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.