Near infra-red fluorescence identification of the thoracic duct to prevent chyle leaks during oesophagectomy.

BACKGROUND: Chyle leaks following oesophagectomy are a frustrating complication of surgery with considerable morbidity. The use of near infra-red (NIR) fluorescence in surgery is an emerging technology and the use of fluorescence to identify the thoracic duct has been demonstrated in animal work and early human case reports. This study evaluated the use mesenteric and enteral administration of indocyanine green (ICG) in humans to identify the thoracic duct during oesophagectomy. METHODS: Patients undergoing oesophagectomy were recruited to the study. Administration of ICG via an enteral route or mesenteric injection was evaluated. Fluorescence was assessed using a NIR fluorescence enabled laparoscope system with a visual scoring system and signal to background ratios. Visualisation of the thoracic duct under white light and NIR fluorescence was compared as well as any identification of active chyle leak. Patients were followed up post-operatively for adverse events and chyle leak. RESULTS: 20 patients received ICG and were included in the study. The enteral route failed to fluoresce the thoracic duct. Mesenteric injection (17 patients) identified the thoracic duct under fluorescence prior to white light in 70% of patients with a mean signal to background ratio of 5.35. In 6 participants, a possible active chyle leak was identified under fluorescence with 4 showing active chyle leak from what was identified as the thoracic duct. CONCLUSION: This study demonstrates that ICG administration via mesenteric injection can highlight the thoracic duct during oesophagectomy and may be a potential technology to reduce chyle leak following surgery. CLINICAL TRIAL REGISTRATION: Clinical trials.gov (NCT03292757).

Fast Pixelated Detectors in Scanning Transmission Electron Microscopy. Part II: Post-Acquisition Data Processing, Visualization, and Structural Characterization.

Fast pixelated detectors incorporating direct electron detection (DED) technology are increasingly being regarded as universal detectors for scanning transmission electron microscopy (STEM), capable of imaging under multiple modes of operation. However, several issues remain around the post-acquisition processing and visualization of the often very large multidimensional STEM datasets produced by them. We discuss these issues and present open source software libraries to enable efficient processing and visualization of such datasets. Throughout, we provide examples of the analysis methodologies presented, utilizing data from a 256 × 256 pixel Medipix3 hybrid DED detector, with a particular focus on the STEM characterization of the structural properties of materials. These include the techniques of virtual detector imaging; higher-order Laue zone analysis; nanobeam electron diffraction; and scanning precession electron diffraction. In the latter, we demonstrate a nanoscale lattice parameter mapping with a fractional precision ≤6 × 10−4 (0.06%).

Bioequivalence and adhesion evaluation of transdermal clonidine following a change in excipient supplier.

OBJECTIVE: To evaluate the bioequivalence (BE), safety, tolerability, and adhesion of Oppanol® polyisobutylene (PIB)-containing transdermal therapeutic system (TTS) formulation (test treatment, T) with VistanexTM PIB-containing TTS formulation (reference treatment, R) of clonidine. METHODS: This randomized, double-blind, 2-way crossover study comprised a 7-day treatment with 0.3 mg clonidine/24 h (T1/R1), a 7-day washout, and another 7-day treatment (R1/T1) period. After a 3-day washout period, subjects used T2 and R2 (each 0.1 mg clonidine/24 h) simultaneously in the 7-day adhesion phase. Primary endpoints were AUC0-168 and Cavg. Secondary endpoints were AUC0-∞ and Cmax. Additional endpoints included adhesion properties for all phases. For the primary endpoint, the geometric mean (gMean) ratios for test/reference treatment were calculated with BE defined as 90% confidence interval (CI) between 80 and 125%. RESULTS: 58 subjects (mean age, 41.3 years) received treatment (T1/R1, n = 29; R1/T1, n = 29); 55 completed the adhesion phase. BE criteria were met for the primary and secondary endpoints. Adjusted gMean ratios for T1/R1 were 102.3% (90% CI: 95.7%, 109.4%) for AUC0-168; 104.3% (90% CI: 98.4%, 110.5%) for Cavg; 102.8% (90% CI: 97.3%, 108.6%) for AUC0-∞; and 104.0% (90% CI: 98.2%, 110.3%) for Cmax. Mean adhesion was greater than 90% for all four patch types when data from all assessment times were included. Most frequently reported adverse events were general disorders and local irritation. CONCLUSIONS: Clonidine Oppanol® PIB-containing TTS formulation was bioequivalent to VistanexTM PIB-containing TTS formulation and had similar adhesive properties. Both doses and formulations of clonidine-TTS were well tolerated.

