Table 2. Stability and activity of tigecycline reconstituted in saline with or without additives.
| Composition (mg/mL) | Tigecycline by HPLC (% of freshly prepared) | Bacterial MIC (ng/mL)* | CellTiter IC50 in TEX cells (μM)* | Alamar blue IC50 in TEX cells (μM)* | Complex IV activity in TEX cells (% of control)† | ||||||||
| Tig | Pyr | CD | AA | Preincubation (days) | Preincubation (days) | ||||||||
| 0 (fresh) | 3 | 4 | 5 | 6 | 7 | 4 | 4 | 5 | 5 | ||||
| 1.0 | — | — | — | 100.0±10.0 | 12.2±0.7 | 15.1±3.2 | 8.4±2.5 | 10.2±2.9 | >2000 | >50.00 | >50.00 | 100.0±6.4 | |
| 2.5 | — | — | — | 100.0±25.0 | 2.7±0.7 | ||||||||
| 5.0 | — | — | — | 100.0±4.2 | 1.9±0.3 | ||||||||
| 1.0 | 60.0 | — | — | 100.0±12.1 | 61.2±8.4 | 42.2±0.2 | |||||||
| 1.0 | — | 50.0 | — | 100.0±5.8 | 33.7±10.8 | 16.2±0.3 | |||||||
| 1.0 | — | — | 0.3 | 100.0±5.8 | 30.2±5.7 | 25.7±5.9 | |||||||
| 1.0 | — | — | 0.6 | 100.0±13.3 | 54.4±7.9 | 46.6±1.5 | 32.1 | ||||||
| 1.0 | — | — | 1.0 | 100.0±2.6 | 78.9±6.2 | 53.2 | |||||||
| 1.0 | — | — | 2.0 | 100.0±4.6 | 62.6±1.1 | 53.2 | |||||||
| 1.0 | — | — | 3.0 | 100.0±10.0 | 67.6±5.6 | 50.6 | 56.7 | ||||||
| 1.0 | 60.0 | — | 0.3 | 100.0±5.6 | 85.5±14.4 | 60.4±1. 1 | 45.5±6.5 | ||||||
| 1.0 | 60.0 | — | 0.6 | 100.0±10.6 | 106.5±6.7 | 87.5±8.4 | 86.8±10.8 | 86.8±3.6 | 76.1±3.2 | ||||
| 1.0 | 60.0 | — | 1.0 | 100.0±3.9 | 95.5±5.1 | 92.8±2.7 | 93.6±2.6 | 77.4±2.6 | 125 | 4.22±0.30 | 35.1±7.7 | ||
| 1.0 | 60.0 | — | 2.0 | 100.0±8.7 | 101.7±10.8 | 97.0±7.5 | 96.0±2.8 | 89.7±8.7 | 125 | 4.74±0.68 | 4.76±0.28 | 37.8±11.5 | |
| 1.0 | 60.0 | — | 3.0 | 100.0±7.1 | 101.2±4.1 | 105.0±7.8 | 99.7±2.3 | 92.5±6.1 | 125 | 5.20±0.59 | 4.25±0.26 | 44.3±10.7 | |
| 2.5 | 60.0 | — | 3.0 | 100.0±18.0 | 114.8±3.1 | 96.4±15.7 | 87.1±18.1 | 59.6±12.0 | 5.00±0.73 | ||||
| 5.0 | 60.0 | — | 3.0 | 100.0±2.3 | 77.3±5.6 | 61.9±9.3 | 48.0±4.8 | 26.4±1.5 | |||||
| 1.0 | — | 50.0 | 0.3 | 100.0±7.5 | 51.1±7.3 | 35.7±5.1 | |||||||
| 1.0 | — | 50.0 | 0.6 | 100.0±11.4 | 65.4 | 56.7 | 48.2 | 48.1 | |||||
| 1.0 | — | 50.0 | 1.0 | 100.0±4.4 | 84.3±6.25 | 71.4±2.9 | 65.2±1.8 | 55.2±5.9 | |||||
| 1.0 | — | 50.0 | 2.0 | 100.0±0.9 | 69.9±4.1 | 58.1±3.8 | 56.2±5.2 | 48.0±22.0 | |||||
| 1.0 | — | 50.0 | 3.0 | 100.0±3.3 | 53.0±8.75 | 66.5±10.7 | 44.7±8.8 | 44.0±29.0 | |||||
| 1.0 | 60.0 | 50.0 | — | 100.0±7.1 | 82.9±9.3 | 67.5±8.1 | 58.6±2.2 | ||||||
| 1.0 | 60.0 | 50.0 | 0.3 | 100.0±10.2 | 72.2±8.6 | 67.4±5.0 | |||||||
| 1.0 | 60.0 | 50.0 | 0.6 | 100.0±13.2 | 90.9±4.0 | 74.8±6.8 | 89.0±0.3 | 75.2±11.9 | 76.5±0.2 | ||||
| 1.0 | 60.0 | 50.0 | 1.0 | 100.0±1.12 | 94.9±9.0 | 98.5±7.5 | 94.0±8.1 | 80.5±7.8 | 125 | 4.50±0.60 | 31.6±11.0 | ||
| 1.0 | 60.0 | 50.0 | 2.0 | 100.0±1.13 | 97.5±7.1 | 99.6±3.5 | 93.6±3.1 | 83.0±8.2 | 125 | 4.16±0.51 | 4.68±0.28 | 40.1±11.9 | |
| 1.0 | 60.0 | 50.0 | 3.0 | 100.0±4.04 | 97.4±2.6 | 99.9±3.1 | 104.5±0.6 | 88.4±8.1 | 125 | 4.62±0.66 | 4.32±0.28 | 33.2±3.5 | |
| 2.5 | 60.0 | 50.0 | 0.3 | 100.0±15.2 | 74.1±1.1 | 51.8±5.8 | |||||||
| 2.5 | 60.0 | 50.0 | 0.6 | 100.0±1.4 | 78.0±0.1 | 56.0±13.1 | |||||||
| 2.5 | 60.0 | 50.0 | 2.0 | 100.0±15.2 | 90.8±13.1 | 93.7±6.1 | 87.1±11.1 | 68.0 | |||||
| 2.5 | 60.0 | 50.0 | 3.0 | 100.0±1.4 | 87.1±22.9 | 88.9±22.5 | 81.0±18.8 | 63.0±17.5 | |||||
*Tigecycline solution was preincubated at room temperature for 4 days, which was then used to determine the minimum inhibitory concentration (MIC) in Escherichia coli (MG1655) and mean half-maximal inhibitory concentration (IC50) in TEX cells using the CellTiter viability assay following incubations with bacteria (24 hours) or cells (72 hours), respectively.
Tigecycline solution was preincubated at room temperature for 5 days, and TEX cells were then incubated with 5 µM tigecycline for 48 hours. The IC50 for cell viability was then assessed by the Alamar blue assay, and respiratory complex IV activity was determined in cell lysates. Activity was normalized to citrate synthase content as a proxy for mitochondrial mass.
All solutions were adjusted to pH 7 and all incubations were performed in the dark. Numbers indicate mean ± standard deviation of at least 3 independent experiments, where applicable.
Tig, tigecycline; Pyr, pyruvate; CD, 2-hydroxypropyl-β-cyclodextrin; AA, ascorbic acid.