Thirteen week toxicity study of dietary l‐tryptophan in rats with a recovery period of 5 weeks

Although l ‐tryptophan is nutritionally important and widely used in medical applications, toxicity data for its oral administration are limited. The purpose of this study was to evaluate the potential toxicity of an experimental diet containing added l ‐tryptophan at doses of 0 (basal diet), 1.25%, 2.5% and 5.0% when administered to Sprague–Dawley rats for 13 weeks. There were no toxicological changes in clinical signs, ophthalmology, urinalysis, hematology, necropsy, organ weight and histopathology between control rats and those fed additional l ‐tryptophan. Body weight gain and food consumption significantly decreased throughout the administration period in males in the 2.5% group and in both sexes in the 5.0% group. At the end of the dosing period, decreases in water intake in males in the 5.0% group and in serum glucose in females in the 5.0% group were observed. The changes described above were considered toxicologically significant; however, they were not observed after a 5 week recovery period, suggesting reversibility. Consequently, the no‐observed‐adverse‐effect level of l ‐tryptophan in the present study was 1.25% for males and 2.5% for females (mean intake of l ‐tryptophan: 779 mg kg^–1 body weight day^–1 [males] and 1765 mg kg^–1 body weight day^–1 [females]). As the basal diet used in this study contained 0.27% of proteinaceous l ‐tryptophan, the no‐observed‐adverse‐effect level of overall l ‐tryptophan was 1.52% for males and 2.77% for females (mean intake of overall l ‐tryptophan: 948 mg kg^–1 body weight day^–1 (males) and 1956 mg kg^–1 body weight day^–1 (females)). We conclude that l ‐tryptophan has a low toxicity profile in terms of human use.     

Preliminary experience with beta-tricalcium phosphate for use in mastoid cavity obliteration after mastoidectomy.

OBJECTIVE: To examine the efficacy and safety of mastoid cavity obliteration using highly purified beta-tricalcium phosphate (beta-TCP) after mastoidectomy in middle ear surgery. PATIENTS: Thirteen patients with cholesteatoma invading the mastoid cavity or showing severe pathologic changes in the mastoid cavity. INTERVENTION: Twelve patients underwent mastoid obliteration with highly purified beta-TCP during the first- and/or second-stage operation of a 2-stage canal-up operation: 5 patients during the first and second stages, and 7 patients during the second stage only. One patient with cholesteatoma underwent mastoid obliteration with highly purified beta-TCP during a 1-stage canal-up operation. In total, beta-TCP was applied in 18 ear operations. MAIN OUTCOME MEASURES: All patients underwent multislice computed tomography (CT) before and after surgery to assess the condition of the middle ear. The amount of residual beta-TCP granules in the mastoid cavity was assessed using the following granular shadow grading scale: Grade 0, no granular shadow in the mastoid cavity; Grade 1, residual granular shadows in part of the mastoid cavity; and Grade 2, granular shadows in most of the mastoid cavity. To assess any harmful effect of beta-TCP implanted in the mastoid cavity, continuous postoperative discharge and delayed wound healing were recorded. In addition, the bone conduction threshold was assessed using pure-tone audiometry, and the patients were asked whether they experienced vertigo or dizziness during the postoperative follow-up. RESULTS: All the patients who underwent multislice CT less than 11.4 months after mastoid cavity obliteration with beta-TCP were Grade 2 on the granular shadow grading scale, whereas all those who underwent multislice CT more than 53.8 months after mastoid obliteration were Grade 0. No patient had continuous postoperative discharge, delayed wound healing, or extrusion of beta-TCP granules. No patient showed deterioration of the bone conduction threshold more than 10 dB after mastoid cavity obliteration with highly purified beta-TCP or complained of postoperative vertigo or dizziness. CONCLUSION: Highly purified beta-TCP may be safe and reliable for mastoid obliteration. Highly purified beta-TCP may also be useful in other surgical procedures, including posterior wall reconstruction of the external auditory canal and scutum plasty.     

