Design, synthesis, and testing of a molecular truck for colonic delivery of 5-aminosalicylic acid

Article abstract of DOI:10.1021/ml300086c

A molecular scaffold bearing eight terminal alkyne groups was synthesized from sucrose. Eight copies of an azide-terminated, azo-linked precursor to 5-aminosalicylic acid were attached to the scaffold via copper(I)-catalyzed azide-alkyne cycloaddition. Th

Full text of DOI:10.1021/ml300086c

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Testing of a Molecular Truck for Colonic
Delivery of 5‑Aminosalicylic Acid
Suryakiran Navath,† Venkataramanarao Rao,† Rita-Marie T. Woodford,‡,§ Monica T. Midura-Kiela,‡
Ali M. Ahad,† Ramesh Alleti,† Pawel R. Kiela,*,‡,∥ and Eugene A. Mash*,†
†Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States
‡Department of Pediatrics, University of Arizona, Tucson, Arizona 85724, United States
§School of Dentistry, Oral Biology Program, University of North Carolina, Chapel Hill, North Carolina 27599, United States
∥Department of Immunobiology, University of Arizona, Tucson, Arizona 85724, United States
S
* Supporting Information
ABSTRACT: A molecular scaffold bearing eight terminal alkyne groups
was synthesized from sucrose. Eight copies of an azide-terminated, azo-
linked precursor to 5-aminosalicylic acid were attached to the scaffold via
copper(I)-catalyzed azide−alkyne cycloaddition. The resulting compound
was evaluated in a DSS model of colitis in BALB/c mice against
sulfasalazine as a control. Two independent studies verified that the novel
pro-drug, administered in a dose calculated to result in an equimolar 5-
ASA yield, outperformed sulfasalazine in terms of protection from mucosal
inflammation and T cell activation. A separate study established that 5-
ASA appeared in feces produced 24−48 h following administration of the
pro-drug. Thus, a new, orally administered pro-drug form of 5-aminosalicylic acid has been developed and successfully
demonstrated.
KEYWORDS: Inflammatory bowel disease, 5-aminosalicylic acid, pro-drug
esalamine (mesalazine, 5-aminosalicylic acid, 5-ASA, 1)
Mremains the compound of choice for the treatment of
inflammatory bowel disease (IBD).1−3 5-ASA is used to induce
remission and/or to prevent relapses, and as a means of
prevention of IBD-associated colon cancer. Among its known
mechanisms of action are inhibition of IL-1β and TNFα
signaling,4−6 downstream inhibition of NF-κB,7,8 and activation
of peroxisome proliferator-activated receptors in colonic
epithelial cells, which are involved in the control of
inflammation, cell proliferation, apoptosis, and metabolic
function.9
In designing a new 5-ASA carrier and pro-drug, we set the
following objectives: (1) the pro-drug should be administered
orally and (2) deliver a highly concentrated payload to the
appropriate region of the gastrointestinal tract; (3) the carrier
should be excreted in the feces with little or no absorption or
breakdown, and (4) have negligible, if any, side effects. With
these goals in mind, the properties of Olestra, a type of “fake
fat” derived by esterification of sucrose with a mixture of fatty
Colon-specific delivery of 5-ASA can be achieved by the use
of enemas and suppositories. However, when administered
orally, rapid absorption of 5-ASA through the mucosa of the
upper gastrointestinal (GI) tract greatly reduces its bioavail-
ability to the colonic mucosa.9 Approaches to enhancing colon-
specific delivery of orally administered 5-ASA include the use of
pH sensitive polymer coatings,10 biodegradable polymer
matrices and hydrogels,11 and pro-drugs. In this last category,
acids, drew our attention. Olestra passes, unmodified and
unabsorbed, through the entire GI tract.14−16 This suggested
bioactivation by reductive cleavage of azo links by bacterial
azoreductases in the gut is exploited in the case of the
prototypical 5-ASA pro-drug, sulfasalazine (2).12 Concomitant
release of the carrier, sulfapyridine (3), is believed to be
responsible for a number of side effects.13 The related pro-
drugs olsalazine (Dipentum) and balsalazide (Colazal) depend
on a similar 5-ASA release strategy.
that a related compound, with metabolism and disposition
similar to that of Olestra, but with fewer side effects owing to
Received: April 6, 2012
Accepted: July 29, 2012
Published: August 1, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Azide 9
a
Reagents and conditions: (a) K2CO3, 1,8-dibromooctane, acetone; (b) 10% Pd/C, H2 (40 psi), EtOH; (c) NaNO2, aq HCl; salicylic acid, aq
NaOH; (d) NaN3, DMSO; (e) K2CO3, (CH3)2SO4, acetone.
a
Scheme 2. Synthesis of Octatriazole Sodium Salt 13
a
Reagents and conditions: (a) CuSO4, sodium ascorbate, THF/water; (b) LiOH, THF/water/methanol; (c) ion exchange chromatography.
structural changes and/or reduction in the administered dose,
would be an excellent candidate for use as a drug transport
scaffold in the GI system. For treatment of IBD, a branched
structure would allow attachment of multiple 5-ASA precursors
to the scaffold, producing a high payload pro-drug that might
provide a means for colon-specific delivery, achieve higher
concentrations of the active drug, and lead to improved healing.
