Intestinally targeted diacylglycerol acyltransferase 1 (DGAT1) inhibitors robustly suppress postprandial triglycerides

Article abstract of DOI:10.1021/ml3000512

High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented.

Full text of DOI:10.1021/ml3000512

Letter
pubs.acs.org/acsmedchemlett
Intestinally Targeted Diacylglycerol Acyltransferase 1 (DGAT1)
Inhibitors Robustly Suppress Postprandial Triglycerides
Michael H. Serrano-Wu,*,† Gary M. Coppola,† Yongjin Gong,† Alan D. Neubert,† Ricardo Chatelain,‡
Kevin B. Clairmont,‡ Renee Commerford,‡ Theresa Cosker,§ Thomas Daniels,‡ Ying Hou,† Monish Jain,§
Marlene Juedes,∥ Lisha Li,‡ Tara Mullarkey,‡ Erik Rocheford,‡ Moo Je Sung,† Andrew Tyler,‡ Qing Yang,‡
Taeyoung Yoon,† and Brian K. Hubbard‡
‡
§
†Departments of Global Discovery Chemistry, Cardiovascular and Metabolism, Metabolism and Pharmacokinetics, and
∥Translational Sciences, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge Massachusetts 02139,
United States
S
* Supporting Information
ABSTRACT: High DGAT1 expression levels in the small
intestine highlight the critical role this enzyme plays in nutrient
absorption. Identification of inhibitors which predominantly
inhibit DGAT1 in the gut is an attractive drug discovery strategy
with anticipated benefits of reduced systemic toxicity. In this
report we describe our discovery and optimization of DGAT1
inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The
impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented.
KEYWORDS: DGAT1, triglyceride synthesis, efflux
rally ingested triglycerides (TG) undergo hydrolysis and
then are reassembled within enterocytes into TG-rich
potential viability of this strategy with regard to efficacy and
safety in multiple preclinical models.
High-throughput screening efforts using recombinant human
DGAT1 enzyme identified the benzimidazole 1 (DGAT1 IC50
= 1.3 μM; DGAT2 IC50 > 20 μM; Figure 1) as a potential
O
chylomicrons destined for systemic circulation. The final
committed step in triglyceride biosynthesis is known to be
mediated by at least two distinct intracellular acyl-coA
diacylglycerol acyltransferases (DGATs), namely DGAT11
and DGAT2.2 Since the development of whole-body knockout
models of these enzymes, there has been intense evaluation of
pharmacological approaches to modulate their activity.3−8 For
DGAT1, this interest is inspired by the favorable metabolic
phenotype of DGAT1−/− mice,9 which are resistant to diet-
induced body weight gain,10 are more insulin-sensitive relative
to wild-type littermates,11 and exhibit a reduced rate of
chylomicrons formation when challenged with lipid nutrients.12
Interestingly, all aspects of this phenotype are lost when
DGAT1 is reintroduced via a tissue-specific promoter into the
intestines of female DGAT1−/− mice, implying that intestinal
DGAT1 plays a crucial role in the effects observed in the
whole-body knockout model.13 Indeed, DGAT1 mRNA
expression levels have been shown to be high in regions of
the small intestine in mice and humans.14,15 Selective inhibition
of intestinal DGAT1 therefore becomes an intriguing drug
discovery approach to recapitulate aspects of the DGAT1−/−
mouse, especially if this gut-specific inhibition reduces the
potential risk of on- and off-target activity for candidate
molecules. Particularly relevant for DGAT1 pharmacological
inhibition is the observation of functional and morphological
abnormalities in the fur and sebaceous glands of DGAT1−/−
mice.16 In this report we describe a novel approach to
specifically inhibit intestinal DGAT1, and demonstrate the
Figure 1. Benzimidazole-based DGAT1 inhibitors.
starting point for optimization. Initial structural modifications
demonstrated that both the ethyl carbamate and the 2,6-
dichlorophenyl substituents on the benzimidazole core could be
replaced without substantial loss of activity (i.e., 2, DGAT1
IC50 = 1.4 μM), and in fact introducing an additional
substituent at the 4-position of the 2,6-dimethylphenyl ring
led to an improvement in potency (3, IC50 = 0.5 μM).
As several reported DGAT1 inhibitor scaffolds feature a
carboxylic acid group, we developed a synthetic strategy to
Received: February 25, 2012
Accepted: April 4, 2012
Published: April 4, 2012
© 2012 American Chemical Society
411
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Assembly of C-4 Functionalized Benzimidazole Derivatives
a
