Relationship between Pharmacokinetics and Pharmacodynamic Responses in Healthy Smokers Informs a Once-Daily Dosing Regimen for Nemiralisib.

Nemiralisib (GSK2269557), a potent inhaled inhibitor of phosphoinositide 3-kinase δ (PI3Kδ), is being developed for the treatment of respiratory disorders including chronic obstructive pulmonary disease. Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n = 56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1-6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-(4,5)-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% confidence interval [CI], 11%-34%) and 36% (90% CI, 11%-64%) after a single dose or after 14 days of treatment with nemiralisib, respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ∼3- to 4.5-fold (peak vs. trough) in plasma exposure after 14 days of nemiralisib treatment. The BAL analysis at trough confirmed higher levels of the drug in the lungs versus plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). A comparison of the drug levels in plasma and the reductions in sputum PIP3 showed a direct relationship between exposure and PIP3 reduction. These results demonstrated target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Utilisation of water soluble iridium catalysts for signal amplification by reversible exchange.

The catalytic hyperpolarisation of pyridine, 3-hydroxypyridine and oxazole by the Signal Amplification By Reversible Exchange (SABRE) process is achieved by a series of water soluble iridium phosphine and N-heterocyclic carbene dihydride complexes. While the efficiency of the SABRE process in methanol-d4 solution or ethanol-d6 solution is high, with over 400-fold (1)H polarisation of pyridine being produced by [Ir(H)2(NCMe)(py)(IMes)(monosulfonated-triphenylphosphine)]BF4, changing to a D2O or a D2O-ethanol solvent mixture leads to dramatically reduced activity which is rationalised in terms of low H2 solubility.

Evaluation of preparative high performance liquid chromatography and cryoprobe-nuclear magnetic resonance spectroscopy for the early quantitative estimation of drug metabolites in human plasma.

Definitive information on the metabolism of a drug candidate in humans is achieved through dosing radiolabelled drug as part of a clinical study, and is typically conducted post-proof of concept in Phase III of the clinical development plan. Here we describe a novel approach, using preparative high performance liquid chromatography and cryoprobe-nuclear magnetic resonance spectroscopy, to determine the human systemic exposure to a drug and its metabolites using samples derived from Phase I clinical studies. Using the described methodology, novel human plasma metabolites, as low as 10 ng/ml can be detected and quantified. This provides an opportunity, early in the development process to understand the potential role of metabolites in the safety and efficacy of drugs in humans.

Utilizing a -100 degrees C microplate CCD Imager, yttrium silicate coated 384-microplates and ultra-performance liquid chromatography for improved profiling of radiolabeled drug metabolites in complex biological samples.

The recent commercial availability of small particle packed columns (<2 microm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography. The improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of ViewLux, a microplate imager, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is assessed. The system demonstrates robustness and sensitivity comparable to a microplate scintillation counter (TopCount) more typically used for off-line metabolite radiodetection. The ViewLux is also used here to undertake successful metabolite profiling of actual samples, for two investigational drug candidates, using both 96- and 384-well yttrium silicate microplates.

Common causes of open-circuit recreational diving fatalities.

Diving fatalities causes were investigated in 947 recreational open-circuit scuba diving deaths from 1992-2003. Where possible, cases were classified at each step of a four step sequence: trigger, disabling agent, disabling injury, cause of death (COD). The most frequent adverse events within each step were: (a) triggers 41% insufficient gas, 20% entrapment, 15% equipment problems; (b) disabling agents--55% emergency ascent, 27% insufficient gas, 13% buoyancy trouble; (c) disabling injuries--33% asphyxia, 29% arterial gas embolism (AGE), 26% cardiac incidents; and (d) COD--70% drowning, 14% AGE, 13% cardiac incidents. We concluded that disabling injuries were more relevant than COD as drowning was often secondary to a disabling injury. Frequencies and/ or associations with risk factors were investigated for each disabling injury by logistic regression. (The reference group for each injury was all other injuries.) Frequencies and/or associations included: (a) asphyxia--40% entrapment (Odds Ratio, OR > or = 30), 32% insufficient gas (OR = 15.9), 17% buoyancy trouble, 15% equipment trouble (OR = 4.5), 11% rough water, drysuit (OR = 4.1), female gender (OR = 2.1); (b) AGE--96% emergency ascent (OR > or = 30), 63% insufficient gas, 17% equipment trouble, 9% entrapment; (c) cardiac incidents--cardiovascular disease (OR = 10.5), age > 40 (OR = 5.9). Minimizing the frequent adverse events would have the greatest impact on reducing diving deaths.