XPF protein levels determine sensitivity of malignant melanoma cells to oxaliplatin chemotherapy: suitability as a biomarker for patient selection.

As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical response. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial.

Exposure-response modelling for empagliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, in patients with type 2 diabetes.

AIMS: To provide model-based clinical development decision support including dose selection guidance for empagliflozin, an orally administered sodium glucose cotransporter 2 inhibitor, through developed exposure-response (E-R) models for efficacy and tolerability in patients with type 2 diabetes mellitus (T2DM). METHODS: Five randomized, placebo-controlled, multiple oral dose studies of empagliflozin in patients with T2DM (n = 974; 1-100 mg once daily, duration ≤12 weeks) were used to develop E-R models for efficacy (glycosylated haemoglobin [HbA1c ], fasting plasma glucose [FPG] and urinary glucose excretion). Two studies (n = 748, 12 weeks) were used to evaluate tolerability E-R. RESULTS: The efficacy model predicted maximal decreases in FPG and HbA1c of 16% and 0.6%, respectively, assuming a baseline FPG concentration of 8 mm (144 mg dl(-1) ) and 10-25 mg every day empagliflozin targeted 80-90% of these maximums. Increases in exposure had no effect on incidence rates of hypoglycaemia (n = 4), urinary tract infection (n = 17) or genital/vulvovaginal-related (n = 16) events, although low prevalence rates may have precluded more accurate evaluation. CONCLUSIONS: E-R analyses indicated that 10 and 25 mg once daily empagliflozin doses achieved near maximal glucose lowering efficacy.

Integration of absorption, distribution, metabolism, and elimination genotyping data into a population pharmacokinetic analysis of nevirapine.

OBJECTIVES: The aim of this analysis was to show the applicability of a newly developed algorithm to assess the influence of genetic variants and other covariates on nevirapine's drug disposition. The algorithm combines high-throughput genotyping data and nonlinear mixed effects modeling methods. METHODS: Patients, who participated in the 2NN pharmacokinetic sub study, were reconsented and reenrolled into a clinical trial for genotyping analysis. Overall, 198 single nucleotide polymorphisms located in 45 absorption, distribution, metabolism, and elimination related genes were genotyped using the Illumina BeadArray technology. Data analysis was performed using NONMEM VI and SAS 9.1.3. RESULTS: Overall, 1260 nevirapine plasma concentrations were obtained from 271 genotyped patients. Plasma concentration-time profiles of nevirapine were best described by a one-compartment model with auto-induced first-order elimination process. Nevirapine clearance was 19.4% reduced in Asian/Black patients, compared with Caucasian/Hispanic patients. For single nucleotide polymorphism rs3745274 (CYP2B6 516G>T) heterozygous patients (GT) showed a 15.3% reduced clearance; patients with homozygous CYP2B6 516TT alleles showed a 30.6% reduced clearance compared to patients with homozygous 516GG alleles. Patients carrying the homozygote genotype of rs12768009 (CYP2C19 8403AA), highly linked to rs4244285 (CYP2C19*2), showed a 26.8% reduced clearance compared with patients with CYP2C19 8403 AG and GG alleles. CONCLUSION: By integration of high-throughput genotyping data into a pharmacometric analysis of nevirapine, the impact of the CYP2B6 516G>T polymorphism on nevirapine's exposure was confirmed and quantified. In addition, a new hypothesis with regard to CYP2C19 involvement in nevirapine metabolism has been generated. The analysis presented might help to optimize and individualize the therapy for patients treated with nevirapine to add to their therapeutic benefit.