Physiologically Based Pharmacokinetic (PBPK) Modeling of Everolimus (RAD001) in Rats Involving Non-Linear Tissue Uptake

Everolimus is a novel macrolide immunosuppressant developed for the prophylaxis of allogeneic renal or cardiac transplant rejection. Treatments with immunosuppressants are often associated with organ toxicity that is linked to high organ exposure. Therefore, gaining insight into the pharmacokinetics of everolimus in various organs is highly desirable especially those organs of therapeutic interest or those that pose safety concerns. The aim of this work was to characterize the disposition kinetics of everolimus in rats by physiologically based pharmacokinetic (PBPK) modeling. Blood and tissue samples were collected from male Wistar rats over 24 hr following intravenous (iv) bolus and iv infusion of 1 mg/kg and 10 mg/kg/2 hr of everolimus. Further blood samples were collected between 1 and 170 hr from a third group of rats, which received iv infusion of 1 mg/kg/2 hr of everolimus. Drug concentrations in blood and tissues were determined by a liquid chromatography reverse dilution method. Distribution of everolimus between blood fractions was determined in vitro at 37°C. The results of the study demonstrated that everolimus exhibited moderate non-linear binding to red blood cells. Also, the tissue-to-blood concentration ratio decreased in all tissues as blood concentration increased. A PBPK model involving non-linear tissue binding was able to successfully describe the observed data in blood and all the organs investigated. The highest binding potential was observed in thymus, lungs, and spleen with the greatest tissue affinity observed in thymus, skin, and muscle as compared to other tissues. Everolimus exhibited a high clearance rate that was limited to the hepatic blood flow (47.2 ml/min/kg). The PBPK model was also able to predict the venous blood concentration reasonably well following oral administration. The oral bioavailability value, as estimated with the PBPK, was 12% and was similar to the value obtained by non-compartmental analysis. In conclusion, A PBPK model has been developed that successfully predicts the time course of everolimus in blood and a variety of organs. This model takes into account the non- linear binding of everolimus to red blood cells and tissues. This model may be used to predict everolimus concentration–time course in organs from other species including humans.     

Excess dietary L-cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs

A comparative species investigation of the relative pharmacologic effects of sulfur amino acids was conducted using young chicks, rats, and pigs. Ingestion of excess Met, Cys, or Cys-Cys supplemented at 2.5-, 5.0-, 7.5-, or 10 times the dietary requirement in a corn-soybean meal diet depressed chick growth to varying degrees. Strikingly, ingestion of excess Cys at 30 g/kg Cys (7.5-times the dietary requirement) caused a chick mortality rate of 50% after only 5 d of feeding. Growth was restored and chick mortality was reduced by supplementing diets containing 25 g/kg excess Cys with KHCO3 at 10 g/kg. Additionally, mortality was prevented by supplementing the drinking water of chicks receiving 25 g/kg supplemental Cys with H2O2 (0.05% final concentration). After young rats and pigs consumed excess Cys or Cys-Cys up to 40 g/kg for 14 d, weight gain was severely depressed, but we observed no mortality. An excess of dietary Cys-Cys>or=48 g/kg caused some mortality in rats. Pigs exhibited rapid recovery from growth-depressing excesses of Cys or Cys-Cys. These results lend credence to the acute toxic effects associated with the ingestion of excess sulfur amino acids and highlight the potential for excess dietary cyst(e)ine to be more pernicious than Met in certain species.     

