Effects of food on the pharmacokinetics of ponatinib in healthy subjects.

WHAT IS KNOWN AND OBJECTIVE: Ponatinib is a potent oral tyrosine kinase inhibitor with activity against BCR-ABL, the primary driver of chronic myeloid leukaemia and Philadelphia chromosome-positive acute lymphoblastic leukaemia. This single-centre, single-dose, randomized, open-label, three-period crossover study evaluated the pharmacokinetics and bioavailability of a single oral dose of ponatinib (45-mg tablet) under fasting conditions and following consumption of high- and low-fat meals by healthy subjects. METHODS: Subjects were randomly assigned to one of the six possible treatment sequences, each evaluating three ponatinib 45-mg treatments: administered under fasting conditions; administered after a high-fat meal; or administered after a standardized low-fat meal. The high-fat meal derived approximately 50% of its total caloric content from fat, with approximately 150, 250 and 500-600 calories derived from protein, carbohydrates and fat, respectively (total of approximately 900-1000 calories). The standardized low-fat meal derived no more than 20% of total caloric content from fat, with approximately 56, 428 and 63 calories derived from protein, carbohydrates and fat, respectively (total of approximately 547 calories). During each of the three treatment periods, blood samples were collected predose and at 13 time points over the 96-h post-dose interval. Plasma concentrations of ponatinib were measured by liquid chromatography/tandem mass spectrometry. Mixed-model analyses of variance (anova) were performed on natural log-transformed PK parameters Cmax and AUC0-∞. RESULTS AND DISCUSSION: Geometric mean maximum plasma concentration (Cmax) values for the fasted, low-fat and high-fat regimens were 54·7, 51·6 and 51·5 ng/mL, respectively. Geometric mean area under the concentration-time curve from time zero to infinity (AUC0-∞) values for the fasted, low-fat and high-fat regimens were 1273, 1244 and 1392 h × ng/mL, respectively. All limits of the 90% CIs of the estimated geometric mean ratios for Cmax and all AUC comparisons fell within the 80%-125% margins. These results indicate that consumption of a high- or low-fat meal within 30 min prior to administration of ponatinib had no effect on the single-dose pharmacokinetics of ponatinib. WHAT IS NEW AND CONCLUSION: Food does not affect the single-dose pharmacokinetics of ponatinib. These data demonstrate that ponatinib may be administered with or without food.

Clearance of Subarachnoid Hemorrhage from the Cerebrospinal Fluid in Computational and In Vitro Models.

Subarachnoid hemorrhage (SAH) mostly occurs following the rupture of cerebral aneurysm causing blood to leak into the cranial subarachnoid space (SAS). Hemorrhage volume has been linked to the development of secondary vasospasm. Therefore, eliminating blood contaminants from the cerebrospinal fluid (CSF) space after the initial hemorrhage could improve patient outcomes and prevent the development of vasospasm. A number of clinical trials demonstrate that lumbar drainage effectively clears hemorrhagic debris from the cranial compartment. The benefits of optimal lumbar drainage rate and patient orientation are difficult to determine by trial-and-error in live patients, because of the invasive nature, limited subject availability and ethical considerations. Therefore, there is a lack of consensus about clinical guidelines for the use of continuous lumbar drainage following the ictus of SAH. A realistic bench-top model which reproduces the anatomy and CSF dynamics of the human central nervous system (CNS) was built to experimentally study contaminant clearance scenarios under lumbar drainage. To mimic a hemorrhagic event, porcine blood was injected at the basal cistern level of the bench-top model and the efficacy of lumbar drains was assessed experimentally for different drainage rates and patient orientations. In addition, the efficacy of blood clearance was predicted with a computational fluid dynamics (CFD) model. Bench-top experiments and CFD simulations identify body position and drainage rates as key parameters for effective blood clearance. The study findings suggest the importance of treatment in upright position to maximize contaminant diversion from the cranial CSF compartment. The bench-top CNS model together with the validated CFD predictions of lumbar drainage systems can serve to optimize subject-specific treatment options for SAH patients.

