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Background: Early initiation of pulmonary arterial hypertension (PAH) therapies is associated with improved long-term outcomes, yet data on the early use of prostacyclin pathway agents are limited. In these post hoc analyses of the Prostacyclin (PGI 2 ) Receptor Agonist In Pulmonary Arterial Hypertension (GRIPHON) study, the largest randomized controlled trial for PAH to date, the prognostic value of time from diagnosis and its impact on treatment response were examined., Research Question: How does time from diagnosis impact morbidity/mortality events and response to selexipag treatment in patients with PAH?, Study Design and Methods: The GRIPHON study randomly assigned 1,156 patients with PAH to selexipag or placebo treatment. Patients were categorized post hoc into a time from diagnosis of ≤ 6 months and > 6 months at randomization. Hazard ratios (selexipag vs placebo) were calculated for the primary end point of morbidity/mortality by time from diagnosis using Cox proportional hazard models., Results: Time from diagnosis was ≤ 6 months in 34.9% and > 6 months in 65.1% of patients. Time from diagnosis was prognostic of morbidity/mortality, with newly diagnosed patients having a poorer long-term outcome than patients diagnosed for longer. Compared with placebo, selexipag reduced the risk of morbidity/mortality in patients with a time from diagnosis of ≤ 6 months and > 6 months, with a more pronounced effect in newly diagnosed patients (hazard ratio, 0.45 [95% CI, 0.33-0.63] and 0.74 [95% CI, 0.57-0.96], respectively; P = .0219 for interaction)., Interpretation: In the GRIPHON study, newly diagnosed PAH patients had a worse prognosis than patients with a longer time from diagnosis. The benefit of selexipag treatment on disease progression was more pronounced in patients treated earlier than in patients treated later., Trial Registry: ClinicalTrials.gov; No.: NCT01106014; URL: www.clinicaltrials.gov., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Approaches to risk assessment in pulmonary arterial hypertension (PAH) include the noninvasive French risk assessment approach (number of low-risk criteria based on the European Society of Cardiology and European Respiratory Society guidelines) and Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL) 2.0 risk calculator. The prognostic and predictive value of these methods for morbidity/mortality was evaluated in the predominantly prevalent population of GRIPHON, the largest randomized controlled trial in PAH., Methods: GRIPHON randomized 1,156 patients with PAH to selexipag or placebo. Post-hoc analyses were performed on the primary composite end-point of morbidity/mortality by the number of low-risk criteria (World Health Organization functional class I-II; 6-minute walk distance >440 m; N-terminal pro-brain natriuretic peptide <300 ng/liter) and REVEAL 2.0 risk category. Hazard ratios and 95% confidence intervals were calculated using Cox proportional hazard models., Results: Both the number of low-risk criteria and the REVEAL 2.0 risk category were prognostic for morbidity/mortality at baseline and any time-point during the study. Patients with 3 low-risk criteria at baseline had a 94% reduced risk of morbidity/mortality compared to patients with 0 low-risk criteria and were all categorized as low-risk by REVEAL 2.0. The treatment effect of selexipag on morbidity/mortality was consistent irrespective of the number of low-risk criteria or the REVEAL 2.0 risk category at any time-point during the study. Selexipag-treated patients were more likely to increase their number of low-risk criteria from baseline to week 26 than placebo-treated patients (odds ratio 1.69, p = 0.0002); similar results were observed for REVEAL 2.0 risk score., Conclusions: These results support the association between risk profile and long-term outcome and suggest that selexipag treatment may improve risk profile., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Parenteral prostacyclin analogs that target the prostacyclin pathway have been used to treat pulmonary arterial hypertension (PAH) since the 1990s. Abrupt discontinuation of parenteral prostacyclin analogs can be associated with acute deterioration of PAH. Less is known about temporary interruption of oral therapies that target the prostacyclin pathway, such as selexipag., Methods: We evaluated the frequency, duration, reasons, and consequences of temporary selexipag interruptions among PAH patients enrolled in the Prostacyclin (PGI 2 ) Receptor Agonist in Pulmonary Arterial Hypertension (GRIPHON) study. In GRIPHON, patients were randomized to selexipag or placebo and titrated to an individualized highest tolerated dose (200 to 1,600 µg twice daily) over 12 weeks, after which patients entered the maintenance phase. Treatment interruptions were allowed; if the interruption was < 3 days, treatment was restarted at the previous highest tolerated dose; if the interruption was ≥ 3 days, retitration from 200 µg twice daily was required. Descriptive analyses were performed., Results: At least 1 treatment interruption occurred in 111 of 574 patients (19.3%) in the selexipag group and in 58 of 582 (10.0%) in the placebo group. Baseline characteristics were similar between patients with and without an interruption. Of the 111 patients in whom selexipag was temporarily interrupted, 94 (85%) were receiving background PAH therapy. Adverse events were the most common reason for selexipag interruption. Selexipag interruptions and reinstitution of treatment were well tolerated. There were no episodes of acute deterioration during treatment interruption., Conclusions: Based on observations from GRIPHON, selexipag interruptions can be expected in clinical practice. However, temporarily interrupting selexipag was well tolerated and manageable., (Copyright © 2018. Published by Elsevier Inc.)

