Sean X. Peng

Application of LC-NMR and LC-MS to the identification of degradation products of a protease inhibitor in dosage formulations

LC-NMR and LC-MS were applied to the characterization of six degradation products of a protease inhibitor, N-hydroxy-1,3-di-[4-ethoxybenzenesulphonyl]-5,5-dimethyl-[1,3]c yclohexyldiazine-2-carboxamide, in a dosage formulation. A reversed-phase HPLC method was developed for the separation of the parent compound and its six degradation products. LC-MS was then utilized to obtain the molecular weight and fragmentation information using an electrospray ionization (ESI) interface in the positive ion mode. LC-NMR was employed to acquire detailed structural information using a selective solvent suppression pulse sequence in the stop flow mode. This work demonstrated the usefulness of this integrated approach for the rapid and unambiguous identification of drug compounds and their degradation products in dosage formulations.

Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

The development of new carboxylic acid-based MMP inhibitors derived from a cyclohexylglycine scaffold.

A series of carboxylic acids was prepared based on cyclohexylglycine scaffolds and tested for potency as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors such as compound 18 display low nanomolar potency for MMP-2 and MMP-13, while selectively sparing MMP-1 and MMP-7.

Direct determination of stability of protease inhibitors in plasma by HPLC with automated column-switching.

Automated column-switching HPLC methods were developed and utilized for the direct analyses of three hydroxamic acid based metalloprotease inhibitors in rat plasma. These column-switching methods involved the use of a restricted-access media (RAM) precolumn and a column-switching valve, allowing the complete automation of sample preparation and HPLC. The plasma samples were directly injected onto a precolumn packed with SPS/ODS stationary phase and then backflushed onto an ODS analytical column using a 6-port column-switching device. The drug stability in rat plasma was determined using both the automated and traditional HPLC methods. The results obtained from the automated column-switching methods were in good agreement with those from traditional methods that involve sequential protein precipitation, liquid extraction, solvent evaporation, and sample reconstitution. In addition to the elimination of labor-intensive and time-consuming sample preparation procedures, the column-switching methods allowed on-line analyte enrichment and accurate determination of drug stability in plasma with detection limits in the range of 10-20 ng/ml(-1). This work represents, for the first time, a drug stability study in plasma by automated column-switching HPLC technique with the use of a RAM column. Our column-switching methods can be readily adapted to any existing HPLC system with minimal hardware modification.

Simultaneous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral high-performance liquid chromatography.

Chiral HPLC methods were developed and utilized for the simultaneous determination of plasma protein binding of enantiomers of two racemic aminohydantoin compounds. Reversed-phase HPLC with the use of a polysaccharide-type chiral stationary phase column was employed for the separation and quantitation of the enantiomers of the two compounds with detection limits in the range 5-10 ng/ml in the plasma matrix. The chiral HPLC methods were selective, sensitive and reproducible. The R and S enantiomers of both compounds were baseline-resolved under the chromatographic conditions employed. Ultrafiltration techniques were applied to determining the plasma protein binding for each enantiomer in rat, dog and human plasma. The results clearly show stereoselective binding of the two enantiomers of each compound with higher protein binding of the R enantiomer than the S enantiomer in rat, dog and human plasma. Binding association constants were also determined to be in the range 1.01-14.0 x 10(4) M(-1) at 37 degrees C. Both the protein binding percentage and binding association constant were enantioselective and species-dependent. Such information is important for a clear understanding of the differences in biological activity as well as in pharmacokinetic and pharmacodynamic properties between the two enantiomers of each compound in the drug discovery and development process.

Separation and identification methods for metalloproteinase inhibitors.

Metalloproteinase inhibitors are being explored for the treatment of a wide variety of human diseases including cancers, arthritis, cardiovascular disorders, human immunodeficiency virus infection, and central nervous system illnesses. This review provides an overview of various analytical sample preparation, separation, detection, and identification techniques employed for the quantitative and qualitative determination of these inhibitor compounds. Special emphasis is placed on biological sample preparation by automated solid-phase extraction, liquid-liquid extraction, and protein precipitation by centrifugation or filtration. Other sample preparation methodologies are also evaluated. Applications of high-performance liquid chromatography. gas chromatography, and capillary electrophoresis to the quantitative determination of metalloproteinase inhibitors are described. Examples of qualitative analysis of metalloproteinase inhibitors by hyphenated liquid chromatography with mass spectrometry and nuclear magnetic resonance are also presented. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.

Permeability of Articular Cartilage to Matrix Metalloprotease Inhibitors

AbstractPurpose. To develop an in vitro cartilage permeation model for cartilage permeability study and to evaluate the effects of molecular hydrophilicity and cartilage location on the permeability of articular cartilage to matrix metalloprotease inhibitors. Methods. An in vitro cartilage permeation model was developed and utilized to determine the permeability of articular cartilage to the matrix metalloprotease inhibitors of different hydrophilicity. Permeability coefficients were obtained by measuring the steady-state flux of the inhibitor compounds. HPLC methods were also developed and employed for the analysis of drug levels in assay media. Results. The relationship between permeability and hydrophilicity of drug molecules was examined. Results indicated that the permeability coefficient increased with increasing hydrophilicity of the molecule. Additionally, the relationship between the permeability and the location of the cartilage section within the animal joint was investigated. Our results showed that the drug molecules penetrated faster in the surface layer cartilage than in the deep layer cartilage. Conclusions. Increasing the hydrophilicity of a molecule would increase its permeability across articular cartilage. The in vitro cartilage permeation model developed could be used to rank order drug compounds according to their cartilage permeability profiles and to aid in drug selection and development.