Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

PURPOSE: A reference reagent, 3-(trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium (TSP), has been used frequently in nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells. MATERIALS AND METHODS: A human osteosarcoma cell line (MG-63) was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM) of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding. RESULTS: In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation) and cell viability. High concentrations of TSP (from 10 to 30 mM) reduced both cell proliferation and viability (to 30% of the control after one week of exposure), but no such effects were found using low concentrations of TSP (0-10 mM). CONCLUSIONS: This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

Increased mortality in groups of cattle administered the ß-adrenergic agonists ractopamine hydrochloride and zilpaterol hydrochloride.

The United States Food and Drug Administration (FDA) approved two ß-adrenergic agonists (ßAA) for in-feed administration to cattle fed in confinement for human consumption. Anecdotal reports have generated concern that administration of ßAA might be associated with an increased incidence of cattle deaths. Our objectives, therefore, were to a) quantify the association between ßAA administration and mortality in feedlot cattle, and b) explore those variables that may confound or modify this association. Three datasets were acquired for analysis: one included information from randomized and controlled clinical trials of the ßAA ractopamine hydrochloride, while the other two were observational data on zilpaterol hydrochloride administration to large numbers of cattle housed, fed, and cared for using routine commercial production practices in the U.S. Various population and time at-risk models were developed to explore potential ßAA relationships with mortality, as well as the extent of confounding and effect modification. Measures of effect were relatively consistent across datasets and models in that the cumulative risk and incidence rate of death was 75 to 90% greater in animals administered the ßAA compared to contemporaneous controls. During the exposure period, 40 to 50% of deaths among groups administered the ßAA were attributed to administration of the drug. None of the available covariates meaningfully confounded the relationship between ßAA and increased mortality. Only month of slaughter, presumably a proxy for climate, consistently modified the effect in that the biological association was generally greatest during the warmer months of the year. While death is a rare event in feedlot cattle, the data reported herein provide compelling evidence that mortality is nevertheless increased in response to administration of FDA-approved ßAA and represents a heretofore unquantified adverse drug event.

Phase I study of TAC-101, an oral synthetic retinoid, in Japanese patients with advanced hepatocellular carcinoma.

Preclinical models have shown that TAC-101 (4-[3,5-bis(trimethylsilyl) benzamide] benzoic acid), an oral synthetic retinoid, has antitumor activity in hepatocellular carcinoma (HCC). We conducted a phase I study in Japanese patients with advanced HCC to examine the pharmacokinetics, recommended dose, safety, and efficacy of TAC-101. The administered dose of TAC-101 was 10 mg/day in four patients (level 1), 20 mg/day in six (level 2), and 30 mg/day in three (level 3). There was no dose-limiting toxicity at level 1. Only one patient each had dose-limiting toxicity at level 2 (grade 2 fatigue, recovery requiring eight or more consecutive days of rest) and at level 3 (grade 3 splenic vein thrombosis). Level 3 (30 mg/day) was considered the maximum tolerated dose and 20 mg/day the recommended dose by a panel of medical experts, placing maximum emphasis on safety. The most frequent adverse events were fatigue, headache, and dermal symptoms such as rash. Pharmacokinetic parameters in Japanese patients with HCC were similar to those in patients in the United States, most of whom were Caucasian. Although no patient had a complete or partial response, the disease control rate was 38.5%. In conclusion, the recommended dose of TAC-101 for patients with HCC is 20 mg/day. TAC-101 had an acceptable toxicity profile, warranting further evaluation in clinical trials.

The efficiency of electrical stimulation to counteract the negative effects of ß-agonists on meat tenderness of feedlot cattle.

Beta agonists used as growth enhancers are known to affect the aging potential of beef muscle negatively. On the other hand, procedures like electrical stimulation could accelerate rigor and the aging process. In this study, 20 out of 40 young steers received no beta agonist (C), the remaining twenty steers received a beta agonist (zilpaterol hydrochloride) (Z) for the 30 days prior to slaughter followed by 4 days withdrawal. After slaughter carcasses were split, the left side electrically stimulated (ES) and the right side not stimulated (NES). Samples were aged for 3 or 14 days post mortem. Parameters included Warner Bratzler shear force (WBSF), myofibril filament length (MFL), sarcomere length and calpastatin and calpain enzyme activity. Zilpaterol resulted in increased (P<0.001) WBSF mainly due to an increased (P<0.001) calpastatin activity. ES improved tenderness (P<0.001) in general by early onset of rigor triggering the activity of calpains. ES also reduced the calpastatin activity (P<0.001), which partially countered the effect of high calpastatin activity on the aging potential of Z loins. ES can therefore be implemented to improve meat tenderness in zilpaterol supplemented steers, although steers without zilpaterol will still have an advantage in final tenderness.

