Determination of a novel bile acid sequestrant in rodent diet by near-infrared spectroscopy.

DMP 504 is a high molecular weight polymer currently under development by The DuPont Merck Pharmaceutical Company as a novel bile acid sequestrant to lower serum cholesterol. To assess its safety, DMP 504 is incorporated into rodent diet for oral administration to rats and mice. An analytical method was developed to determine the accuracy and homogeneity of the blends. Since a physical separation or extraction of DMP 504 from the diet was not feasible, near-infrared spectroscopy (near-IR) was employed. The near-IR method provides accurate and precise results for blends containing 1.5-8.0% of DMP 504. Comparison of results at the 1.5% level with a cholate binding referee method is also presented. Both methods provided equivalent results for the 1.5% level.

Validation of cleaning procedures for highly potent drugs. II. Bisnafide.

The objective of this work was the development and validation of procedures designed to clean glass and stainless steel surfaces after exposure to the experimental anticancer drug, bisnafide. The cleaning procedures, using 5% acetic acid water, Alconox, and water, were validated using a wipe test and an HPLC method developed to quantitate low levels of bisnafide. The procedure developed for cleaning stainless steel is more stringent than that for glass because of the apparent greater affinity of bisnafide for stainless steel. The HPLC method is shown to be linear and reproducible (RSD 4.4% or less), with a detection limit of 4 ng/ml. Recoveries of 95.1, 83.5, and 70.0% were obtained from the wipe pads, glass plates, and stainless steel plates, respectively, at levels of approximately 0.7-1.7 ng/cm2. The cleaning procedures are shown to clean glass and stainless steel plates to less than 0.19 and 0.33 ng bisnafide/cm2, respectively. These results further demonstrate the need to fully characterize the recovery of drugs from surfaces and swabs in order to properly validate cleaning procedures. In addition, they demonstrate the potential need to develop surface-specific cleaning procedures.

Validation of cleaning procedures for highly potent drugs. I. Losoxantrone.

The validation of a procedure designed to clean glass and stainless steel surfaces after exposure to the experimental anticancer drug losoxantrone is described. The cleaning procedure, using water and hypochlorite bleach, was validated using a wipe test and a high-performance liquid chromatography (HPLC) method developed to quantitate low levels of losoxantrone. The HPLC method is shown to be linear and reproducible (relative standard deviation (RSD): 7.1% or less), with a detection limit of 2 ng/ml. Recoveries of 71.0%, 50.1%, and 57.6% were reproducibly obtained from the wipe pads, glass plates, and stainless steel plates, respectively, at levels of 70-140 ng per 100 cm2. The cleaning procedure is shown to clean glass and stainless steel plates to less than 20 ng and 17 ng losoxantrone per 100 cm2, respectively. These results demonstrate the need to fully characterize the recovery of drugs from surfaces and swabs in order to properly validate cleaning procedures.

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.

A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1.

Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form.