Figure Legends
Figure 1. Malonyl-sildenafil is a polar analog with predicted low permeability. (A) Sildenafil is largely hydrophobic and exhibits poor solubility (LogS, circles) and high permeability (LogD, squares) at physiological pH. (B) Substitution of the piperazine methyl group in sildenafil with malonic acid is predicted to increase solubility and reduce permeability at physiological pH. The substitution is indicated by a dashed circle. LogS values from -2 to -4 are poorly soluble, and LogD values 0 to 5 are permeable. (C)In vitro assay for PDE5 inhibitory activity of sildenafil and malonyl-sildenafil. Data shown are mean +/- SD (n=3).
Figure 2. Malonyl-sildenafil retains pharmacological activity but exhibits poor membrane permeability. (A) Cartoon depicting an LS-174T-ll cell-based assay where movement of the PDE5 inhibitor (PDE5i) across the plasma-membrane (dashed line) leads to increased cGMP in colon cancer cells treated with a low dose of linaclotide to stimulate guanylate cyclase-C (GC-C). Increased cGMP levels activate cGMP-dependent protein kinase (PKG) that phosphorylates vasodilator stimulated phosphoprotein (VASP). (C) The LS174T-ll system (as depicted above) was treated with increasing doses of sildenafil or malonyl-sildenafil, and VASP phosphorylated on Ser239 (VASP-P) was determined by immunoblotting. Total VASP (VASP-t), and β-actin are loading controls, whereas PKG2 indicates equal expression of ectopic PKG2. Data in panel A are mean +/- SD (n=4), and results in panel C are representative of 3 independent experiments.
Figure 3. MS/MS Spectra for sildenafil and malonyl-sildenafil.Ion fragment patterns for Sildenafil (A) and Malonyl-sildenafil(B) . Proposed peak identification is illustrated by the dashed lines on the inset structure diagrams. The enlarged bold text indicates common ion peaks between the two compounds, and the asterisk marks the parent compound. The underlined peaks in panel B indicate unique ions generated by the malonyl group.
Figure 4. Pharmacokinetic analysis of malonyl-sildenafil in mice. Increasing doses of Malonyl-sildenafil (circles) or Sildenafil (squares) were administered to mice by gavage. LC-MS/MS was used to measure the compounds in plasma after 1 hour (A) , or in feces collected between 2-4 hours (B) . The compounds were also measured at different time points after administration of a single 9 mg/kg dose in plasma (C) and feces (D) . Data are mean +/- SD (n=6).
Figure 5. Malonyl-sildenafil does not interact with isosorbide mononitrate in mice. Mice were implanted with a carotid artery blood pressure transmitter (Data Science, PA-C10) and real time telemetry was recorded with a DSI PhysioTel system. Mice were randomly administered isosorbide mononitrate by i.p. injection (ISMN; 300 mg/kg) with orally administered vehicle, sildenafil or malonyl-sildenafil (10 mg/kg) as indicated. (A) Blood pressure recording with inset key showing when the drugs were administered. (B) Mean pressure (3 min) before drug (Pre) and after drug (post). Data are mean +/- SD (n=5), and p values are from ANOVA with Tukey’s multiple comparisons test.
Figure 6. Malonyl-sildenafil is well-tolerated in mice. Mice were maintained on water alone (Control), sildenafil (Sild) or malonyl-sildenafil (Mal) dissolved in the drinking water for up to 8 days as indicated. (A) Body mass measurements, (B)representative H&E-stained sections of the distal colon, scale bar is 30 μ. (C) Detection of Sild or Mal in the plasma at 8 days. Data are mean +/- SD (n=5).
Figure 7. Malonyl-sildenafil suppresses proliferation in the colon epithelium of mice. Mice were treated with 9 mg/kg sildenafil (Sild) or malonyl-sildenafil (Mal) in the drinking water, or with water alone (Ctrl). (A) After 8 days the proximal colon epithelium was harvested and subjected to flow cytometry measurement of Ki67+ cells. (B) Quantitation of treatment effects on Ki67+ cells. (C)PCNA+ cells in the distal colon were visualized by IHC, the scale bar is 30 μ. (D) Quantitation of treatment effects on PCNA+ cells per crypt. The plots in panels B and D show mean +/- SD, and p values are from one-way ANOVA followed by Tukey’s multiple comparisons test.