[Establishment of an animal model with thymic lymphoma in mice].

The objective of study was to establish an animal model with thymic lymphoma in mice induced by intraperitoneal injection of DNA alkylating agent N-methyl-N-nitrosourea (MNU). Male and female mice of the C57BL/6 strain were injected by the intraperitoneal route with MNU solution in a dosage of 50 mg/kg body weight. The injection was repeated at week 8. Following injection of MNU, the general status of mice was observed. All mice were sacrificed for autopsy at the 22nd experimental week. Complete gross examination was performed for detection of tumor masses. The results showed that at the 22nd week, the incidence of thymic lymphoma in MNU-treated animals was 67.5% (27/40). No significant sex difference in the incidence of thymic lymphoma was observed. In conclusions, an animal model with thymic lymphoma in mice can be established by twice intraperitoneal administration of MNU. The biological behavior of the induced tumors resembles to those of human thymic lymphoma derived from thymic T-cells.

Effects of dietary zilpaterol hydrochloride on feedlot performance and carcass characteristics of beef steers fed with and without monensin and tylosin.

A feedlot experiment was conducted under commercial conditions in the Texas Panhandle using 3,757 feedlot steers (average of 94 steers/pen) to evaluate the effects of feeding zilpaterol hydrochloride with or without monensin and tylosin on feedlot performance and carcass characteristics. The experiment was conducted using a randomized complete block design. Treatments were arranged as a 2 (no zilpaterol vs. zilpaterol) x 2 (monensin and tylosin withdrawn vs. monensin and tylosin fed during the final 35 d on feed) factorial. Steers were fed for a total of 161 to 167 d, and treatments were administered during the final 35 d that cattle were on feed. When included in the diet, zilpaterol, monensin, and tylosin were supplemented at 8.3, 33.1, and 12.2 mg/kg (DM basis), respectively. Zilpaterol was included in the diet for 30 d at the end of the finishing period and withdrawn from the diet for the last 5 or 6 d cattle were on feed. Cattle were harvested and carcass data collected. There were no zilpaterol x monensin/tylosin interactions (P >or= 0.12) for ADG or G:F. Feeding zilpaterol increased ADG (P < 0.001) by 0.20 kg and G:F (P < 0.001) by 0.029 kg/kg during the last 35 d on feed. Likewise, when feedlot variables were measured throughout the entire 161- to 167-d feeding trial, ADG (3.4%; P < 0.001) and G:F (3.9%; P < 0.001) were increased. Feeding zilpaterol increased (P < 0.001) dressing percent and HCW and decreased (P < 0.001) total liver abscess rate compared with controls. In addition, zilpaterol increased (P < 0.001) LM area by an average of 8.0 cm(2). There was a zilpaterol x monensin/tylosin interaction (P = 0.03) for marbling score. Zilpaterol decreased (P < 0.001) marbling score regardless of monensin and tylosin treatment, although withdrawal of monensin and tylosin for 35 d decreased marbling to a greater extent (31 vs. 17 degrees). Zilpaterol decreased (i.e., improved; P < 0.001) calculated yield grade regardless of monensin and tylosin treatment, but feeding zilpaterol in combination with the withdrawal of monensin and tylosin for 35 d decreased calculated yield grade to a greater extent (0.49 vs. 0.29) compared with the zilpaterol, monensin, and tylosin combination treatment (zilpaterol x monensin/tylosin interaction, P = 0.03). Results suggest that monensin and tylosin can be withdrawn from the diet during the zilpaterol feeding period (final 35 d on feed) with minimal effect on animal performance, although feeding zilpaterol in combination with monensin and tylosin seemed to moderate effects on carcass quality.

Initial clinical trial of oral TAC-101, a novel retinoic acid receptor-alpha selective retinoid, in patients with advanced cancer.

