Chemicals and reagents
Acetonitrile (ACN) and methanol (MeOH) were of HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ, USA). Glacial acetic acid (ACS grade) was purchased from J. T. Baker (Phillipsburg, NJ, USA). Formic acid (99 % pure) was from Acros Organic (New Jersey, USA). Water was purified using the Millipore Milli-Q system (Milford, MA, USA). Mouse plasma for preparation of standards and quality controls (QC) was obtained from The City of Hope Medical Center Animal Center. Py-Im polyamides 1–4 were synthesized by solid-phase synthesis as previously described [21, 22]. For structures of internal standards (INS) 3 and 4, see Online Resource Fig. S1.
Animals for pharmacokinetic studies
Pyrrole–imidazole polyamide pharmacokinetic studies were performed in 10- to 12-week-old female BALB/C mice (Charles River). Polyamides were solubilized in PBS (1) or PBS/DMSO (2) and administered via intravenous (IV) tail vein injection at concentrations of 7.5 and 5 mg/kg, respectively. For each experiment, groups of 3 animals were euthanized at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after injection. Animals designated for the 4, 8, and 24 h time points were housed in metabolic cages for collection of urine and feces as described below. All animals used in the pharmacokinetic experiments were performed under an approved protocol at the City of Hope.
Animals for toxicology studies
Toxicities of polyamides 1 and 2 were measured after SC injections in 8- to 12-week-old female C57BL/6 mice (Jackson Laboratory). In anticipation of future xenograft experiments, subcutaneous injection, which has been shown to be a viable route of polyamide delivery , was chosen as the desired delivery method. A single bolus of polyamide 1 or 2 in PBS/DMSO vehicle was given, and the animals were weighed daily and monitored closely for signs of duress for 7 days. Animals exhibiting >15 % weight loss or signs of distress were euthanized according to regulations outlined by IACUC. Four animals were used in each group unless otherwise noted. This toxicology study was performed under an approved protocol at the California Institute of Technology.
Analytical methods development
Concentrations of polyamides 1 and 2 were analyzed by LC/MS/MS using a Waters Acquity UPLC system (Milford, MA, USA) interfaced with a Waters Quattro Premier XE Mass Spectrometer. HPLC separation was achieved using a Jupiter 4u Proteo 90A 150 × 2.0 mm column (Phenomenex, Torrance, CA, USA) proceeded by a Phenomenex C8 guard column (Torrance, CA, USA). The column temperature was maintained at 30 °C. The mobile phase consisted of A (0.05 % acetic acid in water) and B (0.05 % acetic acid in acetonitrile). The following gradient program was used: 8 % B (0–1 min, 0.3 ml/min), 16 % B (3 min, 0.3 ml/min), 58 % B (6 min, 0.3 ml/min), 90 % B (7 min, 0.3 ml/min), and 8 % B (7.3 min, 0.3 ml/min). The total run time was 11.5 min. The auto-injector temperature was maintained at 5 °C. The strong needle wash solution was 5 % formic acid in MeOH/ACN (2:8) for both compounds, and the weak needle wash solution was 30 % MeOH in water for compound 1 and 50 % ACN in water for compound 2. The electrospray ionization source of the mass spectrometer was operated in positive ion mode with a cone gas flow of 50 L/hr and a desolvation gas flow of 700 L/hr. The capillary voltage was set to 3.2 kV, and the cone and collision cell voltages were optimized to 32 and 27 V for 1 and the INS 3. Voltages were optimized to 31 and 20 V for 2 and the INS 4. The source temperature was 125 °C, and the desolvation temperature was 470 °C. A solvent delay program was used from 0 to 4.0 min and from 6.1 to 11.5 min to minimize the mobile phase to flow to the source. MassLynx version 4.1 software was used for data acquisition and processing.
Positive electrospray ionization of all compounds produced abundant protonated molecular ions (M + 3H) 3+. The fragmentations of these compounds were induced under collision-induced dissociation condition. The precursor → product ion combinations at m/z 453.52 → 206.10 for 1, 454.85 → 210.24 for 3, 467.45 → 238.32 for 2, and 469.9 → 238.4 for 4 were used in multiple reaction monitoring (MRM) mode for determination of these compounds. The use of MRM provided sufficient specificity and sensitivity. MS/MS experimental conditions, such as collision energy and collision cell pressure, were optimized from continuous flow injection sample introduction of standard solutions. Under optimized assay conditions, the retention times for 1 and 3 were 5.0 min, and 5.5 min for 2 and 4.
