Edoxaban Exposure-Response Analysis and Clinical Utility Index Assessment in Patients With Symptomatic Deep-Vein Thrombosis or Pulmonary Embolism.

Edoxaban exposure-response relationships from the phase III study evaluating edoxaban for prevention and treatment of venous thromboembolism (VTE) in patients with acute deep vein thrombosis (DVT) and/or pulmonary embolism (PE) were assessed by parametric time-to-event analysis. Statistical significant exposure-response relationships were recurrent VTE with hazard ratio (HR) based on average edoxaban concentration at steady state (Cav) (HRCav) = 0.98 (i.e., change in the HR with every 1 ng/mL increase of Cav); the composite of recurrent DVT and nonfatal PE with HRCav = 0.99; and the composite of recurrent DVT, nonfatal PE, and all-cause mortality HRCav = 0.98, and all death using maximal edoxaban concentration (Cmax) with HR (Cmax) = 0.99. No statistical significant exposure-response relationships were found for clinically relevant bleeding or major adverse cardiovascular event. Results support the recommendation of once-daily edoxaban 60 mg, and a reduced 30 mg dose in patients with moderate renal impairment, body weight ≤60 kg, or use of P-glycoprotein inhibitors verapamil or quinidine.

Comparisons of Analysis Methods for Proof-of-Concept Trials.

Drug development struggles with high costs and time consuming processes. Hence, a need for new strategies has been accentuated by many stakeholders in drug development. This study proposes the use of pharmacometric models to rationalize drug development. Two simulated examples, within the therapeutic areas of acute stroke and type 2 diabetes, are utilized to compare a pharmacometric model-based analysis to a t-test with respect to study power of proof-of-concept (POC) trials. In all investigated examples and scenarios, the conventional statistical analysis resulted in several fold larger study sizes to achieve 80% power. For a scenario with a parallel design of one placebo group and one active dose arm, the difference between the conventional and pharmacometric approach was 4.3- and 8.4-fold, for the stroke and diabetes example, respectively. Although the model-based power depend on the model assumptions, in these scenarios, the pharmacometric model-based approach was demonstrated to permit drastic streamlining of POC trials.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e23; doi:10.1038/psp.2012.24; advance online publication 16 January 2013.

Diurnal variation in serum levels of cartilage oligomeric matrix protein in patients with knee osteoarthritis or rheumatoid arthritis.

OBJECTIVE: To monitor changes in serum concentrations of cartilage oligomeric matrix protein (COMP) during a 24-h period to determine any diurnal variation, and to estimate the half life of COMP in the circulation in patients with symptomatic knee osteoarthritis and in those with rheumatoid arthritis. METHODS: Serum samples were drawn every 4 h (7 samples/patient over 24 h) in 10 patients with knee osteoarthritis and 14 patients with rheumatoid arthritis. Osteoarthritis was defined radiographically and clinically (American College of Rheumatology (ACR) criteria) and rheumatoid arthritis according to the 1987 ACR criteria. Serum COMP was measured by sandwich ELISA. A statistical model for the diurnal variation in the COMP levels was developed using the computer program NONMEM. RESULTS: No considerable changes in COMP levels were observed during the day between 08:00 and 21:00 in either group. A significant decrease in serum COMP was apparent during bed rest at night, reaching the lowest levels between 04:00 and 05:00 (p<0.03 or better v all other time points) in patients with osteoarthritis and in those with rheumatoid arthritis. From the rate of decreasing serum COMP levels, a putative half life of COMP in the circulation was estimated to be 7.4 h. CONCLUSION: During normal daytime activities, serum COMP levels are constant. The decrease during the night indicates a rapid elimination of COMP once it has reached the circulation. The stable COMP levels during the day suggest that it is not necessary to further standardise the time of serum sampling in clinical practice.

Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations.

Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.

Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography.