Lack of a pharmacokinetic interaction between steady-state tipranavir/ritonavir and single-dose valacyclovir in healthy volunteers.

OBJECTIVE: This study assessed the single-dose pharmacokinetics of the herpes antiviral acyclovir (administered as the pro-drug valacyclovir) alone and in combination with twice-daily 200 mg ritonavir-boosted tipranavir (500 mg) at steady state. METHODS: The study was an open label, one-sequence cross-over pharmacokinetic study in HIV-negative adults. Plasma drug concentrations were measured by validated LC/MS/MS assays; pharmacokinetics (AUC, C(max)) were determined using noncompartmental methods. The geometric mean ratio and 90% confidence interval [GMR, 90% CI] were used to evaluate the drug interaction. RESULTS: Twenty-six of 29 subjects completed the trial. With steady-state tipranavir/ritonavir, acyclovir C(max) decreased 4.9% [0.95, 0.88-1.02] and AUC increased 6.6% [1.07, 1.04-1.09]. The majority of subjects experienced at least one adverse event, most of which were mild gastrointestinal disorders. Three subjects discontinued tipranavir/ritonavir treatment as a result of drug-related increases in ALT/AST, including one subject who experienced mild upper abdominal pain. All subjects recovered without sequelae. CONCLUSIONS: When administered as a single dose of valacyclovir with steady-state tipranavir/ritonavir, there were no clinically important changes in acyclovir pharmacokinetics. This result indicates that valacyclovir can be co-administered safely with no dose adjustments.

Is less clinical research being published by surgeons in the United Kingdom?

INTRODUCTION: Concerns about the decline of surgical research have been expressed in parallel to the debate on the future of academic surgery in the United Kingdom. The aim of this study was to survey the output of UK surgeons in ten high impact general surgical journals between 1987 and 2009 to estimate whether less clinical academic activity is occurring. METHODS: Ten general surgical journals with consistently the highest impact factors (1.2-7.9) were selected for review. All articles excepting letters and news articles were included in the study. The region of origin was identified for each paper and classified as USA, Japan, Europe, UK and Rest of the World (RoW). In articles from multiple centres, the region of the corresponding author was used. RESULTS: Over the study period, the annual number of surgical papers published from the UK dropped both in absolute values (from 286 to 214) and as a percentage of global output (17.7-8.8%). The USA also saw a drop (from 867 to 763/yr), while the output has increased steadily for Europe (from 266 to 822/yr) and RoW (from 151 to 476/yr). In 2009, the percentage of total yearly output was roughly equal for Europe and USA (31-33%), UK and Japan (7-8%). CONCLUSIONS: These data suggests that clinical surgical research in the UK has suffered over the last 20 years. The declining emphasis on research as part of surgical training, the strategic decisions about fund allocation, and the increasing role of centres from Europe and RoW may all play a role. This limited "snapshot" fails to provide information on the output in journals focused on basic science. As more of the current research involves collaborations between clinicians and non-clinicians it is likely that many more papers were published in such journals during the period analysed. It remains to be monitored whether recent initiatives will strengthen academic surgery in the UK in future years.

Pharmacokinetic characterization of three doses of tipranavir boosted with ritonavir on highly active antiretroviral therapy in treatment-experienced HIV-1 patients.