Olestra contains acid-labile and base-labile ester links and is a
complex mixture of compounds. To make our scaffold more
stable and less complex, we elected to employ ether links to
sucrose involving chains of the same constitution. Our recent
work on construction of multivalent compounds17−19 suggested
the use of copper(I)-catalyzed azide−alkyne cycloaddition
(CuAAC)20,21 for attachment of the 5-ASA precursors to the
scaffold. We report herein the design, synthesis, and biological
testing of a sucrose-derived “molecular truck” loaded with eight
copies of 5-ASA in a form releasable by bacterial azoreductases
in the gut.
structure of 11 was confirmed by NMR and MALDI-TOF MS
analyses (see Supporting Information).23
Saponification of 11 using LiOH in 9/3.3/1 THF/water/
methanol gave acid 12, which was converted to the sodium salt
13 by ion exchange chromatography and recovered from
solution by lyophilization. Compound 13 was a nonhydro-
1
scopic bright yellow solid that was characterized by H NMR
and MALDI-TOF analysis (see Supporting Information).
An animal model was utilized to compare the efficacy of 13
in relation to 2, a medicine used in treatment of IBD. For this in
vivo model, 8−10 week-old male BALB/c mice were given a 5
day treatment of drinking water containing 4% dextran sulfate
sodium salt (DSS), which causes damage to and inflammation
of the epithelial lining of the colon. Mice were then given
regular drinking water and were monitored for a total of 12
days. During the study, mice were given daily gastric gavage
feedings of (a) placebo suspension medium (Ora-Blend SF,
Paddock Laboratories), (b) suspension medium containing 2,
or (c) suspension medium containing 13. A dose of 0.28 mmol
of 5-ASA equivalent/(kg day) was chosen based on published
literature.24−26 Thus, the amounts of 2 (111.5 mg/(kg day))
and 13 (155.7 mg/(kg day)) were calculated to give the same
theoretical yield of 5-ASA after azo reduction, assuming 100%
efficiency. Control groups included mice given regular drinking
water and placebo, as well as mice treated with DSS and
placebo.
The results of the first study demonstrated decreased
morbidity and mortality for mice treated with 13 when
compared to mice treated with either DSS and placebo or DSS
and 2 (Figure 1). In general, mice treated with 13 had a much
better appearance and displayed significantly less behavioral
depression, as evidenced by posture, mobility, grooming, and
social behavior (data not shown). Mice treated with 13
exhibited less weight loss (Figure 1A) and colon shortening
(Figure 1B) as a measure of inflammation. Survival (Figure 1C)
was equal among mice treated with 13 and untreated control
The synthesis of 5-ASA derivative 9 is depicted in Scheme 1.
Reaction of p-nitrophenol (4) with potassium carbonate and 5
equiv of 1,8-dibromooctane in acetone gave ether 5 in 89%
yield.22 Nitro reduction was effected by hydrogenation using
10% Pd/C catalyst, producing aniline 6 in 93% yield.
Treatment of 6 with sodium nitrite in aqueous hydrochloric
acid gave the corresponding diazonium salt, which was allowed
to react with salicylic acid in an aqueous alkaline solution. This
gave the azobenzene derivative 7 in 40% yield. Reaction of
bromide 7 with sodium azide in DMSO produced the
corresponding azide 8 in 91% yield. Treatment of 8 with
potassium carbonate and dimethyl sulfate in refluxing acetone
gave ester 9 in 50% yield.
CuAAC reaction20,21 of sucrose-derived octaalkyne 1019 with
10 equiv of azide 9 using CuSO4 and sodium ascorbate in
deoxygenated (with argon) 9/1 THF/water at room temper-
ature for 12 h produced, after column chromatography,
octatriazole octaester 11 in 68% yield (Scheme 2). The
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Figure 1. (A) Body weight loss in 4% DSS colitis. Data were analyzed statistically with AVOVA followed by a Fisher PLSD posthoc test. *
differences between DSS and DSS + 2 or DSS + 13 at day 7, and between DSS + 13 and DSS or DSS + 2 at days 8−12. (B) Changes in colon length
in DSS colitis. Data were analyzed statistically with AVOVA followed by a Fisher PLSD posthoc test. P values indicate differences between all groups.
Treatment with 2 did not result in significant improvement in colon length in DSS-treated mice (n.s.). (C) Survival of control (blue line), 4% DSS-
treated (red line), DSS + 2 treated (green line), and DSS + 13 treated (purple line) mice.
