards, patient groups, physician organizations, and health assurances) to discuss if targeted antenatal Rh prophylaxis should be introduced in German-speaking countries or which additional data are required to make a decision and how these additional studies should be funded.

Cadazolid for the treatment of Clostridium difficile infection: results of two double-blind, placebo-controlled, non-inferiority, randomised phase 3 trials.

BACKGROUND: Cadazolid is a novel quinoxolidinone antibiotic developed for treating Clostridium difficile infection. We aimed to investigate the safety and efficacy of cadazolid compared with vancomycin in patients with C difficile infection. METHODS: IMPACT 1 and IMPACT 2 were identically designed, multicentre, double-blind, placebo-controlled, non-inferiority, randomised phase 3 trials. IMPACT 1 was done in Australia, Brazil, Canada, France, Germany, Italy, the Netherlands, Peru, Poland, Romania, Spain, and the USA, and IMPACT 2 was done in Argentina, Belgium, Brazil, Canada, Chile, Croatia, Czech Republic, Greece, Hungary, Israel, Romania, Slovakia, South Korea, the UK, and the USA. Patients (aged 18 years or older) with mild-to-moderate or severe C difficile infection (diarrhoea with positive glutamate dehydrogenase and toxin A or B enzyme immunoassays) were randomly assigned (1:1) with a randomisation list stratified by centre and C difficile infection episode type (block size of four), and allocation was masked to investigators and participants. Patients received either oral cadazolid 250 mg twice daily with vancomycin-matching placebo capsule four times daily or oral vancomycin 125 mg four times a day with cadazolid-matching placebo suspension twice daily for 10 days, with 30 days of follow-up. The primary efficacy outcome was non-inferiority (margin -10%) of cadazolid versus vancomycin for clinical cure in the modified intention-to-treat and per-protocol populations. Clinical cure was defined as resolution of diarrhoea with no additional treatment for C difficile infection. These trials are registered with ClinicalTrials.gov, numbers NCT01987895 (IMPACT 1) and NCT01983683 (IMPACT 2). FINDINGS: Between March 28, 2014, and March 24, 2017, for IMPACT 1, and Dec 13, 2013, and May 2, 2017, for IMPACT 2, 1263 participants were randomly assigned to receive cadazolid (306 in IMPACT 1 and 298 in IMPACT 2) or vancomycin (326 in IMPACT 1 and 311 in IMPACT 2). In the modified intention-to-treat population in IMPACT 1, 253 (84%) of 302 had clinical cure in the cadazolid group versus 271 (85%) of 318 in the vancomycin group. In IMPACT 2, 235 (81%) of 290 versus 258 (86%) of 301 had clinical cure. In the per-protocol population, 247 (88%) of 282 versus 264 (92%) of 288 had clinical cure in IMPACT 1 and 214 (87%) of 247 versus 237 (92%) of 259 in IMPACT 2. Non-inferiority for clinical cure to vancomycin was shown in IMPACT 1 but not in IMPACT 2 (IMPACT 1 treatment difference: -1·4 [95% CI -7·2 to 4·3] for modified intention to treat and -4·1 [-9·2 to 1·0] for per protocol; IMPACT 2: -4·7 [-10·7 to 1·3] for modified intention to treat and -4·9 [-10·4 to 0·6] for per protocol). The safety and tolerability profiles of the two antibiotics were similar. INTERPRETATION: Cadazolid was safe and well tolerated but did not achieve its primary endpoint of non-inferiority to vancomycin for clinical cure in one of two phase 3 C difficile infection trials. Therefore, further commercial development of cadazolid for C difficile infection is unlikely. FUNDING: Actelion Pharmaceuticals.

The bifunctional flavokinase/flavin adenine dinucleotide synthetase from Streptomyces davawensis produces inactive flavin cofactors and is not involved in resistance to the antibiotic roseoflavin.

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5'-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, kcat/Km = 1.7 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 7.5 10(-3) microM(-1) s(-1)). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1)). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

Significant correlation between maternal body mass index at delivery and in the second trimester, and second trimester circulating total cell-free DNA levels.

OBJECTIVES: The use of cell-free fetal deoxyribonucleic acid (DNA) requires the knowledge of variables that may influence the levels of cell-free DNA, such as maternal body mass index (BMI). MATERIAL AND METHODS: In this study, using 406 maternal blood samples from the second trimester of pregnancy, cell-free fetal DNA specific for the SRY and DYS14 loci and glyceraldehyde-3-phosphate dehydrogenase sequence were quantified by real-time polymerase chain reaction. RESULTS: No significant correlation was seen between the levels of cell-free fetal DNA and maternal BMI, whereas total cell-free DNA was significantly associated with maternal BMI at 20 to 21 weeks of gestation (P = .034) and at the end of pregnancy (R2 regression: 0.016, P = .014). CONCLUSION: Quantitative levels of cell-free fetal DNA are not affected by maternal BMI, whereas total DNA levels in the second trimester significantly correlate with maternal BMI at the moment of blood drawing and at delivery.