PURPOSE: This study characterized the pharmacokinetic effects, safety, and antiretroviral activity of three different doses of the nonpeptidic protease inhibitor tipranavir, in combination with ritonavir administered twice daily for 28 days, on a number of triple-combination regimens containing a nonnucleoside reverse transcriptase inhibitor (efavirenz or nevirapine) plus two nucleoside reverse transcriptase inhibitors (abacavir, didanosine, lamivudine, stavudine, and zidovudine) or a three nucleoside reverse transcriptase inhibitor combination (zidovudine, lamivudine, and abacavir). METHODS: The study enrolled 208 HIV-1-positive patients who had been on stable antiretroviral treatment for at least 12 weeks prior to study entry and had an HIV-1 RNA load of delta 20,000 copies/mL. The patients were randomized to receive one of three dose combinations of tipranavir and ritonavir (1250/100 mg, 750/100 mg, and 250/200 mg) in addition to their antiretroviral (ARV) regimen for the next 22 days. The effects of twice-daily tipranavir and ritonavir combinations on the steady-state pharmacokinetics of the antiretrovirals were assessed by comparing pharmacokinetic parameters at baseline and after 3 weeks of coadministration. RESULTS: No clinically relevant changes were observed in the Cmin, Cmax, or AUC parameters for nevirapine, efavirenz, lamivudine, stavudine, or didanosine, when coadministered with tipranavir and ritonavir at the dose combinations studied. All three dose combinations of tipranavir and ritonavir decreased the systemic exposure of abacavir (by 35% to 44%) and zidovudine (by 31% to 42%). Consistent with previous tipranavir studies, gastrointestinal adverse events were those most frequently observed. These reactions tended to be mild, with the majority being of Grade 1, and only 8 being of Grade 3 or 4 in intensity. Virologic response improved from 40.4% of participants at baseline with <50 copies/mL to 67.6% at Day 28 of study following addition of tipranavir and ritonavir. CONCLUSIONS: Tipranavir coadministered with ritonavir has been demonstrated to be safe, effective, and pose little potential for clinically meaningful drug interactions when added to the highly active antiretroviral therapy regimens containing nevirapine, efavirenz, lamivudine, stavudine, or didanosine.

Pharmacokinetics of BILR 355 after multiple oral doses coadministered with a low dose of ritonavir.

The pharmacokinetics and safety of BILR 355 following oral repeated dosing coadministered with low doses of ritonavir (RTV) were investigated in 12 cohorts of healthy male volunteers with a ratio of 6 to 2 for BILR 355 versus the placebo. BILR 355 was given once a day (QD) coadministered with 100 mg RTV (BILR 355/r) at 5 to 50 mg in a polyethylene glycol solution or at 50 to 250 mg as tablets. BILR 355 tablets were also dosed at 150 mg twice a day (BID) coadministered with 100 mg RTV QD or BID. Following oral dosing, BILR 355 was rapidly absorbed, with the mean time to maximum concentration of drug in serum reached within 1.3 to 5 h and a mean half-life of 16 to 20 h. BILR 355 exhibited an approximately linear pharmacokinetics for doses of 5 to 50 mg when given as a solution; in contrast, when given as tablets, BILR 355 displayed a dose-proportional pharmacokinetics, with a dose range of 50 to 100 mg; from 100 to 150 mg, a slightly downward nonlinear pharmacokinetics occurred. The exposure to BILR 355 was maximized at 150 mg and higher due to a saturated dissolution/absorption process. After oral dosing of BILR 355/r, 150/100 mg BID, the values for the maximum concentration of drug in plasma at steady state, the area under the concentration-time curve from 0 to the dose interval at steady state, and the minimum concentration of drug in serum at steady state were 1,500 ng/ml, 12,500 h.ng/ml, and 570 ng/ml, respectively, providing sufficient suppressive concentration toward human immunodeficiency virus type 1. Based on pharmacokinetic modeling along with the in vitro virologic data, several BILR 355 doses were selected for phase II trials using Monte Carlo simulations. Throughout the study, BILR 355 was safe and well tolerated.

Pharmacokinetic assessment of nevirapine and metabolites in human immunodeficiency virus type 1-infected patients with hepatic fibrosis.

