Figure 1.
Ascorbic acid and pyruvate stabilize tigecycline in saline solution.
Tigecyline (1 mg/mL) was dissolved in saline solution with or without supplementation with various excipients and incubated in the light. Tigecycline concentrations were detected over time by HPLC and expressed as a relative percentage of that detected immediately following fresh dilution in saline ( = 100%). Tigecycline was dissolved in (A) saline with or without 0.6 mg/mL ascorbic acid, 6 mg/mL pyruvate, 50 mg/mL 2-hydroxypropyl-β-cyclodextrin (HPCD), 0.3 U/mL Oxyrase with 20 mM sodium lactate, 6 mg/mL EDTA sodium, 5% (w/v) glucose, 5% (w/v) mannitol, 10% (v/v) ethanol, 10 mM sodium phosphate (Na-PO4) buffer pH 6.9, or 10 mM sodium bicarbonate (Na-HCO3) buffer pH 6.8; (B) saline containing 0–30 mg/mL ascorbic acid; or (C) saline containing 0–300 mg/mL pyruvate; (D) Iscove's modified Dulbecco's medium (IMDM). In all panels, data indicate the mean ± standard deviation of 3 independent experiments.
Table 1.
Activity of tigecycline (1 mg/mL) when freshly prepared in saline.
Table 2.
Stability and activity of tigecycline reconstituted in saline with or without additives.
Figure 2.
Tigecycline is stabilized in solution under novel formulation conditions for up to 7 days.
Following dissolution in saline or a novel stabilizing formulation containing ascorbic acid (3 mg/mL) and pyruvate (60 mg/mL) in saline, adjusted to pH 7, tigecycline concentrations were detected by HPLC and expressed as a relative percentage of that detected following fresh dilution in saline ( = 100%). (A) Tigecycline (1 mg/mL) dissolved in saline or the novel formulation was incubated under light or dark conditions, demonstrating light sensitivity. (B) Increasing concentrations of tigecycline were dissolved in the novel formulation and incubated in the dark. Tigecycline stability in solution was reduced at concentrations greater than 1 mg/mL. In all panels, data indicate the mean ± standard deviation of 3 independent experiments.
Table 3.
Antileukemic activity of tigecycline reconstituted in saline with pyruvate and ascorbic acid.
Figure 3.
The novel ascorbic acid- and pyruvate-containing formulation displays efficacy in AML cells grown in vivo.
(A) Mice were administered 50/kg tigecycline or 50 mg/kg novel tigecycline formulation by intraperitoneal injection and plasma was collected at increasing times after treatment. Plasma tigecycline concentration was determined using HPLC. The peak plasma concentration (Cmax), the terminal half-life (t1/2), area under the plasma concentration-time curve (AUC), clearance (CL) and volume of distribution (Vz) were evaluated using WinNonlin 6.2.1. Data represent the mean ± standard deviation of a representative experiment with 3 mice per group. Human leukemia OCI-AML2 cells were injected subcutaneously into the flank of NOD/SCID mice. Eleven days after injection, once tumors were palpable, mice were treated with 50 mg/kg of tigecycline, novel formulation of tigecycline, or vehicle controls (saline or formulation) by intraperitoneal injection twice a day for 11 days (n = 9 per group). Tigecycline in each formulation was prepared fresh twice a day. (B) Tumor volume was monitored over time. Eleven days after injection, mice were sacrificed and tumors excised. (C) Tumor weight was measured. ** indicates p<0.01 and * indicates p<0.05 as determined by Tukey's post-test and one-way ANOVA analysis. Lines represent median. (D) Total proteins were extracted and analyzed by immunoblotting for Cox-1, Cox-2 and Cox-4 expression. PVDF membrane was stained with 0.1% Amido Black.
Figure 4.
The novel ascorbic acid- and pyruvate-containing formulation displays similar tolerability in mice to saline.
NOD/SCID mice (n = 3 per group) were administered saline or the novel formulation (60 mg/mL pyruvate (Pyr), 3 mg/mL ascorbic acid (AA) in saline, pH 7.0) by intraperitoneal injection 5 of 7 days over 3 weeks. At the end of the experiment, mice were sacrificed and serum and organs were collected. (A) Serum levels of total bilirubin, aspartate transaminase (AST) and alkaline phosphatase (ALP) were measured as indicators of liver function, while creatine kinase levels were measured as an indicator of muscle, heart or brain injury. (B) Heart, liver, kidney and muscle organs were sectioned and stained with hematoxylin and eosin. Representative sections from organs are shown of 1 section from 1 of 3 mice per group. Images were collected using a ScanScope XT microscope at 10× magnification. Scale bars are 100 µm.