Induction of human monocyte motility by lysyl oxidase.

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10(-10) M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with beta-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant response to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.

Standardized surveillance of hemodialysis vascular access infections: 18-month experience at an outpatient, multifacility hemodialysis center.

OBJECTIVE: To develop a standardized surveillance system for monitoring hemodialysis vascular-access infections in order to compare infection rates between outpatient sites and to assess the effectiveness of infection control interventions. DESIGN: Prospective descriptive analysis of incidence infection rates. SETTING: An outpatient hemodialysis center with facilities in Idaho and Oregon. PATIENTS: All outpatients receiving chronic outpatient hemodialysis. RESULTS: There were 38,096 hemodialysis sessions (31,603 via permanent fistulae or grafts, 5,060 via permanent tunneled central catheters, and 1,433 via temporary catheters) during an 18-month study period in 1997 to 1998. We identified 176 total infections, for a rate of 4.62/1,000 dialysis sessions (ds). Of the 176, 80 involved permanent fistulae or grafts (2.53/1,000 ds), 69 involved permanent tunneled central catheter infections (13.64/1,000 ds), and 27 involved temporary catheter infections (18.84/1,000 ds). There were 35 blood-stream infections (0.92/1,000 ds) and 10 episodes of clinical sepsis (0.26 /1,000 ds). One hundred thirty-one vascular-site infections without bacteremia were identified (3.44/1,000 ds), including 65 permanent fistulae or graft infections (2.06/1,000 ds), 42 permanent tunneled central catheter infections (8.3/1,000 ds), and 24 temporary catheter infections (16.75/1,000 ds). CONCLUSIONS: Infection rates were highest among temporary catheters and lowest among permanent native arteriovenous fistulae or synthetic grafts. This represents the first report of extensive incidence data on hemodialysis vascular access infections and represents a standardized surveillance and data-collection system that could be implemented in hemodialysis facilities to allow for reliable data comparison and benchmarking.

Transmembrane pressures generated by filtrate line suction maneuvers and predilution fluid replacement during in vitro continuous arteriovenous hemofiltration.

A recirculating in vitro CAVH system was designed which generated pulsatile blood and filtrate flows. Monitors recorded hydrostatic pressures simultaneously in the arterial, venous and filtrate lines during varying plasma or blood flow rates and predilution (vs postdilution) replacement fluid flow rates. Similar hydrostatic pressure monitoring was carried out during multiple maneuvers to generate suction on the filtrate side of the hemofilter (Amicon D-20's and Renaflo's). With a plasma flow (Qp) of 100 cc/min and predilution replacement fluid infusion rate of 500 cc/hr, the arterial pressure was 5% greater than during postdilution (p less than 0.05). With a blood flow (Qb) of 50 cc/min, predilution fluid replacement rates of 500 and 1000 cc/hr, and vacuum suction applied to the filtrate compartment, the arterial pressure was 33% lower than during postdilution fluid replacement (p less than 0.03). Nonetheless, the ultrafiltration rate (UFR) was 10 to 30% higher (p less than 0.03). At many other combinations of Qp, Qb and replacement rates and modes, there were no significant changes in arterial pressure. Despite these arterial pressure changes, greater than 70% of the transmembrane hydrostatic pressure (TMP) was due to the negative pressure induced by filtrate suction (gravity, Gomco, wall suction, IMED). The actual pressure in the filtrate compartment measured during Gomco or wall suction was 3/4 of that stated by their gauges, presumably due to leakage. Maximum wall suction never generated TMP's greater than 150 mmHg.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA.

The ATP concentrations allowing transcription of both heavy- and light-strand of human mtDNA in a HeLa cell mitochondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p[NH]ppA), an ATP analog with a nonhydrolyzable beta-gamma bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous ATP. Evidence was obtained indicating that p[NH]ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process.

Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination.