TopCount coupled to ultra-performance liquid chromatography for the profiling of radiolabeled drug metabolites in complex biological samples.

The recent commercial availability of small particle packed columns (<2microm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography (UPLC). It has recently been shown that the improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of TopCount, a microplate scintillation counter, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is tested. TopCount, has innumerable benefits over more traditional on-line radioactivity flow detection methods, when dealing with the narrow peak widths and small peak volumes associated with the enhanced efficiency of sub-2microm columns. The system is tested for robustness and sensitivity, and then used to undertake successful metabolite profiling of actual samples, and the data compared to traditional HPLC with on-line radioactivity flow detector.

Plasma glucose response to recreational diving in novice teenage divers with insulin-requiring diabetes mellitus.

A growing number of individuals with insulin-requiring diabetes mellitus (IRDM) dive, but data on plasma glucose (PG) response to diving are limited, particularly for adolescents. We report on seven 16-17 year old novice divers with IRDM participating in a tropical diving camp who had recent at least moderate PG control (HbA1c 7.3 +/- 1.1%) (mean +/- SD). PG was measured at 60, 30 and 10 min pre-dive and immediately following 42 dives. Maximum depth (17 +/- 6 msw) and total underwater times (44 +/- 14 min) were not extreme. Pre-dive PG exceeded 16.7 mmol x L(-1) (300 mg x dL(-1)) in 22% of dives. Males had significantly higher pre-dive levels (15.4 +/- 5.6 mmol x L(-1) [277 +/- 100 mg x dL(-1)] vs. 12.8 +/- 2.9 mmol x L(-1) [230 +/- 52 mg x dL(-1)], respectively) and greater pre-post-dive changes (-4.3 +/- 4.4 mmol x L(-1) [-78 +/- 79 mg x dL(-1)] vs. -0.5 +/- 4.3 mmol x L(-1) [-9 +/- 77 mg x dL(-1)], respectively). Post-dive PG was < 4.4 mmol x L(-1) [< 80 mg x dL(-1)] in two dives by two different males (3.4 and 3.9 mmol x L(-1) [61 and 70 mg x dL(-1)]). No symptoms or complications of hypoglycemia were reported. These data show that in a closely monitored situation, and with benign diving conditions, some diabetic adolescents with good control and no secondary complications may be able to dive safely. The impact of purposeful elevation of PG to protect against hypoglycemia during diving remains to be determined.

Ultra-performance liquid chromatography coupled to linear ion trap mass spectrometry for the identification of drug metabolites in biological samples.

The coupling of ultra-performance liquid chromatography, operating at elevated pressures, to a linear ion trap mass spectrometer provides a high-performance system suitable for drug metabolite characterisation. This system demonstrates improved chromatographic efficiency and sensitivity and at the same time provides diagnostic MSn data often critical for metabolite structural assignment. The linear ion trap was capable of dealing with the high chromatographic efficiencies and hence narrow peak widths associated with 1.7 microm particle-packed column separations. Polarity switching and data-dependent MSn data were generated with ease, and applied to the identification of metabolites found in human plasma.

The determination of nicotinic acid in plasma by mixed-mode liquid chromatography-tandem mass spectrometry following ion exchange solid phase extraction.

An assay for nicotinic acid in plasma samples has been developed using ion exchange solid phase extraction in 96-well format followed by mixed-mode ion exchange/reversed-phase liquid chromatography with positive ion tandem mass spectrometry detection. The assay avoids the need for time-consuming derivatisation procedures or involatile ion-pair chromatography reagents. The assay is linear over the wide range 0.05-20 microg/mL, based on a 100 microL sample (correlation coefficient>0.99). The assay is accurate and precise (bias and coefficient of variation<18%) over this calibration range.

Characterization of the metabolites of alosetron in experimental animals and human.

The metabolism of radiolabelled alosetron was studied in rat, dog, rabbit, mouse and human. The metabolism in rat and dog was studied at a low and an elevated dose designed to generate sufficient quantities of metabolite for definitive identification. A strategy for the characterization of metabolites in cases of extensive metabolism was developed and demonstrated for alosetron. Semi-preparative high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and liquid chromatography-nuclear magnetic resonance (HPLC-NMR) enabled the isolation and characterization of 28 metabolites of alosetron. The characterization of the metabolites in animal excreta facilitated the identification of human systemic metabolites.

