Physical stability of salmon calcitonin spray-dried powders for inhalation.

The effects of excipient crystallinity and water content on the physical stability of salmon calcitonin (sCT) in a spray-dried powder for inhalation have been investigated. sCT was dissolved in water with and without mannitol and then spray dried using a Büchi 190 spray dryer. The spray dried powders were stored for 5 days at 0, 29, 51, 58, 69, and 84% relative humidity at ambient temperature. The crystalline content, water content, secondary structure, and aggregation rates were determined for each powder immediately following spray drying and after storage at various relative humidities. In addition, the water sorption isotherms and reactivity to water vapor were determined using DVS and isothermal calorimetry, respectively. No sCT aggregation occurred during the spray drying process. Crystallinity depended on the amount of mannitol in the formulation. Powders containing up to 50% mannitol were fully amorphous, and those containing 70 and 90% mannitol contained some crystalline polyol. The powders remained aggregate free for over 2 years when stored below the critical RH (e.g., <20% for the powder containing 30% mannitol). Above this RH, sCT aggregation increased as a function of time. The amount of aggregate observed correlates with the amount of intermolecular beta-sheet formed, determined by FTIR. The sCT aggregation rate in powders containing 70% mannitol was significantly lower than that in powders containing 30% mannitol at all RH tested, presumably because of a higher ratio of amorphous mannitol to sCT, which inhibits the formation of beta-sheet structure. Moisture-induced crystallization of mannitol was observed in all powders stored at RH >50%. The moisture induced thermal activity trace (MITAT) offers a useful description on the physical stability of the spray dried powders. In conclusion, spray drying sCT and sCT/mannitol mixtures yields dry powders that contain physically intact peptide. In addition, sCT aggregation and mannitol crystallization in spray dried powders can be prevented during long-term storage if stored in low humidity environments, which can be easily assessed by MITAT.

Quantification of 2'-fluoro-2'-deoxyuridine and 2'-fluoro-2'-deoxycytidine in DNA and RNA isolated from rats and woodchucks using LC/MS/MS.

Apatmers are synthesized using 2'-fluoropyrimdines in place of normal pyrmidines to stabilize them against enzymatic degradation, and thereby improve their therapeutic efficacy. Despite this stabilizing effect, the apatmers can still be degraded by nucleases in the blood. Primer template extension studies have demonstrated that mammalian DNA polymerases can incorporate these 2'-fluoropyrimidines into growing strands of DNA. The toxicologic effects of these compounds have been examined in rats and woodchucks, animals known to be susceptible to the toxic effects of other modified pyrimidines. Whether these nucleosides can be incorporated into DNA in vivo has not been established. These studies report the development of methodologies and the results of studies designed to determine if and to what extent 2'-fluoropyrimidines are incorporated into tissue DNA following long-term treatment. Rats were dosed intravenously with either 2'-fluorouridine (2'-FU) or 2'-fluorocytidine (2'-FC) at doses of 5, 50, and 500 mg/kg/day for 90 days. Woodchucks were dosed intravenously with either 2'-FU or 2'-FC at doses of 0.75 or 7.5 mg/kg/day for 90 days. The amounts of 2'-FU or 2'-FC in DNA and RNA were quantified using newly developed LC/MS/MS methodologies. Administration of 2'-FU to rats and woodchucks resulted in incorporation of the compound into DNA from liver, spleen, testis, muscle, and kidney. Incorporation also occurred in RNA from rat liver (only tissue examined). Similarly, administration of 2'-FC to rats and woodchucks resulted in incorporation into liver DNA (only tissue examined). These data demonstrate that 2'-fluoropyrimidines are incorporated into DNA and RNA of various tissues of rats and woodchucks following long-term administration.