In a transcription system using a HeLa cell mitochondrial lysate programmed by mitochondrial DNA constructs containing the main heavy strand promoter and the transcription termination site at the 16S rRNA/leucyl-tRNA boundary, an appreciable fraction of the heavy strand transcripts terminates at this site, as it does in vivo. A DNA binding protein(s) that protects a 28 bp region immediately adjacent and downstream of the 3' ends of the in vivo and in vitro transcripts has been identified in the lysate and highly purified on an oligodeoxynucleotide affinity column. An activity promoting specific termination of heavy strand transcripts copurified with the DNA binding protein(s), pointing to the involvement of this protein in transcription termination. A distinctive modification of the footprint not correlated with terminating activity has also been observed.

Stereoselective metabolism and pharmacokinetics of tegafur.

OBJECTIVES: UFT is composed of racemic tegafur (FT), a prodrug of 5-fluorouracil (5-FU), and uracil in a fixed molar combination (1:4). FT contains a chiral center and has two stereoisomers, R-FT and S-FT. The objectives of this study were to assess the stereoselectivity in the metabolism of FT to 5-FU in vitro and to determine stereoselective differences in the disposition of FT in vivo. METHODOLOGY: R-FT, S-FT, and racemic FT were incubated with pooled human liver microsomes and S-9 fraction for a period of up to 30 min for in vitro studies. For pharmacokinetics, plasma samples were obtained from fasted cancer patients over a period of 24 h after oral administration of 200 mg UFT. Samples from in vitro studies and patient plasma samples were analyzed for FT using a validated achiral and a chiral assay, and for 5-FU using a validated GC/MS assay. RESULTS: R-FT was metabolized at a rate 5.6-times faster than S-FT by human liver microsomes. Similarly, stereoselective metabolism of R-FT was also seen in the S-9 incubations. In cancer patients, the peak plasma concentrations (C(max)) and the time to reach C(max) (T(max)) were similar for the two isomers after the administration of UFT suggesting no apparent differences in their absorption kinetics. However, the area under the curve from zero to infinity [AUC(INF)] and the terminal elimination half-life (T-HALF) values for R-FT were about 4.6- and 4.4-fold lower compared to S-FT, respectively, suggesting the preferential elimination of R-FT. The T-HALF of racemic FT (8.3 h) was comparable to the T-HALF of S-FT (10.3 h) which indicated that the kinetics of the racemate are governed by S-FT. The active cytotoxic moiety, 5-FU, exhibited formation rate limited kinetics from R-FT because the T-HALF of 5-FU (3.4 h) was similar to that of R-FT (2.4 h). CONCLUSIONS: The R-isomer of FT is preferentially metabolized to 5-FU compared to the S-isomer in vitro. The distinct kinetic profiles of the stereoisomers of FT following the administration of UFT is apparently due to the stereoselective disposition of the R-isomer relative to the S-isomer. These data suggest that the R-isomer of FT is worthy of further preclinical and clinical evaluation for safety, efficacy, and pharmacokinetics.

Effects of chemical and enzymic probes on microsomal covalent binding of bromobenzene and derivatives. Evidence for quinones as reactive metabolites.

1. The chemical reactivity of bromobenzene metabolite(s) responsible for its protein covalent binding was investigated by determining the effects of many chemical and enzymic probes on the metabolism and covalent binding of [3,5-3H]bromobenzene with rat liver microsomes in vitro. 2. Classical cytochrome P-450 enzyme inhibitors decreased both metabolism and binding in parallel, whereas scavenging agents for reactive oxygen species and free radicals exhibited little or no effect. Sulphur nucleophiles were extremely efficient in decreasing binding with little or no effect on metabolism. Reducing agents such as ascorbate and diaphorase decreased binding slightly more than metabolism. 3. UDP-Glucuronic acid inhibited neither metabolism nor binding, but all three mono-bromophenols decreased binding more than metabolism. Trichloropropene oxide was unique in decreasing metabolism more than binding. 4. The effects of ascorbate, glutathione, bisulphite and butylated hydroxytoluene (BHT) on metabolism and binding of five ortho-substituted bromobenzene derivatives (o-BrC6H4X; X = OCH3, CH3, Br, CF3, and CN) were similar to their effects on the metabolism and binding of bromobenzene. 5. Collectively these results support a major role for quinones as the reactive metabolites responsible for the majority of the protein covalent binding of bromobenzene and its ortho-substituted derivatives in microsomal systems in vitro.