Nevirapine is a nonnucleoside reverse transcriptase inhibitor used as part of combination therapy for human immunodeficiency virus (HIV) infection. Nevirapine may be prescribed for patients with hepatic fibrosis and cirrhosis. Significant autoinduction of cytochrome P450 3A4 and 2B6 following multiple dosing prompted an assessment of the metabolic profiles in patients with liver disease receiving chronic nevirapine therapy. HIV-infected patients with hepatic fibrosis who were receiving a stable antiretroviral regimen containing nevirapine for > or = 6 weeks had liver biopsy specimens assessed by Ishak histologic scoring and were grouped by severity (group 1, Ishak scores of 1 and 2; group 2, Ishak scores of 3 and 4; group 3, Ishak scores of 5 and 6). Steady-state trough nevirapine levels were determined for all patients, and additional measurements were obtained at 1, 2, and 4 h following nevirapine dosing for a subset of patients. The pharmacokinetics of nevirapine and its five metabolites were characterized, and a comparison of the results for the different Ishak groups was performed. Among 51 patients with hepatic fibrosis, the majority of whom were coinfected with hepatitis C virus or hepatitis B virus, differences between the maximum and the minimum observed plasma concentrations demonstrated a statistically significant flattening of the systemic exposure curves with progression from Ishak group 1 to Ishak group 2 or 3, suggesting a decrease in systemic clearance with the progression of liver disease. However, there were no significant differences in the trough and the maximum nevirapine concentrations between the Ishak groups. The metabolite profiles were also comparable across the Ishak groups. In HIV-infected patients who were chronically treated with nevirapine and who had various degrees of hepatic fibrosis, including cirrhosis, trough plasma nevirapine concentrations were not significantly increased, and thus, no dose adjustment is warranted.

In vitro-in vivo correlation for nevirapine extended release tablets.

An in vitro-in vivo correlation (IVIVC) for four nevirapine extended release tablets with varying polymer contents was developed. The pharmacokinetics of extended release formulations were assessed in a parallel group study with healthy volunteers and compared with corresponding in vitro dissolution data obtained using a USP apparatus type 1. In vitro samples were analysed using HPLC with UV detection and in vivo samples were analysed using a HPLC-MS/MS assay; the IVIVC analyses comparing the two results were performed using WinNonlin. A Double Weibull model optimally fits the in vitro data. A unit impulse response (UIR) was assessed using the fastest ER formulation as a reference. The deconvolution of the in vivo concentration time data was performed using the UIR to estimate an in vivo drug release profile. A linear model with a time-scaling factor clarified the relationship between in vitro and in vivo data. The predictability of the final model was consistent based on internal validation. Average percent prediction errors for pharmacokinetic parameters were <10% and individual values for all formulations were <15%. Therefore, a Level A IVIVC was developed and validated for nevirapine extended release formulations providing robust predictions of in vivo profiles based on in vitro dissolution profiles.

Pharmacokinetic and safety evaluation of BILR 355, a second-generation nonnucleoside reverse transcriptase inhibitor, in healthy volunteers.

BILR 355 is a second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) under clinical development for the treatment of human immunodeficiency virus infection, particularly in those who harbor virus resistant to the currently available NNRTIs. Two single-center, double-blinded, placebo-controlled, parallel dose-escalation studies were conducted to evaluate the pharmacokinetics and safety of oral BILR 355 administration alone and after coadministration with ritonavir (RTV) at 100 mg. Following a single dose of BILR 355 in oral solution, the mean half life (t(1/2)) was 2 to 4 h, with peak concentrations occurring at 0.5 to 1 h postadministration. The mean apparent clearance (CL/F) ranged from 79.2 to 246 liters/h for administered doses of 12.5 mg to 100 mg. This observed nonlinearity in CL/F resulted from the increased bioavailability attributed to a saturated absorption and/or elimination process at higher doses. In contrast, after the coadministration of single doses of 5 mg to 87.5 mg of BILR 355 with RTV, the mean CL/F ranged from 5.88 to 8.47 liters/h. Over the dose range (5 to 87.5 mg) studied, systemic BILR 355 exposures were approximately proportional to the doses administered when they were coadministered with RTV. With RTV coadministration, the mean t(1/2) increased to 10 to 16 h, and the mean time of the maximum concentration in plasma lengthened to 1.5 to 5 h. Compared to the values for BILR 355 given alone, the mean area under the concentration-time curve from time zero to infinity, the maximum concentration in plasma, and the t(1/2) of BILR 355 achieved after coadministration with RTV increased 15- to 30-fold, 2- to 5-fold, and 3- to 5-fold, respectively. In both studies, BILR 355 appeared to be safe and well tolerated in healthy volunteers when the outcomes in the treated volunteers were compared with those in the placebo group.