Plasma sample preparation
Plasma and urine samples were prepared for LC/MS/MS analysis by mixing 30 μL of plasma with 20 μL of 50 % MeOH and 50 % aqueous 1 % HOAc. The mixture was vortexed and mixed with an additional 120 μL of 0.5 % HOAc in MeOH/ACN (4:6) and 20 μL of 6.0 μg/mL INS in MeOH/1 % aqueous HOAc (1:1). The mixture was vortexed again for 2 min and centrifuged at the highest setting for 4 min. Next, 20 μL of the supernatant was transferred to a new tube and mixed with 180 μL of 50 % MeOH/ACN (4:6) and 50 % aqueous 1 % HOAc.
Standard curves were prepared mixing untreated plasma with 20 μL of 50 % MeOH and 50 % aqueous 1 % HOAc prepared with various concentrations of 1 and 2. Internal standards were added as described above. The standard curves, as determined by linear regression, displayed good linearity (r
2 > 0.99) over the range tested for 1 (0.1–30 μg/mL) and 2 (0.2–20 μg/mL).
Urine and fecal sample preparation
Urine and fecal samples were collected using metabolic cages (Ancare, Techniplast Metabolic Rack, 12 cages by Nalgene). Urine samples were collected at 3 time points over 24 h, and fecal samples were collected at 8 and 24 h time points. Py-Im polyamides were extracted from urine according to the plasma extraction procedure described above.
Fecal samples were first dried at room temperature and then weighed and grounded to a powder. Approximately 100 mg of powder was weighed out and reconstituted in distilled water (6 μL/mg powder). The fecal sample was then homogenized in a TissueLyser (Qiagen) for 2 min at 30 Hz twice, and an additional 6 μL/mg of distilled water was added. Next, 30 μL of the fecal homogenate was mixed with 50 μL distilled water and 20 μL of 50 % MeOH and 50 % aqueous 1 % HOAc. The mixture was then vortex mixed with 0.1 mL 0.5 % HOAc in MeOH/ACN (2:8) and 20 μL of 6.0 μg/mL INS in MeOH/1 % aqueous HOAc (1:1) for 10 min and centrifuged at the highest setting for an additional 10 min. The supernatant was diluted with 50 % MeOH/ACN (4:6) and 50 % aqueous 1 % HOAc.
Tissue sample preparation
Distribution of polyamides 1 and 2 was determined in the liver, kidneys, and lungs. The organs were harvested post-euthanasia and prepared via similar processes. A piece of the mouse organ was weighed and mixed with distilled water (3 μL/mg tissue). The tissue was then homogenized by pulsing three times on a TissueLyser for 2 min each at 30 Hz. Next, 30 μL of the tissue homogenate was mixed with 20 μL of 50 % MeOH and 50 % aqueous 1 % HOAc. The mixture was then vortex mixed with 0.12 mL 0.5 % HOAc in MeOH/ACN (2:8) and 20 μL of 6.0 μg/mL INS in MeOH/1 % aqueous HOAc (1:1) for 10 min and centrifuged at the highest setting for an additional 10 min. Samples treated with polyamide 1 were then diluted with 50 % MeOH/ACN (4:6) and 50 % aqueous 1 % HOAc. Samples treated with polyamide 2 were diluted with 50 % MeOH/ACN (4:6) and 50 % aqueous 3 % FA.
Pharmacokinetic data analysis
Plasma pharmacokinetic parameters were derived from polyamide concentration profiles using both non-compartmental and compartmental methods. Non-compartmental analysis was performed according to statistical moment theory and the rule of linear trapezoids, while compartmental analysis was performed in ADAPT II . Pharmacokinetic parameters estimated from the non-compartmental analysis include the maximum concentration (C
max), the terminal elimination half-life (t
1/2), the mean residence time (MRT), the area under the concentration curve (AUC0–24h), the AUC extrapolated to infinity (AUC0–∞), and the clearance (CL). Additional plasma pharmacokinetic parameters determined from the compartmental analysis include the alpha and beta half-lives (t
1/2) and the apparent volume of distribution (V
d). Tissue pharmacokinetic parameters were determined non-compartmentally and included the C
max and AUC0–24h. Urinary and fecal excretion data were expressed as the cumulative percentage of the administered dose.
pH stability analysis
The pH stability of Py-Im polyamides was analyzed as previously described . In summary, 15 μL of a 10 μM solution of polyamide 1 or 2 in DMSO was incubated with 85 μL of buffer with pH of 2.5, 4, 7, or 10 (Fluka) at 37 °C for 24 h. After incubation, the sample was mixed with an equal volume of N,N-dimethylformamide and sonicated briefly. Next, 20 μL of the sample solution was mixed with 180 μL of aqueous buffer containing 100 mM NH4OAc and 25 μM methyl 4-nitro-1H-pyrrole-2-carboxylate as an internal standard. Analytical HPLC analysis was performed on a Beckman analytical HPLC.