Discussion and Conclusions
Sildenafil epitomizes the hallmark Rule of 5 (Ro5) properties (Lipinski,
2004; Lipinski et al. , 2001) regarding overall size and lack of
polarity that allow near-complete systemic delivery into the circulation
(Hong et al. , 2017; Walker et al. , 1999). Extensive
structural studies of sildenafil have identified the piperazine nitrogen
as amenable to modifications that affect pharmacokinetic properties
without dramatically affecting pharmacological efficacy (Dunn, 2005; Kimet al. , 2001b; Kim et al. , 2001c; Yoo et al. ,
2007). To develop a non-systemic PDE5i we chose to replace the methyl
group of sildenafil with malonate, which is predicted to dramatically
reduce permeability at post-gastric pH values between 5-7. This
modification did not affect the inhibitor potency as malonyl-sildenafil
was equally potent as sildenafil toward human PDE5a. The
IC50 value for sildenafil determined using the assay
conditions in the present study were slightly higher than previously
reported (Gupta et al. , 2005). However, the results are congruent
with a previous study suggesting that the carboxylate mimics the
phosphate of cGMP to increase affinity (Kim et al., 2001a). We used a
novel assay to assess cell permeability, whereby entry of PDE5i into
colon cancer cells amplifies GC-C activity that is driven by a low level
of the synthetic agonist linaclotide. We used ELISA to measure cGMP
levels in our system, which demonstrated a dramatic reduction in
EC50 for malonyl-sildenafil compared to the parent
compound sildenafil. This observation was supported using activation of
cGMP-dependent protein kinase (PKG2) as a readout for the ability of
PDE5i to enter cells and increase cGMP levels. As the IC50 toward
purified PDE5 was similar, these results support the idea that the
carboxylic acid group impeded diffusion of malonyl-sildenafil across the
plasma-membrane. However, a differential susceptibility of the compounds
to efflux pumps (e.g., MDR1) might also have contributed to the
exclusion but was not investigated here.
Sildenafil exhibits near-complete systemic delivery (Hong et al. ,
2017), and in circulation it undergoes hepatic modification into
desmethyl-sildenafil that retains some bioactivity (Kim et al., 2003).
The prominent desmethyl-sildenafil peak in the MS/MS ion spectrum is due
to the ionization energy as it was not detected in the liquid
chromatography elution profile using pure compound, or in the plasma
from malonyl-sildenafil treated mice (not shown). One of the most
significant findings is that oral administration of malonyl-sildenafil
even at concentrations up to 36 mg/kg exhibited minimal leakage into the
circulation but was excreted at high levels in the feces. This
contrasted sharply with sildenafil, which peaked at 1-2 hours in the
plasma and was not detected in the feces. The typical human dose of
sildenafil is 0.7-1.4 mg/kg that is equivalent to consumption of a 50 or
100 mg pill (respectively). The 36 mg/kg dose used here is 26 times the
maximum human dose, and 2-4-fold the human dose with allometric scaling.
This clearly demonstrates that malonyl-sildenafil has very poor
permeability in vivo and is largely retained in the gut lumen.
The typical transit time for these mice is between 2-4 hours (Sharman et
al., 2017), so the continued excretion of malonyl-sildenafil in the
feces 8 hours after oral gavage was surprising. One possibility for
future exploration is that the drug is retained in the mucus layer long
after excretion of the luminal contents. Regardless, this property is
ostensibly ideal for a drug that targets the colon epithelium. A major
drawback to the use of contemporary PDE5i for colon cancer prevention is
the contraindication for patients taking nitrates to treat stable angina
due to synergistic hypotensive effects (Lee et al. , 2004). It was
shown here that malonyl-sildenafil did not significantly affect blood
pressure in mice exposed to ISMN. This supports the pharmacokinetic data
and is congruent with the notion that bioactive lipophilic metabolites
were not generated from malonyl-sildenafil.
Taken together the pharmacokinetic results demonstrated that
malonyl-sildenafil did not enter the circulation, but it was unclear
whether it could target the colon epithelium. Previous studies have
demonstrated that PDE5i treatment of mice can alter intestinal
homeostasis by suppressing proliferation and apoptosis while promoting
differentiation (Sharman et al., 2017; Wang et al., 2014). The
suppression of proliferation in the preneoplastic epithelium has been
proposed to mediate the CRC-prevention effects of PDE5i (Browning, 2019;
Islam et al. , 2018) because this proliferative compartment is
where replication errors and genotoxic stress drive neoplastic
transformation (Chamorro-Jorganes et al. , 2010; Tomasettiet al. , 2017). Two different approaches were used to demonstrate
that oral administration of malonyl-sildenafil to mice was equally
effective as sildenafil at suppressing the proliferative compartment in
the colon. This result was surprising because the 18-fold reduction in
cell penetration for malonyl-sildenafil (EC50) would
predict a lesser response in vivo . It is plausible that
malonyl-sildenafil levels became concentrated in the mucosal region over
time. However, a more plausible explanation is that the lengthy exposure
of malonyl-sildenafil to the colon epithelium compared to transient
delivery of sildenafil by systemic delivery through the vasculature,
produced a larger area under the curve to compensate. These
possibilities might be explored in future studies with this compound.
In summary, with the goal of creating a non-systemic PDE5i that targets
the colon epithelium, we have synthesized a novel analog of sildenafil
containing a carboxylic acid group derived from malonic acid.
Malonyl-sildenafil remains in the colon for many hours without entering
the circulation and is equally effective as sildenafil at suppressing
epithelial proliferation. The results shown here are proof of principle
that will enable the development of drugs for the prevention and
treatment of intestinal disorders, with fewer side-effects and drug-drug
interactions.
Acknowledgement: This work was supported by a grant from
the Georgia Research Alliance, USA (GRA.VL21.E1 to DDB), and from the
National Institutes of Health (1 R15 CA264555-01 to IL).
Conflict of interest statement: DDB and IL are cofounders
of a startup biotechnology company (Targut Biotechnologies Inc.) that
has the goal of developing drugs to target intestinal diseases.