Orthogonal HPLC methods for quantitating related substances and degradation products of pramlintide.

Pramlintide is a 37-amino acid peptide that is being evaluated as a drug candidate for treating people with type 1 and insulin-using type 2 diabetes. Two high-performance liquid chromatography (HPLC) methods were developed for quantitating related substance impurities in pramlintide drug substance as well as degradation products of pramlintide formulated for parenteral administration. The methods differ with respect to separation mode and therefore provide orthogonal information concerning related substances and degradation products. One method uses a reverse phase (RP) separation mode, and the other involves a strong cation exchange (SCX) separation. Method performance testing showed that the RP- and SCX-HPLC methods both afford a high degree of selectivity, accuracy, precision, and sensitivity. The limit of quantitation for determining spiked authentic samples of degradation products was shown to be approximately 0.1% (relative to intact pramlintide) for both methods. Relative retention times for known pramlintide degradation products were determined for both the RP- and SCX-HPLC methods, demonstrating the selectivities of the 2 methods as well as the orthogonality of the information. The methods were also shown to be diastereospecific with respect to separating pramlintide from authentic samples of D-isomers at Ala5, Ala8, Ala5-Ala8, and Leu12. The methods did not resolve pramlintide, however, from diastereomers with D-isomers near the C- and N-termini, namely Lys1,Cys2, and Tyr37.

Development and characterization of a lyophilized dosage form of IL-1 beta.

The development and characterization of a lyophilized dosage form for recombinant Interleukin-1 beta is described. Included in the evaluation of the drug product are accelerated and long-term stability studies utilizing a number of biophysical techniques (reverse-phase HPLC, SDS-PAGE, isoelectric focusing and ELISA). Data collected with these methods were examined for correlations with biological activity assessments provided by an in-vitro cell culture system (mouse thymocyte proliferation). Results of these studies demonstrate that a lyophilized dosage form of Interleukin-1 beta can be prepared which retains its potency for at least 1 year when stored at ambient temperature. The analytical methodology used to assess the physicochemical integrity of the protein provided a sensitive and reproducible means of predicting changes in biological activity.

Chromatographic separation of the diastereomers of a dihydropyridine-type calcium channel antagonist as the bis-3,5-dinitrobenzoates.

The diastereomeric components of the calcium channel antagonist RS-93522-004 are separated as the bis-3,5-dinitrobenzoate ester by reversed-phase high-performance liquid chromatography. The diastereomer ratio determination is shown to be precise, accurate and sensitive, and is not affected by the reaction yield of the derivatization with 3,5-dinitrobenzoyl chloride. The four individual stereoisomers of RS-93522-004 were independently shown to have equal reactivity toward 3,5-dinitrobenzoyl chloride.

Separation of the stereoisomers of an allenic E-type prostaglandin.

Enprostil (I) is a synthetic dehydro-prostaglandin E2 containing a chiral allene moiety which is unresolved relative to the four remaining chiral centers. The relative configuration of the four remaining chiral centers is consistent with that of the naturally occurring E series of prostaglandins. Thus, enprostil exists as enantiomeric pairs of two allenic epimers. An analytical procedure has been developed that separates the four optical isomers present in enprostil. This procedure involves, first, the acid-catalyzed dehydration of enprostil to its corresponding prostaglandin A analogue followed by derivatization with beta-naphthylsulfonyl-L-prolyl chloride. The resulting diastereomeric sulfonate esters are separated on an achiral silica gel high-performance liquid chromatographic column. This procedure has been applied to the analysis of both enprostil drug substance and enprostil formulated in a propylene carbonate solution from soft elastic gelatin capsules. An efficient procedure for the recovery of enprostil from the solution formulation is also described.

"Atmospheric" Epoxidation of Benzo[a]pyrene by Ozone: Formation of the Metabolite Benzo[a]pyrene-4,5-Oxide.

Benzo[a]pyrene deposited on a glass fiber filter reacts rapidly in the dark or light with ambient levels of ozone to yield a mixture of products that display strong direct mutagenicity in the Ames assay. The major stable contributor to this activity has been identified as benzo[a]pyrene-4,5-oxide, a DNA-binding metabolite in biological systems, known to be a strong direct mutagen with Salmonella typhimurium strain TA98.

Mechanism of thermal rearrangement of the spiro bicyclo[2.1.0]-pentane-5,2'-methylenecyclopropanes to 6- and 7- methylenebicyclo[3.2.0]hept-1-enes.

The thermal rearrangements of the bicyclo[2.1.0]pentane-5,2'-methylenecyclopropanes fall into two classes. The first occurs near 80 degrees C and consists of a double epimerization ("bridge flip") which is initiated by cleavage of the bridge bond. An alternative mechanism by way of a trimethylenemethane intermediate is ruled out by an isotopic position-marking experiment. The second rearrangement begins to be detected above 120 degrees C. It gives the isomeric 6- and 7-methylenebicyclo[3.2.0]hept-1-enes. Two possible mechanisms can operate in this complex change, but a choice between them is not yet possible.