PURPOSE: The goals of this study were to determine the safety, toxicity, and pharmacokinetics of TAC-101, a novel synthetic retinoic acid receptor-alpha (RAR-alpha) selective retinoid, in patients with advanced cancer. PATIENTS AND METHODS: Twenty-nine patients at two centers received oral TAC-101 at doses ranging from 12 to 34 mg/m(2)/d. Pharmacokinetic sampling was performed on days 1 and 28. RESULTS: The most frequent toxicities were myalgia/arthralgia, fatigue, and triglyceridemia. No dose-limiting toxicities were observed within the first 28 days up to 28 mg/m(2). However, seven of 21 patients experienced venous thromboembolic events (VTEs) during TAC-101 treatment. Eight additional patients who received 34 mg/m(2) were treated after a hypercoagulable work-up to exclude potential risk factors for VTE, and two of eight patients subsequently experienced VTEs. The maximum tolerated dose was exceeded at 34 mg/m(2)/d within the first 28 days, with one grade 3 hypertriglyceridemia, two grade 3 myalgia/arthralgia, and one grade 3 fatigue. One patient with advanced non-small-cell lung cancer had a complete response. No other responses were observed. No autoinduction of metabolism was observed with dosing over 28 days. CONCLUSION: This is the first human clinical study with TAC-101, a RAR-alpha selective retinoid. Musculoskeletal toxicity and hypertriglyceridemia were observed characteristics of previously studied retinoids. The recommended phase II dose is 24 mg/m(2) with this treatment schedule. Alternative treatment schedules and prospective evaluation of thrombotic risk will be investigated in subsequent studies.

Acetylcholinesterase inhibition by zifrosilone: pharmacokinetics and pharmacodynamics.

OBJECTIVE: To determine the pharmacokinetics, pharmacodynamics and safety of the acetylcholinesterase inhibitor zifrosilone in healthy male volunteers. METHODS: Pharmacokinetics, pharmacodynamics, and tolerance of zifrosilone were studied in a double-blind, sequential, single-escalating-dose, randomized panel design. Each panel consisted of six subjects, with four subjects receiving zifrosilone (10, 30, 60, 90, 120, 150, 200, 250, and 300 mg orally) and two subjects receiving matching placebo. Serial blood samples were obtained for zifrosilone plasma concentrations and red blood cell acetylcholinesterase and butyrylcholinesterase activities. Participating subjects (n = 54) were men between the ages of 18 and 45 years. Each subject had a normal physical examination, electrocardiogram, serum chemistries, hematology, urinalysis, and test for human immunodeficiency virus at screening. RESULTS: A greater than proportionate increase in mean plasma concentration values for area under the curve from time zero to infinity was observed over the 200 to 300 mg dose range groups. Red blood cell acetylcholinesterase showed a dose-inhibition relationship, with a mean maximum inhibition of 20.9% at 10 mg that increased to 62.1% at 300 mg. Butyrylcholinesterase activity was relatively unaffected by zifrosilone (< 20% inhibition at 300 mg). For doses > or = 200 mg, an Emax pharmacodynamic model was used to describe the relationship between zifrosilone plasma concentration and red blood cell acetylcholinesterase inhibition (Emax = 83.8%; EC50 = 0.65 ng/ml). CONCLUSIONS: Zifrosilone showed dose-dependent pharmacokinetics after oral administration and was effective in causing selective inhibition of red blood cell acetylcholinesterase.

[Combination chemotherapy of multiple myeloma--BCNU.cyclophosphamide.procarbazine.prednisolone and MCNU.cyclophosphamide.procarbazine.prednisolone therapy].

Forty-four patients with progressive symptomatic multiple myeloma were treated with a protocol combining cyclophosphamide (10 mg/kg weekly), procarbazine (2 mg/kg daily), prednisolone (20 mg daily), and either BCNU (1 mg/kg weekly) (BCPP protocol) or MCNU (0.8-1 mg/kg) (MCPP protocol). Of these, 34 patients were treated with the former, and 10 patients with the latter. With BCPP protocol, 12 achieved a complete response, 11 evidenced a 75% response, and 7 displayed a 50% response. With MCPP protocol, on the other hand 2 achieved a complete response, 6 showed a 75% response, and 1 exhibited a 50% response. The median tumor halving time in both groups was 77 days and 57 days respectively. The 5-yr disease-free survival of patients treated with BCPP protocol was 62.0 +/- 10.8%. In MCPP group, 8 of 10 patients are alive with more than 49-87 weeks survival. Although myelotoxicity was moderate, other toxicities were moderate. Toxicity requiring dose modification and discontinuation of the scheduled therapy was observed.