Figure 2. (A) Body weight loss in 3% DSS colitis. Data were analyzed statistically with AVOVA followed by a Fisher PLSD posthoc test. *
differences between DSS + 2 and DSS + 13; statistical trend p < 0.1. (B) Changes in the appearance of rectal bleeding expressed as a percent of mice
in the respective groups. (C) Colon length in 3% DSS colitis. Data were analyzed statistically with AVOVA followed by a Fisher PLSD posthoc test.
P values indicate differences between all groups. Slight, but statistically significant improvement was observed in DSS + 13 compared to mice
receiving DSS + 2. Treatment with 2 did not result in significant improvement in colon length in DSS-treated mice (n.s.). (D) Colon length/weight
ratio in 3% DSS study. Data analyzed statistically as in panel C.
mice. Overall it appeared that 13 had a beneficial and
therapeutic effect in the context of intestinal inflammation.
However, an unexpectedly high mortality rate was observed
among DSS-treated animals (Figure 1C). As the efficacy of DSS
treatment varies by the preparation lot, the mouse strain, and
the dosage, the study was repeated using 3% DSS (vide infra).
Equal mortality was observed among all treated groups in the
study with 3% DSS (9 of 10 mice survived per DSS-treated
group, and 10 of 10 survived in the control group). The results
of the second study were consistent with the first study and
demonstrated decreased morbidity and mortality for mice
treated with 13 when compared to the other DSS-treated
groups (Figure 2). Unexpectedly, mice treated with DSS + 2
fared worse than mice treated with DSS and placebo based on
body weight loss (Figure 2A). Mice treated with 13 lost
significantly less weight than those receiving 2 (Figure 2A).
That is, mice treated with 13 were closer to a normal body
weight and, thus, were healthier. Compound 13 was also
superior in terms of delaying the onset and decreasing the
persistence of rectal bleeding (Figure 2B). Colon length was
not a good indicator of improvement, although a positive trend
was seen for mice treated with 13 (p = 0.08), but not for mice
treated with 2 (Figure 2C). No difference was observed in the
colon weight/length ratio between the groups treated with 13
and 2 (Figure 2D).
Inflammation was measured by the release of pro-
inflammatory cytokines from lymphoid cells of the mesenteric
lymph nodes (MLNs) (Figure 3). MLN cells were collected
from mice and exposed to CD3/CD28 activation ligand-bound
Dynabeads in order to expand the T cell population. We then
utilized a mouse multiplex xMAP panel designed for detection
of IFN-γ, IL-17, TNF-α, IL-1ß, IL-6, and MMP8 (neutrophil
collagenase, a surrogate marker of mucosal neutrophil
infiltration). Multiplex cytokine analysis showed a trend toward
a better response in mice treated with 13 compared to mice
treated with 2 (Figure 3A). Quantitative real-time reverse
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ACS Medicinal Chemistry Letters
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Figure 3. (A) Cytokine release from MLN cells stimulated with CD3/CD28 activation beads. (B) qRT-PCR analysis of colonic cytokine mRNA
expression.
transcription PCR (qRT-PCR) analysis of colonic gene
expression demonstrated generally poor and inconsistent
responses to 2. However, a consistently reduced colonic
expression of IFNγ, IL-6, IL-1β, and MMP8 was evident in
mice treated with 13 (Figure 3B), suggesting that mice treated
with 13 produced less pro-inflammatory cytokines compared to
the other DSS-treated groups.
In a preliminary study of metabolism and disposition, 13
(155.7 mg/(kg day)) was administered by gastric gavage to
seven male 129 strain wild-type control mice and the fecal
matter collected daily from the cage bedding. Feces were
extracted with methanol and the extracts analyzed by high
resolution ESI mass spectrometry.27 5-ASA was detected in
feces produced between 24 and 48 h after administration of 13
(see Supporting Information for details).
preliminary metabolism and disposition study. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
* P.R.K.: telephone, 520-626-9687; fax, 520-626-4141; e-mail,
pkiela@peds.arizona.edu. E.A.M.: telephone, 520-621-6321; fax,
520-621-8407; e-mail, emash@email.arizona.edu.
Funding
This work was supported by Grants R33 CA 95944, RO1 CA
97360, RO1 CA 123547, and P30 CA 23074 from NCI and by
RO1 DK 067286 from NIDDK.
Notes
The authors declare no competing financial interest.
These studies with an acute in vivo model of epithelial-injury
mediated colitis strongly suggest that 5-ASA pro-drug 13 may
be superior to 2 and potentially to other drugs currently used
for IBD treatment. We plan to evaluate the efficacy of 13 and of
related constructs in more depth in follow-up studies with this
and other IBD models.
ABBREVIATIONS
■
5-ASA, 5-aminosalicylic acid; CuAAC, copper(I)-catalyzed
azide−alkyne cycloaddition; DSS, dextran sulfate sodium salt;
MLN, mesenteric lymph node
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* Supporting Information
Experimental details of the synthesis and characterization of
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1
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NMR spectra of compounds 5−12, the H NMR spectrum of
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