Association between the nuclear to cytoplasmic ratio of p27 and the efficacy of adjuvant polychemotherapy in early breast cancer.

BACKGROUND: The purpose of this study was to evaluate the prognostic and predictive value of p27 expression in patients with early breast cancer. PATIENTS AND METHODS: Quantitative immunofluorescence assays for p27 were done on a tissue microarray that included 823 samples from patients randomized between anthracycline-based chemotherapy and no chemotherapy. Quantification of p27 was done using the AQUA® system (HistoRx, Inc., Branford, CT). Both p27 nuclear expression and the nuclear to cytoplasmic ratio were assessed. RESULTS: Nuclear p27 expression was not predictive for the efficacy of anthracycline-based chemotherapy [adjusted P=0.18 for disease-free survival (DFS)] nor prognostic [95% confidence interval (CI) 0.99-1.01, P=0.49]. However, p27 nuclear/cytoplasmic ratio was predictive for the efficacy of adjuvant chemotherapy (adjusted P=0.016 DFS). The adjusted hazard ratio (HR) for relapse associated with adjuvant chemotherapy was 0.56 (95% CI 0.37-0.84, P=0.005) and 1.06 (95% CI 0.76-1.47, P=0.74) for patients with high and low nuclear/cytoplasmic ratio, respectively. p27 N/C ratio was prognostic in patients treated with chemotherapy (HR for relapse or death for a 1 unit increase in p27 N/C ratio was 0.30, 95% CI 0.12-0.77) but not in the untreated arm (HR for relapse or death was 1.27, 95% CI 0.58-2.8). CONCLUSIONS: This study did not confirm the role of p27 nuclear expression as a prognostic parameter. However, the p27 nuclear/cytoplasmic ratio was predictive in patients treated with anthracycline-based chemotherapy.

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein.

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.

Determination of bis(tributyltin) oxide by GC--MS with on-line hydride derivatization: application to drug substance analysis.

We report the determination of residual bis(tributyltin) oxide in a drug substance by GC-MS after extraction and on-line conversion to tributyltin hydride. Gas chromatography was performed using a 15 m x 0.25 mm i.d. DB-5 HT column with a temperature program from 100 to 160 degrees C at 15 degrees C min(-1). A mass range of 165-185 amu was monitored with the MS detector. Hydride generation is performed by placing a small amount of solid sodium borohydride in the injection port of a gas chromatograph and injecting samples and standards through this material. Conversion to tributyltin hydride is shown to be quantitative and linear for levels of bis(tributyltin) oxide between 1 and 100 ppm in the drug substance. The use of GC-MS provides sensitive and selective detection of tin containing species and the tin isotope pattern allows for confirmation of the presence of tin in chromatographic peaks. Recovery at 6 ppm was 89% with an injection precision of 6%. The limit of detection for bis(tributyltin) oxide in drug substance is 1 ppm.

Mechanistic aspects of chiral discrimination on modified cellulose.

Cellulose and cellulose derivatives are biopolymers which are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, we attempt to elucidate the types of interactions occurring between a diol intermediate for a LTD(4) antagonist and a tris(4-methylbenzoate)-derivatized cellulose stationary phase. Thermodynamic studies indicate that, at low temperatures, the enantioselectivity is entropy driven. At higher temperatures, the separation is enthalpy driven. DSC and IR experiments reveal that the transitions between the enthalpic and the entropic regions of the van't Hoff plots are a result of a change in conformation of the stationary phase. Investigation of chromatographic kinetic parameters reveals that, at low temperature, the second eluted enantiomer undergoes sluggish inclusion interactions. Subtle changes in the structure of the analyte indicates that π-π interactions do not contribute to enantioselectivity. Finally, molecular modeling of (R)- and (S)-diol and the stationary phase suggests that hydrogen bonding is a primary factor in the separation, and the calculated energy values obtained from the molecular modeling correlate well with the chromatographic elution order.

Drug substance manufacture process control: application of flow injection analysis and HPLC for monitoring an enantiospecific synthesis.

Effective process control can only be achieved through an understanding of the operating issues of the reaction. The development and use of effective and rugged analytical methods is necessary to monitor these parameters. The intent of this paper is to present some key analytical issues encountered in the synthesis of MK-0679, an LTD4 antagonist. In a key step of the compound's synthesis, a prochiral diester intermediate undergoes an enantioselective enzymatic hydrolysis (in the presence of Triton X-100) leading to the (S)-ester acid. Subsequent processing transforms the ester acid into the final product. The residual amount of the detergent in the final product, the rapid determination of the enzymatic activity and the optical purity of the final product emerged as key issues in the control of the reaction. As a solution, two techniques were utilized and are presented: flow injection analysis and HPLC.