Plasma glucose responses in recreational divers with insulin-requiring diabetes.

Insulin-requiring diabetes mellitus (IRDM) is commonly described as an absolute contraindication to scuba diving. A 1993 Divers Alert Network survey, however, identified many active IRDM divers. We report on the plasma glucose response to recreational diving in IRDM divers. Plasma glucose values were collected before and after diving in IRDM and healthy control divers. Time/depth profiles of 555 dives in IRDM divers were recorded. IRDM divers had been diving for a mean of almost nine years and had diabetes for a mean of over 15 years. No symptoms or complications related to hypoglycemia were reported (or observed). Post-dive plasma glucose fell below 70 mg x dL(-1) in 7% (37/555) of the IRDM group dives compared to 1% (6/504) of the controls (p<0.05). Moderate levels of hyperglycemia were also noted in 23 divers with IRDM on 84 occasions. While large plasma glucose swings from pre-dive to post-dive were noted, our observations indicate that plasma glucose levels, in moderately-controlled IRDM, can be managed to avoid hypoglycemia during routine recreational dives under ordinary environmental conditions and low risk decompression profiles.

Effects of age and exercise on physiological dead space during simulated dives at 2.8 ATA.

Physiological dead space (Vds), end-tidal CO(2) (Pet(CO(2))), and arterial CO(2) (Pa(CO(2))) were measured at 1 and 2.8 ATA in a dry hyperbaric chamber in 10 older (58-74 yr) and 10 younger (19-39 yr) air-breathing subjects during rest and two levels of upright exercise on a cycle ergometer. At pressure, Vd (liters btps) increased from 0.34 +/- 0.09 (mean +/- SD of all subjects for normally distributed data, median +/- interquartile range otherwise) to 0.40 +/- 0.09 (P = 0.0060) at rest, 0.35 +/- 0.13 to 0.45 +/- 0.11 (P = 0.0003) during light exercise, and 0.38 +/- 0.17 to 0.45 +/- 0.13 (P = 0.0497) during heavier exercise. During these conditions, Pa(CO(2)) (Torr) increased from 33.8 +/- 4.2 to 35.7 +/- 4.4 (P = 0.0059), 35.3 +/- 3.2 to 39.4 +/- 3.1 (P < 0.0001), and 29.6 +/- 5.6 to 37.4 +/- 6.5 (P < 0.0001), respectively. During exercise, Pet(CO(2)) overestimated Pa(CO(2)), although the absolute difference was less at pressure. Capnography poorly estimated Pa(CO(2)) during exercise at 1 and 2.8 ATA because of wide variability. Older subjects had higher Vd at 1 ATA but similar changes in Vd, Pa(CO(2)), and Pet(CO(2)) at pressure. These results are consistent with an effect of increased gas density.

N-O glucuronidation: a major human metabolic pathway in the elimination of two novel anti-convulsant drug candidates.

1. The urinary metabolites of the anti-convulsant compound 4-amino-1-(2,6-difluorobenzyl)-1H-1,2,3-triazolo[4,5-c]-pyridine hydrochloride (GI265080) obtained following a single oral dose to man have been detected and quantified relative to each other using 19F-NMR spectroscopy. 2. The human urinary metabolites of GI265080 were isolated using semipreparative HPLC and unequivocally characterized using 1H-NMR spectroscopy, two-dimensional heteronuclear NMR spectroscopy and mass spectrometry. The assignments of the N-(5)-oxide and the N-(5)-O-glucuronide metabolites of GI265080 were further confirmed by independent synthesis. The urinary metabolites obtained following single oral doses to dog and rat have also been isolated and characterized. 3. The human urinary metabolites of GI265080 comprise the N-(5)-oxide, the quaternary N+-(5)-glucuronide, the 7-hydroxy glucuronide and a glucuronide conjugate of the N-(5)-oxide. The N-(5)-O-glucuronide conjugate is a novel species in human metabolism and is a significant route of elimination of GI265080 in man. 4. The urinary metabolites of the potential anti-convulsant GW273293 (6-amino-3-(2,3,5-trichlorophenyl)pyrazin-2-ylamine) obtained following a single oral dose to man have also been isolated and characterized. The formation of a novel N-O-glucuronide was also observed and was shown to constitute a significant route of elimination of GW273293 in man.

Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review.

1. The development of bio-analysis of drug molecules over the last 10 years is reviewed, focusing on advances in sample preparation, liquid chromatography and detection. 2. Developments have led to improvements in detection sensitivity, enhancements in specificity and increased capacity. 3. Emerging technologies such as monolithic column chromatography and miniaturized chip-based systems are discussed.

Direct analysis of pharmaceutical compounds in human plasma with chromatographic resolution using an alkyl-bonded silica rod column.

Monolithic columns have been successfully used with steep gradient and high flow rates for the direct analysis of a candidate pharmaceutical compound in human plasma. The monolithic columns showed excellent robustness with nearly 300 20-microL injections of plasma (diluted 1:1 with water) being made onto one column without significant deterioration in performance. The system gave excellent sensitivity with a limit of quantification of 5 ng/mL being achieved. Unlike previous methods of direct analysis the monolithic columns showed excellent resolution even after nearly 300 plasma injections. The column performance was measured before and after the analysis of the plasma samples.

Formation of a defluorinated metabolite of a quinoxaline antiviral drug catalysed by human cytochrome P450 1A2.

The in-vitro metabolism of GW420867X ((S)-2-ethyl-7-fluoro-3-oxo-3, 4-dihydro-2H-quinoxaline-1-carboxylic acid isopropyl ester), a quinoxaline drug for the potential treatment of HIV, has been studied with singly expressed human cytochromes P450 (CYP 450). No biotransformation of [14C]GW420867X was evident in the presence of any of the CYP 450 isoforms, with the exception of CYP 450 1A2, where a single metabolite was observed in the HPLC radiochromatograms of enzyme incubations with the test compound. The structure of this metabolite was determined by nuclear magnetic resonance spectroscopy and mass spectrometry, and was shown to correspond to the replacement of the aromatic fluorine of GW420867X with a hydroxyl group. Thus, it appeared that CYP 450 1A2 catalysed the specific defluorination of GW420867X, presumably during formation of an arene oxide intermediate during aromatic hydroxylation.

Determination of 4-hydroxytamoxifen in mouse plasma in the pg/mL range by gradient capillary liquid chromatography/tandem mass spectrometry.

Capillary high-performance liquid chromatography (HPLC; 300 microm i.d.) coupled to tandem mass spectrometry has been used to determine the concentration of 4-hydroxytamoxifen in mouse plasma in the pg/mL range following the administration of Tamoxifen. A limit of quantification (LOQ) of 100 pg/mL was achieved using only 25 microL of plasma. The on-column sensitivity was determined to be 100 fg. The column performance was determined isocratically before and after the assay and showed only a 15% reduction in performance after 70 injections of plasma extract. No significant peak band broadening was observed due to the mass spectrometer interface using a standard TurboIonspray source.

How much are patients willing to pay to avoid postoperative nausea and vomiting?

Postoperative nausea and vomiting (PONV) are unpleasant experiences. However, there is no drug that is completely effective in preventing PONV. Whereas cost effectiveness analyses rely on specific health outcomes (e.g., years of life saved), cost-benefit analyses assess the cost and benefit of medical therapy in terms of dollars. We hypothesized that patients were willing to pay for a hypothetical new drug that would eliminate PONV. Eighty elective day surgical patients using general anesthesia participated in the study. After their recovery in the postanesthetic care unit, they were asked to complete an interactive computer questionnaire on demographics, the value of avoiding PONV, and their willingness to pay for an antiemetic. Patients were willing to pay US$56 (US$26--US$97; median, 25%--75%) for an antiemetic that would completely prevent PONV. Patients who developed nausea (n = 21; 26%) and vomiting (n = 9; 11%) were willing to pay US$73 (US$44--US$110) and $100 (US$61--US$200; median, 25%--75%), respectively (P < 0.05). Seventy-six percent of patients considered avoiding postoperative nausea and 78% of patients considered avoiding vomiting as important (> or = 50 mm on a 0--100-mm visual analog scale). Nausea or vomiting in the postanesthetic care unit, greater patient income, previous history of PONV, more importance placed on avoiding nausea and vomiting, increasing age, and being married are independent covariates that increase the willingness to pay estimates. Patients associated a value with the avoidance of PONV and were willing to pay between US$56 and US$100 for a completely effective antiemetic.