Identification of genes encoding glycosyltransferases involved in lipopolysaccharide synthesis in Porphyromonas gingivalis

Porphyromonas gingivalis can synthesize both A‐LPS and O‐LPS lipopolysaccharides, which contain anionic O‐polysaccharides and conventional O‐polysaccharides, respectively. A‐LPS can anchor virulence proteins to the cell surface, so elucidating the mechanism of A‐LPS synthesis is important for understanding the pathogenicity of this bacterium. To identify the genes involved in LPS synthesis, we focused on uncharacterized genes encoding the glycosyltransferases, PGN_0361, PGN_1239, PGN_1240 and PGN_1668, which were tentatively named gtfC, gtfD, gtfE and gtfF, respectively, and characterized their mutants. We found that disruption of gtfC and gtfF resulted in A‐LPS deficiency. In addition, a gtfD mutant had abnormal A‐LPS synthesis, and a gtfE mutant exhibited a rough‐type LPS that possesses a short oligosaccharide with lipid A‐core. We then constructed a gtfC and gtfD double mutant, because their amino acid sequences were very similar, and this mutant similarly possessed a rough‐type LPS. Cross‐complementation analysis revealed that the GtfD protein is a functional homologue of the Escherichia coli WbbL protein, which is a rhamnosyltransferase. These results suggested that the GtfE protein is essential for the synthesis of both O‐LPS and A‐LPS, and that GtfC and GtfD proteins may work together to synthesize the two kinds of LPS. In addition, the GtfF protein was essential for A‐LPS synthesis, although this may be achieved in a strain‐specific manner.     

Homozygous familial hypercholesterolemia in Japan

Fifty-one homozygous patients with familial hypercholesterolemia, including our six patients, are described in this paper. Twenty were men and 31 were women. Their ages ranged between two and 52 years, with a mean of 16.8 years. Six patients exceeded the third decade. The mean age at death in seven patients was 17 years. The serum cholesterol levels were between 508 and 1,108 mg/dl. The mean and standard deviation (SD) of serum cholesterol were 713 ± 142 mg/dl. The serum cholesterol levels in the 35 parents (obligate heterozygotes) were between 246 and 571 mg/dl, except in one patient in whom the serum cholesterol level (936 mg/dl) was suggestive of homozygous familial hypercholesterolemia, considering the serum cholesterol level (354 mg/dl) of her heterozygous husband. The mean and SD of serum cholesterol levels of the 34 heterozygote parents were 342 ± 79 mg/dl. The mean and SD of serum cholesterol in 119 normal subjects were 187 ± 30 mg/dl. Thus, trimodai distribution was evident in the serum cholesterol levels of normal subjects, the heterozygotes and the homozygotes in Japan. The frequency of parental consanguinity was at least 33 per cent. The frequencies of ischemic heart disease in the age groups 0–9, 10–19 and above 20 years were 25 per cent (four of 16 patients), 33 per cent (six of 18 patients) and 53 per cent (nine of 17 patients), respectively. The frequency of homozygotes in Japan was in close accordance with those of Western countries. Therefore, the treatment of hypercholesterolemia and prevention of premature coronary heart disease in familial hypercholesterolemic patients are very important problems in Japan as well as in the West.     

Mechanical accuracy of a stereotactic irradiation system using a micro multi-leaf collimator

Mechanical accuracy of a stereotactic irradiation system using a micro multi-leaf collimator (mMLC), Elekta DMLC, has been evaluated. Measurements were made to obtain transmission, leakage, penumbra, and positioning accuracy of the DMLC leaf for a 6 MV photon beam. Mechanical accuracy and long term stability of a linac isocenter was also evaluated. The resulting transmission, along a line perpendicular to the leaf movement, was 0.31±0.01%, and the leakage from the closed opposing leaf pairs was 0.39±0.01%. The measured penumbra, at a depth incurring maximum dose, was 2.37±0.16 mm toward the leaf end and 2.14±0.18 mm toward the leaf side for various field sizes. The leaf gap width error, of 0.10±0.08 mm, was obtained by analyzing picket fence test results. The maximum leaf positioning error, of 0.14±0.06 mm, was obtained by analyzing the log file for a various gantry angles during an arc delivery. The isocenter accuracy was within a radius of 1 mm, without any recalibration for two years. In conclusion, our stereotactic irradiation system using DMLC was capable of providing accurate stereotactic treatment.     

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.