Thermodynamic and circular dichroism studies differentiate inhibitor interactions with the stromelysin S(1)-S(3) and S(')(1)-S(')(3) subsites.

Interactions of stromelysin with a series of inhibitors representative of three chemical templates with distinct binding modes were examined. Unfolding temperatures for inhibitor complexes were 10 degrees C to 15 degrees C greater than for apo stromelysin. Minor changes in ellipticity in the far-UV CD spectra of complexes indicated that ligand-induced conformational changes were localized to the binding site and did not involve gross changes in protein folding. Isothermal titrating calorimetry of thiadiazole-containing inhibitors, which bind in the S(1)-S(3) subsites of stromelysin, indicated that the binding interaction was exothermic and only slightly favorable entropically. Near-UV CD spectra showed large positive ellipticity increases from 250 to 300 nm, consistent with an interaction between the benzene ring of the inhibitor and stromelysin residues Tyr155 and Tyr168. Interactions between stromelysin and amide-hydroxamate ligands, which bind in the S(')(1)-S(')(3) subsites, were found to be both enthalpically and entropically driven. Binding of this class of ligands resulted in modest negative ellipticity changes at 260-285 nm and positive increases at 292 nm. Stromelysin complexed to a lactam-hydroxamate inhibitor with structure extending into both the S(1)-S(3) and S(')(1)-S(')(3) subsites showed increased ellipticity at 245 nm and negative changes at 260-285 and 295 nm.

Micellar electrokinetic chromatography as a generalized alternative to high-performance liquid chromatography for purity determination of a class of investigational antibacterial drugs.

Micellar electrokinetic chromatography (MEKC) was successfully used to provide purity data for a number of oxazolidinone antibacterials. A run buffer of 100 mM SDS and 40 mM HEPES (pH 7.5, NaOH) separated fifteen different materials of neutral and cationic, anionic or zwitterionic character, usually with efficiencies ten-fold of those observed for HPLC. Different HPLC conditions were required for compounds with different structural characteristics. While the high efficiency and finite migration window of MEKC may allow observation of impurities not seen by HPLC, general use of this method for purity screening of combinatorial compounds will require micellar solutions tolerant of high amounts of organic, in order to accommodate materials of low aqueous solubility.

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids.

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.

Solution chemistry and analytical characterization of ormaplatin.

Ormaplatin is a racemic, platinum(IV), anti-cancer drug which is currently involved in phase 1 clinical trials. Characterizing the purity of ormaplatin represents an analytical challenge for several reasons. These include the lack of solution stability of ormaplatin, its process impurities and solution decomposition products, the strong concentration and pH dependence of various decomposition pathways and solution equilibria and the absence of chromatographic reproducibility. Two independent types of aqueous decomposition pathways have been observed. The first pathway involves the generation of soluble PtIV containing species which remain in equilibrium with the parent material in solution. The second pathway involves the generation of PtII containing materials which tend to be less soluble. The most interesting material in this latter class is probably the chloride bridged mixed valence material which has been characterized structurally by X-ray diffraction. Liquid chromatography and sample handling procedures have been developed which allow for the accurate determination of bulk drug purity, as well as, an understanding of numerous reversible equilibria.

Applications of a simultaneous atomic emission mass spectral gas chromatography detector in drug analysis.

For gas chromatographic eluents a microwave induced plasma (MIP) emission detector has two important features for a wide range of nonmetals. These features are (1) elemental selectivity and (2) the ability to determine elemental composition. These capabilities, used individually or in combination, can provide important information which is largely complementary to mass spectral data. Simultaneous determination of the MIP emission and mass spectral data for individual chromatographic peaks can be very useful in resolving a variety of problems encountered in pharmaceutical analysis. Several of these possible applications are illustrated with specific examples.

Paldimycins A and B and antibiotics 273a2 alpha and 273a2 beta. Synthesis and characterization.

Paldimycin (antibiotic 273a1) and antibiotic 273a2 as well as their individual components, paldimycins A (273a1 alpha) and B (273a1 beta) and antibiotics 273a2 alpha and 273a2 beta were synthesized from paulomycin, paulomycin A and paulomycin B, respectively, by reacting with N-acetyl-L-cysteine. The semisynthetic antibiotics had chromatographic behavior (TLC, HPLC) and physical and chemical properties identical to the properties of the corresponding antibiotics produced by Streptomyces paulus.

High-performance liquid chromatographic determination of acid-catalyzed degradation products of methyl carboprost in a polymeric controlled-release device.

A normal-phase high-performance liquid chromatographic method was used for the determination of methyl carboprost and acid-catalyzed degradation products in a polymer-based, controlled release dosage form. A reversed-phase method was used to isolate sufficient quantities of the degradation products to determine their identity. Degradation of methyl carboprost under acidic conditions results in epimerization and dehydration, to several isomers, at the tertiary allylic hydroxyl group. Mass balance was 94% for a sample allowed to degrade 50%. These compounds were observed to form in the polymer-based, controlled release dosage form. For the determination of methyl carboprost in the dosage form, the method was found to be linear, precise with a relative standard deviation of 2% and to have an average recovery of 99.2%.