Objectives: It is unclear why duration of type 2 diabetes (T2D) is associated with increased cognitive compromise. High hemoglobin A1c (HbA1c) has also been associated with dementia, and is the primary contributor to T2D complications. Here we investigated whether the association of duration of T2D with cognitive functioning is modulated by HbA1C levels., Methods: This study examined nondemented community-dwelling T2D elderly (N = 897) participating in the Israel Diabetes and Cognitive Decline study, who were assessed with a broad neuropsychological battery. Subjects were all from the Maccabi Healthcare Services, which has a Diabetes Registry with complete HbA1c measurements since 1998. Partial correlations were performed to examine the modulating effect of HbA1c on the relationship of duration of T2D with five cognitive measures, controlling for sociodemographic and cardiovascular risk factors., Results: An interaction of duration of T2D with HbA1c was associated with executive functioning (p = 0.006), semantic categorization (p = 0.019), attention/working memory (p = 0.011), and overall cognition (p = 0.006), such that the associations between duration of T2D and cognitive impairment increased as HbA1c levels increased-but not for episodic memory (p = 0.984)., Conclusions: Because duration of T2D was associated with cognition in higher HbA1c levels and overall no associations were found in lower HbA1c levels, our results suggest that individuals with T2D may limit their risk of future cognitive decline by maintaining long-term good glycemic control., (Copyright © 2014 American Association for Geriatric Psychiatry. All rights reserved.)

Aim: To assess whether the APOE4 genotype affects the relationship of long-term glycemic control with cognitive function in elderly with type 2 diabetes (T2D)., Methods: Participants were cognitively normal and pertained to a Diabetes Registry which provided access to HbA1c levels and other T2D related factors since 1998. Glycemic control was defined as the mean of all HbA1c measurements available (averaging 18 measurements) per subject. Four cognitive domains (episodic memory, semantic categorization, attention/working memory and executive function), based on factor analysis and an overall cognitive score (the sum of the 4 cognitive domains) were the outcome measures., Results: The analysis included 808 subjects; 107 (11.9%) subjects had ≥1ApoE4 allele. In ApoE4 carriers, higher mean HbA1c level was significantly associated with lower scores on all cognitive measures except attention/working memory (p-values ranging from 0.047 to 0.003). In ApoE4 non-carriers, higher mean HbA1c level was significantly associated with lower scores on executive function, but not with other cognitive measures-despite the larger sample size. Compared to non-carriers, there were significantly stronger associations in ApoE4 carriers for overall cognition (p=0.02), semantic categorization (p=0.03) and episodic memory (p=0.02), and the difference for executive function approached statistical significance (p=0.06)., Conclusion: In this cross-sectional study of cognitively normal T2D subjects, higher mean HbA1c levels were generally associated with lower cognitive performance in ApoE4 carriers, but not in non-carriers, suggesting that ApoE4 affects the relationship between long-term glycemic control and cognition, so APOE4 carriers may be more vulnerable to the insults of poor glycemic control., (Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.)

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.

Voriconazole is a widely used triazole antifungal agent with a broad spectrum including Aspergillus species. A simple, sensitive and selective high-performance liquid chromatography method for the determination of voriconazole in human plasma and saliva was developed. Drug and internal standard (UK-115 794) were extracted from alkaline plasma and saliva with n-hexane-ethyl acetate (3:1, v/v) and analyzed on a Luna C 18 column with fluorimetric detection set at excitation and emission wavelengths of 254 and 372 nm, respectively. The calibration curve was linear through the range of 0.1-10 microg/ml using a 0.3 ml sample volume. The intra- and inter-day precisions were all below 6.1% for plasma and below 9.1% for saliva. Accuracies ranged from 94 to 109% for both matrices. Mean recovery was 86+/-4% for voriconazole. The method showed acceptable values for precision, recovery and sensitivity and is well suited for routine analysis work and for pharmacokinetic studies.

