Glutathione

Example 8

Characterization of Absorption, Distribution, Metabolism, and Excretion of Oral [¹⁴C]Vorasidenib with Concomitant Intravenous Microdose Administration of [¹³C₃¹⁵N₃]Vorasidenib in Humans

Metabolite profiling and identification of vorasidenib (AG-881) was performed in plasma, urine, and fecal samples collected from five healthy subjects after a single 50-mg (100 μCi) oral dose of [14C]AG-881 and concomitant intravenous microdose of [¹³C3 ¹⁵N3]AG-881.

Plasma samples collected at selected time points from 0 through 336 hour postdose were pooled across subjects to generate 0—to 72 and 96-336-hour area under the concentration-time curve (AUC)-representative samples. Urine and feces samples were pooled by subject to generate individual urine and fecal pools. Plasma, urine, and feces samples were extracted, as appropriate, the extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectrometry (LC-MS and/or LC-MS/MS) analysis and by comparison of retention time with reference standards, when available.

Due to low radioactivity in samples, plasma metabolite profiling was performed by using accelerator mass spectrometry (AMS). In plasma, AG-881 was accounted for 66.24 and 29.47% of the total radioactivity in the pooled AUC_{0-72 h} and AUC_{96-336 h} plasma, respectively. The most abundant radioactive peak (P7; M458) represented 0.10 and 43.92% of total radioactivity for pooled AUC_0-72 and AUC_{96-336 h} plasma, respectively. All other radioactive peaks accounted for less than 6% of the total plasma radioactivity and were not identified.

The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), while no AG-881 was detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of dose in feces and for approximately 4% of dose in urine. M515, M460-1, M499, M516/M460-2, and M472/M476 were the most abundant metabolites in feces, and each accounted for approximately 2 to 5% of the radioactive dose, while M266 was the most abundant metabolite identified in urine and accounted for a mean of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for <1% of the dose.

Overall, the data presented indicate [¹⁴C]AG-881 underwent moderate metabolism after a single oral dose of 50-mg (100 μCi) and was eliminated in humans via a combination of metabolism and excretion of unchanged parent. AG-881 metabolism involved the oxidation and conjugation with glutathione (GSH) by displacement of the chlorine at the chloropyridine moiety. Subsequent biotransformation of GSH intermediates resulted in elimination of both glutamic acid and glycine to form the cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further converted by a series of biotransformation reactions such as oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation and N-dealkylation resulting in the formation multiple metabolites.

A summary of the metabolites observed is included in Table 2

Example 3

Cultivations were harvested on day 14 by centrifugation at 8000 g for 40 min. Filtered supernatants were purified for further characterization of IL-2 mutein Ala-M1. The supernatant was concentrated and loaded onto a Superdex 75 prep grade column (GE Healthcare Life Sciences, now Cytiva) equilibrated in 25 mM Tris, 200 mM NaCl pH 8.0. Fractions were analyzed by SDS-PAGE and fractions containing target protein were pooled. Further purification was done by cation exchange chromatography on an SP Sepharose HP column (GE Healthcare Life Sciences, now Cytiva) equilibrated in 25 mM Na-acetate pH 5.5. Before loading, the protein pool was pH adjusted to 5.5 and diluted with water. Elution was done by linear salt gradient elution. Fractions were analyzed by SDS-PAGE and those containing target protein were pooled to give purified IL-2 mutein Ala-M1 1 (SEQ ID NO:14 with an additional cysteine or glutathione connected to the thiol group of the cysteine at position 38 via a disulfide bridge).

Example 2

Recombinant human fascin 1 was expressed as a GST fusion protein in BL21 Escherichia coli. One liter of 2YT medium with ampicillin was inoculated overnight with 3 mL of BL21/DE3 culture transformed with pGEX4T-fascin 1 plasmid and grown at 37° C. until attenuance at 600 nm (D₆₀₀) reached about 0.8. The culture was then transferred to 18° C. and induced by the addition of 0.1 mM isopropyl β-d-thiogalactoside (IPTG) for 12 h. Bacteria were harvested by centrifugation at 5,000 r.p.m. for 10 min. The pellets were suspended in 30 mL of PBS supplemented with 0.2 mM PMSF, 1 mM DTT, 1% (v/v) Triton X-100 and 1 mM EDTA. After sonication, the suspension was centrifuged at 15,000 r.p.m. for 30 min to remove the cell debris. The supernatant was then incubated for 2 h with 4 mL of glutathione beads (Sigma) at 4° C. After extensive washing with PBS, the beads were resuspended in 10 mL of thrombin cleavage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl₂, 1 mM DTT). Fascin was released from the beads by incubation overnight with 40-100 U of thrombin at 4° C. After centrifugation, 0.2 mM PMSF was added to the supernatant to inactivate the remnant thrombin activity. The fascin protein was further concentrated with a Centricon® (Boca Raton, FL) filter to about 50 mg/mL.