Digital PCR: a powerful new tool for noninvasive prenatal diagnosis?

Recent reports have indicated that digital PCR may be useful for the noninvasive detection of fetal aneuploidies by the analysis of cell-free DNA and RNA in maternal plasma or serum. In this review we provide an insight into the underlying technology and its previous application in the determination of the allelic frequencies of oncogenic alterations in cancer specimens. We also provide an indication of how this new technology may prove useful for the detection of fetal aneuploidies and single gene Mendelian disorders.

Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum.

Riboflavin (vitamin B(2)) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin (K(m) 11 microM). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.

Identification and characterization of two Streptomyces davawensis riboflavin biosynthesis gene clusters.

In Streptomyces davawensis roseoflavin is synthesized from GTP and ribulose-5-phosphate through riboflavin. As a first step towards the molecular analysis of flavin metabolism in S. davawensis the genes involved in riboflavin biosynthesis were cloned by hybridization of heterologous probes to a genomic library on a high-density colony-array. The genes ribB (riboflavin synthase, alpha-chain; EC 2.5.1.9), ribM (putative membrane protein), ribA (bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase; EC 3.5.4.25) and ribH (lumazine synthase; EC 2.5.1.9) are organized in an operon-like cluster. Northern blot analysis of this cluster revealed two transcripts of 1.7 and 3.1 kb, respectively. The gene ribB was overexpressed in Escherichia coli. The specific riboflavin synthase activity in a cell-free extract of a recombinant strain was 0.246 nmol mg(-1 )min(-1). Overexpression of ribM enhanced the transport of riboflavin in the corresponding recombinant E. coli strain. Furthermore, overexpression of ribM increased roseoflavin sensitivity of E. coli. On another subgenomic fragment a putative S. davawensis ribG gene coding for the missing pyrimidine deaminase/reductase (EC 3.5.4.26 and EC 1.1.1.193) of the riboflavin biosynthetic pathway and ribY coding for a second (monofunctional) GTP cyclohydrolase II were identified.

Riboflavin analogs and inhibitors of riboflavin biosynthesis.

Flavins are active components of many enzymes. In most cases, riboflavin (vitamin B(2)) as a coenzyme represents the catalytic part of the holoenzyme. Riboflavin is an amphiphatic molecule and allows a large variety of different interactions with the enzyme itself and also with the substrate. A great number of active riboflavin analogs can readily be synthesized by chemical methods and, thus, a large number of possible inhibitors for many different enzyme targets is conceivable. As mammalian and especially human biochemistry depends on flavins as well, the target of the inhibiting flavin analog has to be carefully selected to avoid unwanted effects. In addition to flavoproteins, enzymes, which are involved in the biosynthesis of flavins, are possible targets for anti-infectives. Only a few flavin analogs or inhibitors of flavin biosynthesis have been subjected to detailed studies to evaluate their biological activity. Nevertheless, flavin analogs certainly have the potential to serve as basic structures for the development of novel anti-infectives and it is possible that, in the future, the urgent need for new molecules to fight multiresistant microorganisms will be met.

HMGB6 from Arabidopsis thaliana specifies a novel type of plant chromosomal HMGB protein.

The high-mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in various nucleoprotein structures, which regulate DNA-dependent processes such as transcription and recombination. Database analyses revealed that in addition to the previously identified HMGB1-HMGB5 proteins, the Arabidopsis genome encodes at least three other family members having the typical overall structure of a central HMG-box DNA binding domain, which is flanked by basic and acidic regions. These novel HMGB proteins display some structural differences, when compared to HMGB1-HMGB5. Therefore, a representative of the identified proteins, now termed HMGB6, was further analyzed. The HMGB6 protein of approximately 27 kDa is the largest plant HMGB protein identified so far. This is essentially due to its unusually extended N-terminal domain of 109 amino acid residues. Subcellular localization experiments demonstrate that it is a nuclear protein. According to CD measurements, HMGB6 has an alpha-helical HMG-box domain. HMGB6 can bind DNA structure-specifically, and it is a substrate for the protein kinase CK2alpha. Because of these features, HMGB6, and presumably its relatives, can be considered members of the plant HMGB protein family. Hence, eight different chromosomal HMGB proteins are expressed in Arabidopsis, and they may serve specialized architectural functions assisting various DNA-dependent processes.