Case-control exploration of relationships between early rash or liver toxicity and plasma concentrations of nevirapine and primary metabolites.

OBJECTIVE: This investigation measured trough nevirapine and five oxidative metabolite concentrations in plasma specimens collected from patients who exhibited a rash or liver function abnormality during the first 6 weeks of treatment. METHOD: Patient selection came from three clinical trials, totaling 1,357 patients, from which frozen specimens had been stored and were available for assay. The control patients were matched according to trial, steroid use, CD4 cell count, gender, race, and hepatitis B/C status. Observed plasma metabolite concentrations were compared using signed rank tests. RESULTS: A total of 49 case-control pairs were studied. Women had significantly greater exposure than men to nevirapine and four of the five metabolites at week 2, but the plasma concentrations were comparable by week 4. Steroid (prednisone) co-medication produced significantly different plasma nevirapine and metabolite concentrations for the majority of case-control comparisons at week 3, a week after cessation of steroid treatment, but only occasionally produced a measurable difference at other weeks. CONCLUSION: During the first 6 weeks of nevirapine therapy, the rashes and liver enzyme elevations that occurred appear to be idiosyncratic. There were no strong relationships observed between the plasma concentrations of nevirapine or any of its five metabolites to a casedefining event. The systemic exposure of the metabolite 12-hydroxynevirapine and its successor 4-carboxynevirapine, hypothesized in the skin rash female Brown Norway rat model as reactive intermediates for idiosyncratic immune-mediated adverse reactions, were comparable between case and control samples and were comparable in proportion to the precursor nevirapine exposure.

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry.

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.

Differential clonal evolution in oesophageal cancers in response to neo-adjuvant chemotherapy.

How chemotherapy affects carcinoma genomes is largely unknown. Here we report whole-exome and deep sequencing of 30 paired oesophageal adenocarcinomas sampled before and after neo-adjuvant chemotherapy. Most, but not all, good responders pass through genetic bottlenecks, a feature associated with higher mutation burden pre-treatment. Some poor responders pass through bottlenecks, but re-grow by the time of surgical resection, suggesting a missed therapeutic opportunity. Cancers often show major changes in driver mutation presence or frequency after treatment, owing to outgrowth persistence or loss of sub-clones, copy number changes, polyclonality and/or spatial genetic heterogeneity. Post-therapy mutation spectrum shifts are also common, particularly C>A and TT>CT changes in good responders or bottleneckers. Post-treatment samples may also acquire mutations in known cancer driver genes (for example, SF3B1, TAF1 and CCND2) that are absent from the paired pre-treatment sample. Neo-adjuvant chemotherapy can rapidly and profoundly affect the oesophageal adenocarcinoma genome. Monitoring molecular changes during treatment may be clinically useful.

Clinical Trial of Oral Nelfinavir before and during Radiation Therapy for Advanced Rectal Cancer.