Two large clusters with thirty-seven transfer RNA genes adjacent to ribosomal RNA gene sets in Bacillus subtilis. Sequence and organization of trrnD and trrnE gene clusters.

Sequences of two large tRNA gene clusters (trrnD and trrnE) in Bacillus subtilis 168 revealed 16 and 21 tRNA genes, respectively, as identified by anticodon assignments. Each cluster contains upstream flanking 23 and 5 S rRNA sequences. The 23-5 S intergenic space in trrnE corresponds exactly to the analogous space in trrnB, which was previously sequenced (Wawrousek, E.F., and Hansen, J.N. (1983) J.Biol. Chem. 258, 291-298). The 5 S rRNA genes in trrnB and trrnE are B. subtilis major species; but trrnD possesses a minor species (Raue, H.A., and Planta, R. J. (1977) Mol. Gen. Genet. 156, 185-193) gene with a putative promoter that may allow differential expression with respect to the upstream rRNA gene set. Most of the tRNA genes are probably expressed as large transcriptional units, except for a LeuTTG tRNA in the trrnD cluster that appears to constitute its own operon with putative promoter and terminator sequences. Although all the amino acids are represented among the tRNA anticodons, there are few repeats of amino acid types within clusters; trrnD with 16 tRNA genes has anticodons corresponding to 15 amino acids. About two-thirds of the tRNA genes encode a 3'-terminal-CCA, and these are intermingled with those that do not, with no apparent pattern.

Catalytic properties and structural components of lysyl oxidase.

Key aspects of the biosynthesis and catalytic specificity of lysyl oxidase (LO) have been explored. Oxidation of peptidyl lysine in synthetic oligopeptides is markedly sensitive to the presence of vicinal dicarboxylic ami/no acid residues. Optimal activity is obtained with the -Glu-Lys- sequence within a polyglycine 11-mer, whereas the -Lys-Glu- sequence is much less efficiently oxidized. The -Asp-Glu-Lys- sequence is a very poor substrate, although this sequence is oxidized in type I collagen fibrils. These results are considered in the light of a model requiring collagen to be assembled as fibrils prior to oxidation by LO. An in vitro system for the expression of catalytically active LO has been devised. Deletion or inclusion of the cDNA coding for the propeptide region in the expressed construct results in apparently identical, catalytically active enzyme products, indicating the lack of essentiality of this region for active enzyme production. These effects are considered with respect to the conservation of the amino acid sequence of LO produced by different species.

Clinical characteristics and diagnostic considerations in acquired renal cystic disease.

Acquired multiple bilateral cystic transformation of kidneys has been increasingly noted in patients with long-standing renal failure treated by chronic dialysis. To study the clinical characteristics of this newly described disease and assess the utility of available diagnostic methods, 130 patients with chronic renal failure (100 on dialysis, 30 nondialyzed) were studied with ultrasonography and/or computerized tomography (CT). Among patients on dialysis, 22% had acquired renal cystic disease (ARCD), an additional 30% had one to three solitary cysts, and 48% had no cysts. In nondialyzed patients, 7% had ARCD, 53% had one to three solitary cysts, and 40% had no cysts. Among these 130 chronic renal failure patients (nondialyzed and dialyzed), 21 of 86 males compared to 1 of 44 females had ARCD (P less than 0.001). Duration of dialysis therapy and age were greater in patients with ARCD (49.8 +/- 8 months, 55 +/- 4 years, respectively) compared to those with solitary cysts (28 +/- 6 months, 45 +/- 2 years) or no cysts (15 +/- 3 months, 42 +/- 2 years). The diagnostic accuracy of ultrasound (US) was compared to CT. CT is purportedly 100% accurate in the characterization of renal cysts. We are disappointed at the low level of diagnostic accuracy for both CT and US in the detection of renal cysts in chronic uremia. It appears both a negative CT and ultrasound are necessary to absolutely exclude either ARCD or solitary cyst.