Besides hepatic P450 (cytochrome P450) metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP2C8, CYP2C9, CYP2C18, and CYP2C19) by RT (reverse transcriptase)-polymerase chain reaction (PCR) and of CYP2C9 by Western blotting in 10 different breast tumor samples. Since P450 may be down regulated by interleukin (IL) IL-6, the receptor (R) for IL-6 was analyzed by RT-PCR and IL-6 in supernatants was calculated from ELISA data. None of the breast tumors was positive for CYP2C18 and CYP2C19 mRNA, whereas CYP2C8 and CYP2C9 were detected in all 10 breast tumors. Correspondingly, all breast tumors tested (9 of 10) revealed low, but nevertheless positive, staining of the CYP2C9 protein. All 10 samples were positive for the IL-6 receptor mRNA. ELISA measurement of IL-6 cytokine in supernatants revealed that all measured samples (8 of 10) were producing IL-6, the amounts ranging from 0.004 to 3.1 ng/g(tumor tissue). In summary, we have demonstrated that tumors of the breast express two out of four members of the CYP2C family, indicating that activation of such prodrugs as oxazaphosphorines may take place intratumorally. The presence of the IL-6 receptor and of IL-6 cytokine, which is produced in an autocrine manner, opens up the possibility that the well-known down regulating effect of IL-6 also takes place in breast tumors and might explain the weak or even absent expression of different CYP2C members.

An isocratic reversed-phase HPLC method for the simultaneous quantitation of alpha-lipoic acid and five of its metabolites in human plasma as well as in human urine employing solid-phase extraction and pulsed amperometric detection was developed and validated. The method was found to be sufficiently precise and accurate for the measurement of alpha-lipoic acid and its metabolites 6,8-bis(methylthio)octanoic acid, 4,6-bis(methylthio)hexanoic acid and 2,4-bis(methylthio)butanoic acid in plasma and urine samples, obtained from patients suffering from diabetic neuropathy as well as from healthy volunteers following daily oral administration of 600 mg alpha-lipoic acid. The quantitation of the metabolite bisnorlipoic acid was often impaired by interferences caused by an unidentified metabolite. However, bisnorlipoic acid was detected in few test samples and the concentrations were consistently low. Despite the poor recovery of the metabolite tetranorlipoic acid from plasma, reproducibility and accuracy were found to be from acceptable magnitude to assess concentration time courses. Thus, the obtained analytical results are considered as reliable and well suited for pharmacokinetic studies of alpha-lipoic acid and its metabolites.

This article presents an overview of the methods of bioanalysis of oxazaphosphorines, in particular, cyclophosphamide, ifosfamide, and trofosfamide as well as their metabolites. The metabolism of oxazaphosphorines is complex and leads to a large variety of metabolites and therefore the spectrum of methods used is relatively broad. The various methods used are shown in a table and the particularly important assays are described.

The cytokine interleukin (IL)-1 is known to be involved in angiogenesis and metastasis. A prerequisite for IL-1 signalling is the presence of its receptor. Previously we have shown that glioblastoma cells express the IL-1 receptor type I (IL-1RI). In this study we analysed 11 breast tumour specimens for IL-1RI expression using the reverse transcriptase (RT) polymerase chain reaction (PCR). We found all the 11 breast tumours were positive for IL-1RI. This suggests that paracrine or autocrine produced IL-1 mediated signalling via IL-IRI might take place in breast tumours to control the production of pro-tumourigenic factors such as angiogenic factors and support further progression of tumour growth and metastasis.

A sensitive HPLC-MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C18 cartridges followed by HPLC-MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.

A selective and sensitive method for the determination of lipoic acid in human plasma samples has been developed. After enzymatic hydrolysis of the sample, the liberated lipoic acid was extracted by a solid-phase cartridge and measured by HPLC using electrochemical detection. The detection limit was 1 ng/ml lipoic acid in plasma. The calibration curve was non-linear in the range 0.01-50 microgram/ml but could be described by a power function. The average extraction recoveries were 82.5 and 85.1% at the 25 and 2500 ng/ml levels, respectively. Coefficients of variation for both within-day and day-to-day analysis were between 2.1 and 9.4%. The assay method is sensitive, reproducible and suitable for disposition studies of lipoic acid in humans.