PURPOSE: Nelfinavir, a PI3K pathway inhibitor, is a radiosensitizer that increases tumor blood flow in preclinical models. We conducted an early-phase study to demonstrate the safety of nelfinavir combined with hypofractionated radiotherapy (RT) and to develop biomarkers of tumor perfusion and radiosensitization for this combinatorial approach. EXPERIMENTAL DESIGN: Ten patients with T3-4 N0-2 M1 rectal cancer received 7 days of oral nelfinavir (1,250 mg b.i.d.) and a further 7 days of nelfinavir during pelvic RT (25 Gy/5 fractions/7 days). Perfusion CT (p-CT) and DCE-MRI scans were performed pretreatment, after 7 days of nelfinavir and prior to the last fraction of RT. Biopsies taken pretreatment and 7 days after the last fraction of RT were analyzed for tumor cell density (TCD). RESULTS: There were 3 drug-related grade 3 adverse events: diarrhea, rash, and lymphopenia. On DCE-MRI, there was a mean 42% increase in medianKtrans, and a corresponding median 30% increase in mean blood flow on p-CT during RT in combination with nelfinavir. Median TCD decreased from 24.3% at baseline to 9.2% in biopsies taken 7 days after RT (P= 0.01). Overall, 5 of 9 evaluable patients exhibited good tumor regression on MRI assessed by tumor regression grade (mrTRG). CONCLUSIONS: This is the first study to evaluate nelfinavir in combination with RT without concurrent chemotherapy. It has shown that nelfinavir-RT is well tolerated and is associated with increased blood flow to rectal tumors. The efficacy of nelfinavir-RT versus RT alone merits clinical evaluation, including measurement of tumor blood flow.

Nevirapine and efavirenz pharmacokinetics and covariate analysis in the 2NN study.

OBJECTIVE: The aim of this 2NN pharmacokinetic substudy was to investigate the population pharmacokinetics of nevirapine and efavirenz. METHODS: Treatment-naive, HIV-1-infected patients received nevirapine (once or twice daily), efavirenz or a combination with lamivudine and stavudine. Blood samples were collected on day 3 and weeks 1, 2, 4, 24 and 48. Using non-linear mixed effects modelling, pharmacokinetics of nevirapine and efavirenz and factors involved in the inter-individual variability were investigated. RESULTS: Clearance of nevirapine in the induction phase (<14 days) and at steady state (>28 days) were 2.02 I/h and 2.81 I/h, respectively. Volume of distribution and absorption rate constant were 77.0 l and 1.66 h(-1), respectively. Clearance of nevirapine was lower in females (13.8%) and in patients with hepatitis B (19.5%). Patients from South America and Western countries had higher clearance of nevirapine compared with Thai and South African patients. The clearances of efavirenz in the induction phase and at steady state were 7.95 l/h and 8.82 l/h, respectively. The volume of distribution and absorption rate constant were 4181 and 0.287 h(-1), respectively. Concomitant use of nevirapine increased clearance of efavirenz (43%). Patients from Thailand had lower clearance than the rest of the population. CONCLUSIONS: The population pharmacokinetics of nevirapine and efavirenz were assessed in the 2NN trial. For both drugs, an induction phase was distinguished from the steady-state phase. Gender, hepatitis B and geographical region were involved in the variability of the pharmacokinetics of nevirapine. Region and concomitantly used nevirapine were determinants of the pharmacokinetics of efavirenz.

Are adverse events of nevirapine and efavirenz related to plasma concentrations?