Glycosylated human plasminogen activator inhibitor type 1 (PAI-1), produced in Chinese hamster ovary (CHO) cells, showed a variety of compounds with different molecular weights when subjected to electrospray mass spectrometry (ES-MS), owing to the heterogeneity of the carbohydrate chains. However, non-glycosylated human PAI-1, produced in E. coli, gave rise to a prominent species with a molecular weight of 42,774, consistent with the amino-acid sequence. A non-glycosylated mutant of the proteinase domain (B-chain) of tissue-type plasminogen activator (tPA) produced in C 127 cells, had a molecular weight of 28,168. Full-length, glycosylated, tPA showed a large heterogeneity in molecular mass. For a mass study, a tPA-PAI-1 complex was formed, composed of non-glycosylated PAI-1 and non-glycosylated B-chain. This complex was remarkably stable at room temperature in buffer with a neutral pH. The mass spectrum of the complex provided two main species, a peptide with a mass of 3803 and a dominating species of 67,133. These masses are consistent with a complex where PAI-1 is cleaved at the P1-P1' position. A trace of a species with a molecular mass of 70,942 was also found, corresponding to the complete, non-dissociated complex with PAI-1. Separation of the cleaved peptide, corresponding to the hydrophobic C-terminal 33 amino-acid residues of PAI-1, from the complex, was achieved by size-exclusion chromatography in the presence of 30% acetonitrile. Thus, in the complex between tPA and PAI-1, the proteins are held together by a tight covalent bond, but the C-terminal cleaved peptide of PAI-1 is only bound to the complex by hydrophobic forces. To assess whether this is specific to the tPA B-chain alone, experiments with the complex of full-length, glycosylated tPA and glycosylated PAI-1 were also performed, and it was possible to demonstrate the release of the C-terminal PAI-1 peptide by chromatography, mass spectrometry, as well as by SDS-PAGE.

Identification of a new by-product detected in metoprolol tartrate.

A new impurity has been found in some batches of metoprolol tartrate. As the amount exceeded 0.1% it was of interest to deduce the structure. Techniques involved in solving the problem were LC, LC-MS and GC-MS. The LC systems showed that the impurity and metoprolol behaved differently to modifications of the mobile phase, indicating that there were differences in the functional groups. LC-MS was used to determine the molecular weight, which was 74 mass units higher than metoprolol. A hydrogen-deuterium shift technique using micro column LC-MS gave the information that three hydrogen atoms were bound to heteroatoms, i.e. one more than in metoprolol. This led to the conclusion that the impurity had three extra carbon and two extra oxygen atoms. It was supposedly a by-product in the synthesis. Knowledge of the synthesis steps for beta-receptor blocking drugs suggested three possible structures. Two were independently synthesized and one of these was found to be identical to the impurity.

A metabolic route of omeprazole involving conjugation with glutathione identified in the rat.

A metabolic route of omeprazole involving glutathione has been established through identification of endproducts excreted in the urine of rats after oral administration of 400 mumol/kg of a mixture of [3H]- and [14C]omeprazole. The labeled positions enabled facile tracing of metabolites that were formed through fission of omeprazole, producing [3H]pyridine and [14C]benzimidazole metabolites. The structures of the metabolites were established by HPLC thermospray MS and MS/MS. Two of the metabolites were isolated and characterized by 1H NMR studies. The fact that the N-acetylcysteine derivative of the benzimidazole was one of the endproducts indicated that the initial reaction involved glutathione. Three metabolites reflecting the fate of the pyridine moiety were identified. Their proposed formation route is via initial reduction to the pyridylmethylthiol compound followed by S-methylation and S-oxidation to the corresponding sulfoxide or sulfone. The quantity of metabolites formed via the glutathione route identified in urine was about 10% of the dose given, both in male and female rats. The male and female rats excreted the same cleaved metabolites and approximately equal quantities thereof.

Direct derivatization of drugs in untreated biological samples for gas chromatographic analysis.

The possibilities to derivatize an analyte directly in the biological sample are reviewed with examples from our own experiences and from the literature. Techniques, such as extractive acylation, alkylation and benzoylation, are frequently used. Improvement of the extractability of the drug from the matrix is a common feature, especially with hydrophilic compounds, where sometimes cyclizing reactions can be employed. Several analytes are reactive or labile in the sample and can be trapped in derivatization reactions in situ. In many cases, two-phase reactions lead to milder derivatization conditions (e.g. dealkylation of tertiary amines), which is favourable from a clean-up point of view.

New biochemical separations using precolumn derivatization and microcolumn liquid chromatography.

Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve mixtures of biologically important steroids and prostaglandins. In order to enhance detection sensitivity, the samples are first derivatized with a suitable chromophore-yielding agent. Hydroxy steroids were derivatized with either benzoyl chloride or 7-(chlorocarbonylmethoxy)-4-methylcoumarin, while Dns hydrazine was employed to react with the ketonic groups of certain steroidal conjugates. Bile acids can also be derivatized with bromomethylcoumarin to yield fluorescent products. The use of microcolumns permits both a high degree of component resolution and enhanced detection sensitivity.

Two-phase derivatization of amitriptyline and structurally related tertiary amines for gas chromatography with electron-capture detection.

A new derivatization technique for tertiary amines has been developed. The amine is extracted at ambient temperature after addition of sodium iodide with methylene chloride containing an aryl chloroformate ester. By use of two new reagents, 2,4-dichlorophenyl and pentafluorophenyl chloroformate, the carbamates formed have high electron-capture response. The derivatization is rapid, often completed within 5 min. Amitriptyline was determined down to 6 ng/ml directly in a spiked plasma sample. A method for selective derivatization of a tertiary amine in the presence of the corresponding secondary amine is given.