Insect antifeedant and growth-regulating activities of Salannin and other c-seco limonoids from neem oil in relation to Azadirachtin.

The antifeedant and insect growth-regulating activities of salannin, nimbin, and 6-deacetylnimbin, in comparison with azadirachtin-A, have been studied againstSpodoptera litura, Pericallia ricini, andOxya fuscovittata. Salannin deterred feeding, delayed molt by increasing larval duration, caused larval and pupal mortalities, and decreased pupal weights in the two lepidopterans. Salannin also caused molt delays and nymphal mortalities inOxya fuscovittata. The role of salannin and other compounds in conferring bioactivity, along with azadirachtin-A, to neem oil/neem seed extracts is emphasized.

In vitro metabolism and covalent binding among ortho-substituted bromobenzenes of varying hepatotoxicity.

A series of ortho-substituted bromobenzene derivatives with widely differing hepatotoxicities were investigated for their tendency to undergo oxidative metabolism and protein covalent binding in the presence of rat liver microsomes in vitro. Compounds studied included o-bromobenzonitrile, o-dibromobenzene, bromobenzene, o-bromoanisole, and o-bromotoluene (names in order of decreasing hepatotoxicity and increasing rate of in vitro metabolism). No correlation was found between net covalent binding and toxicity. However, a good rank order correlation was observed between toxicity and the relative binding index of each compounds, defined as (picomoles covalently bound/nmol metabolized), with values ranging from a low of 7 with o-bromotoluene to a high of 365 with o-bromobenzonitrile, Extensive side chain metabolism was observed with o-bromotoluene (92-95%) and o-bromoanisole (26-42%), which accounts for their high rate of total metabolism and low relative binding index. The extent of tritium loss, relative to C-14, was assessed for each compound as an index of the "average oxidation state" of those metabolites, presumably epoxides and/or quinones, which covalently bound to protein. Tritium retention ranged from a high of 84% with o-bromobenzonitrile to a low of 21% with o-bromoanisole, and generally paralleled toxicity. These results show that introduction of ortho-substituents onto bromobenzene leads to qualitative and quantitative changes in overall oxidative metabolism, as well as important qualitative changes in the nature of reactive metabolites formed. Nevertheless, the relative binding index computed for each compound appears to reconcile these changes to a large degree, giving an in vitro index which correlates well with toxicity.

Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. II. Unique properties of an operon containing a minor 5 S rRNA gene.

This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes. The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J. (1988) J. Biol. Chem. 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B. subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA. Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B. subtilis 168 genome. S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter. No promoters have been found immediately 5' to any of the major 5 S rRNA species in B. subtilis rRNA operons. S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism. S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon. A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation. A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs. Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter. However, the last tRNA gene is primarily under the control of its own unique promoter.

Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites.

1. The metabolism and covalent binding of [3H/14C]bromobenzene has been investigated using liver microsomes from untreated and phenobarbital (PB)-pretreated rats. A model has been developed to relate the observed 3H/14C ratios in the covalently bound residues to the type of metabolite (epoxide versus quinone) responsible for their formation. 2. With control microsomes metabolism was linear for 60 minutes, but with PB microsomes the time course showed a short-lived burst of rapid metabolism followed by a long phase with an overall rate comparable to control. With both types of microsomes covalent binding was synchronous with metabolism. 3. The normalized 3H/14C ratios of recovered substrate and water-soluble metabolites was 1.0, whereas that of the covalently bound material was only 0.5. Such extensive loss of tritium implies that a considerable portion of the covalent binding arises from bromobenzene metabolites more highly oxidized than an epoxide (e.g. quinones). 4. The normalized 3H/14C ratios for bromobenzene metabolites covalently bound to liver proteins in vivo (total and microsomal) was the same as with microsomes in vitro (0.5). However, for the lung and kidney the 3H/14C ratios were considerably higher (0.71 and 0.62), indicating that differences between tissues in vivo may be greater than between liver microsomes in vitro and in vivo.