OBJECTIVE: The relationships between adverse events (AEs) and plasma concentrations of nevirapine (NVP) and efavirenz (EFV) were investigated as part of the large, international, randomized 2NN study. METHODS: Treatment-naive, HIV-1-infected patients received NVP (once or twice daily), EFV or their combination, each in combination with lamivudine and stavudine. Blood samples were collected on day 3 and weeks 1, 2, 4, 24 and 48. Concentrations of NVP and EFV were quantitatively assessed by a validated HPLC assay. Individual Bayesian estimates of the area under the plasma concentration-time curve over 24 h (AUC24h), and minimum and maximum plasma concentrations (Cmin and Cmax) as measures for drug exposure of NVP and EFV, were generated using a previously developed population pharmacokinetic model. Pharmacokinetic parameters were compared for patients with and without central nervous system (CNS) and psychiatric AEs, hepatic events, liver enzyme elevations (LEEs) and rash. Furthermore, it was investigated whether a clear cut-off for a pharmacokinetic parameter could be identified above which the incidence of AEs was clearly increased. AEs were also related to demographic parameters and baseline characteristics. RESULTS: In total, from 1077 patients, NVP (3024 samples) and EFV (1694 samples) plasma concentrations and AE data (825 observations) were available. For all patients Cmin, Cmax and AUC24h were determined. When corrected for known covariates of gender, CD4 cell count at baseline, region, hepatitis coinfection and possible interactions between these factors, no significant associations between AEs and any tested exposure parameter of NVP was observed. Also, no target Cmin value, above which patients were at increased risk for AEs, could be established. On the other hand, geographical region, hepatitis coinfection, CD4 cell count and gender were found to be significantly related with the incidence of CNS and psychiatric AEs, hepatic events, LEEs and rash during the treatment with NVP. The occurrence of elevated liver enzymes during the first 6 weeks in the EFV-containing arm was significantly (P = 0.036) correlated to the exposure of EFV (Cmin). Only hepatitis coinfection impacted on LEEs during the first 6 weeks of treatment. With an EFV Cmin above 2.18 mg/l during the induction phase, patients were 4.4 (range 1.3-15.5) times more at risk for elevated liver enzymes. No other correlations between AEs and EFV pharmacokinetics or patient characteristics could be identified. CONCLUSIONS: Pharmacokinetic parameters of NVP did not have a relationship to AEs in the 2NN trial when corrected for known covariates. The value of periodical drug monitoring of NVP as a way to prevent toxicity is therefore limited. Treating physicians should instead focus on factors that are more predictive of AEs (gender, CD4 count and hepatitis coinfection). High EFV Cmin levels resulted in elevated liver enzyme values during the first 6 weeks of treatment. Regular measurement of EFV levels and liver enzymes at the start of therapy may therefore be advised.

Pharmacokinetics of nevirapine: once-daily versus twice-daily dosing in the 2NN study.

OBJECTIVE: As part of the large international, randomized 2NN trial, the pharmacokinetics of nevirapine in once-daily 400 mg and twice-daily 200 mg dosing regimens were investigated. METHOD: Treatment-naive HIV-1-infected patients were randomized to receive nevirapine 400 mg once daily or 200 mg twice daily, in combination with lamivudine and stavudine. Blood samples were collected at several time-points (day 3, weeks 1, 2, 4, 24, and 48). Differences in pharmacokinetics between once- versus twice-daily dosing were investigated with nonlinear mixed effects modelling (NONMEM). RESULTS: In total, 2,899 nevirapine plasma concentrations were available from 578 patients. Dosage and dosing frequency did not influence clearance or volume of distribution of nevirapine, indicating linear pharmacokinetic behavior of nevirapine whether given as a single daily dose or as divided doses over 24 hours. During steady state, the Cmin was lower (3.26 mg/L vs. 4.44 mg/L; p < .001) and the Cmax was higher (7.88 mg/L vs. 6.55 mg/L; p < .001) in the once-daily arm. However, compared to total variability in nevirapine levels for both treatments, these differences were minor. During steady state, total exposure, measured as AUC24h, was comparable for both regimens (133 mg/L*h vs. 133 mg/L*h; p = .084). CONCLUSION: The daily exposure to nevirapine (AUC24h) was similar for the 400 mg once-daily and the 200 mg twice-daily dosing regimens. The Cmin of nevirapine is lower and the Cmax of nevirapine is higher for the once-daily regimen as compared to the twice-daily regimen. As a result, 200 mg nevirapine dosed twice daily may be preferred over 400 mg nevirapine dosed once daily.