A comparison of three injectable corticosteroids for the treatment of patients with seasonal hay fever.

Three injectable corticosteroids, betamethasone dipropionate, beta-methasone disodium phosphate and betamethasone acetate, and methylprednisolone acetate, were compared for onset and duration of action in patients with severe seasonal allergic rhinoconjunctivitis. The sixty patients who were entered into the trial had been well-studied in our allergy clinic. They were assigned, on the basis of a random number code, to treatment with one of the corticosteroids. Following a single intramuscular injection with one of the preparations, plasma cortisol and blood glucose concentrations also were compared at days 1, 2--3, 5--7 and 14. All three preparations improved the nasal symptoms. There were no individual differences with respect to onset or to duration of action. However, there were some differences in the effects on endogenous cortisol production and on blood glucose levels. Two of the preparations, betamethasone dipropionate and methylprednisolone acetate, suppressed endogenous cortisol for more than 14 days, while betamethasone phosphate/acetate did not suppress cortisol beyond 12 days. Beta-methasone dipropionate produced a moderate increase in blood glucose concentrations for the first two days after administration; betamethasone phosphate/acetate caused an increase for one day and methylprednisolone acetate had no effect.

A selected ion monitoring method for the determination of pethidine and norpethidine in plasma. Comparison with a gas chromatographic method using electron capture detection.

An analytical procedure for the simultaneous determination of pethidine and norpethidine in plasma by selected ion monitoring is described. The method has a capacity of 15--20 analyses per hour and can be used to determine pethidine and norpethidine down to 25 ng ml(-1) and 5 ng ml(-1), respectively. The selected ion monitoring method has been compared with a method based on electron capture detection after analysis of pethidine and norpethidine in spiked plasma samples and in plasma from patients. The two methods are capable of performing selective and accurate determinations of pethidine and norpethidine, in the concentration ranges obtained in man after a single therapeutic dose of pethidine.

Simultaneous determination of therapeutic plasma concentrations of pethidine and norpethidine in man by electron capture gas chromatography.

An analytical procedure for the simultaneous determination of pethidine and its N-Demethylated metabolite, norpethidine, in plasma is described. Pethidine and norpethidine are separated by partition chromatography, converted to the trichloroethyl carbamate with trichloroethyl chloroformate and determined by electron capture gas chromatography. The smallest amounts of pethidine and norpethidine determined by the method were 10 and 2 ng, respectively, in 0.1 ml plasma. The relative standard deviation in the determination of 50 ng of pethidine and 40 ng of norpethidine in 0.1 ml plasma wwere 5.8% (n = 8) and 6.3% (n = 10), respectively. The method was used to determine plasma levels of pethidine and norpethidine in three patients who received subcutaneous doses of pethidine 50-75 mg for postoperative pain. The peak levels of pethidine were found to be in the range 200-400 ng/ml, with a plasma half-life of the order of 4 hours. The levels of norpethidine were low.

Determination of pethidine in plasma by electron capture gas chromatography after reaction with trichloroethyl chloroformate.

Pethidine (meperidine) is determined in plasma by electron capture gas chromatography after derivatization with trichloroethyl chloroformate. The analytical procedure involves extraction of pethidine and the internal standard from plasma and their separation from metabolites by partition chromatography. After purification of the eluate, the derivatization is accomplished with trichloroethyl chloroformate in the presence of anhydrous sodium carbonate. The reaction mixture is further purified with methanolic alkali before gas chromatographic analysis. Optimum conditions for extraction and derivatization, as well as the sensitivity and selectivity of the method are discussed. Owing to the high sensitivity the pethidine levels are determined in 0.1 ml of plasma. The smallest amount of pethidine determined by the method was 100 ng/ml. The relative standard deviation at the 50-ng level of pethidine added to 0.1 ml of plasma was 5.8%(n =8).

A computer program for an open two compartment system distinguishing three models.

A digital computer program is described for the estimation of parameters in an open two compartment system distinguishing 3 models. The mathematical function to be fitted to a given set of experimental data is given initial estimates for the parameters of the function. The program uses an iterative procedure to adjust the parameters until the sum of squares of residuals has converged to a minimum. Assuming that a given substance is introduced into compartment 1, the function can be fitted to the set of experiment data of that compartment. A set of experimental data from compartment 2 can also be included in the minimizing function. Two optional weighting functions are presented. From the constants of the two-exponential functions the physiological parameters of the three models are determined. Experimental observations of compartment 1 only allow for calculation of physiological parameters of two of the models.