Reagents and condtions: (a) MeLi/LiBr, THF, s-BuLi, DMF; (b) 1 N NaOH, MeOH; (c) 48% HBF4, NaNO2, water, Pd(OAc)2, MeOH, methyl
acrylate; (d) H2, Pd/C, CH2Cl2, 1 atm; (e) BrCH2COOMe, K2CO3, DMF; (f) 11, 13, or 14, Yb(OTf)3, DMSO.
access a variety of carboxylic acid trajectories via the synthetic
routes described in Scheme 1. Transmetalation of 8a followed
by reaction with DMF furnished aldehyde 8b. After hydrolytic
removal of the trifluoroacetate group, diazotization of aniline 9
was immediately followed by Heck coupling with methyl
acrylate and then hydrogenation to afford 11.17 The aniline 9
could also be alkylated with methyl bromoacetate to afford 13,
and the oxygen-linked analogue 14 was prepared in a similar
fashion.18 A convergent assembly of the 1,2-phenylene diamine
15 and C4-functionalized benzaldehyde derivatives under
oxidative cyclization conditions afforded the fully elaborated
benzimidazole carboxylic acid targets 4−6.19,20
The introduction of a carboxylic acid at the 4-position of the
2,6-dimethylphenyl ring led to a dramatic enhancement of
inhibitor potency, with the oxyacetic acid derivative 4 among
the most potent analogues in this series (IC50 = 28 nM, Table
1). Despite exceptional biochemical potency, however,
carboxylic acid derivatives 4−6 only weakly inhibited
triglyceride synthesis in a C2C12 mouse myoblast cellular
assay, whereas more permeable inhibitors such as 3 (PAMPA;
cFA = 89%) showed comparable if not improved cellular
potency relative to the enzymatic assay. Carboxylate inhibitors
including 4 (measured pKa21 = 4.8 and 10.7) are zwitterionic at
physiological pH and presumably unable to passively penetrate
cell membranes (PAMPA; cFA < 32%).
However, additional in vitro profiling of 4−6 revealed an
unanticipated difference among these benzimidazole carbox-
ylate inhibitors. In the bidirectional Caco-2 monolayer assay,
compounds 4 and 5 showed approximately equal permeability
in the absorptive (A−B) and nonabsorptive (B−A) directions,
whereas the carbon-linked analogue 6 uniquely exhibited 18-
fold higher permeability in the nonabsorptive direction. The
high (B−A)/(A−B) ratio suggests the potential for transporter-
mediated efflux to influence the intestinal disposition of 6,
whereas compounds 4 and 5 are predicted by the Caco-2 assay
to be passively transported. It is remarkable that a single atom
replacement (O and N vs C) leading to a subtle but measurable
increase in carboxylic acid pKa (pKa difference of +0.5 and +0.8
with respect to 6) can potentially account for such dramatic
differences in the Caco-2 permeability assay.
The pharmacological relevance of this distinct Caco-2
permeability data was assessed in an acute rat model of
intestinal DGAT1 activity (Figure 2). Male Sprague−Dawley
rats were dosed with inhibitors 4−6 (5 mg/kg) 2 h prior to
receiving an oral Intralipid challenge (10 mL/kg). In vehicle-
treated control animals, a robust plasma triglyceride excursion
was observed with peak levels reached 2 h after caloric
challenge (see the Supporting Information for the time course).
Interestingly, only compound 6 blunted the Intralipid-induced
triglyceride excursion (73% reduction relative to vehicle at t = 2
h) whereas plasma triglyceride levels following oral admin-
istration of 4 or 5 were indistinguishable from vehicle. Higher
doses of 4 or 5 were not investigated in this model.
Table 1. In Vitro Potency and Permeability Profile of
Compounds 3-6
3
4
5
6
DGAT1 IC50 (nM)
Cell IC50 (nM)
500
120
89
28
56
28
>10,000
17
2,000
<32
0.65
1.2
6,500
22
a
PAMPA cFA (%)
Caco-2 (A−B) (10−6 cm/s)
Caco-2 (B−A) (10−6 cm/s)
pKa
1.3
0.55
10.0
4.3
2.1
A potential explanation for these divergent pharmacody-
namic effects is that transporter-mediated efflux effectively
recycles 6 in the intestine, leading to significantly higher local
concentrations of 6 compared to compound 5 in DGAT1-
4.8
5.1
a
cFA = calculated fraction absorbed. See the Supporting Information
for a description of the cellular TG synthesis assay.
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sustained enterocyte levels of 7 even 17 h following compound
oral administration support the notion of repeated cycling of
the compound throughout the length of the small intestine.
Interestingly, concentrations of 7 were uniformly higher in
enterocytes harvested from the jejunum, providing additional
evidence for recycling of 7. The high lipophilicity of 7 (log D21
= 2.6 at pH 6.8) may enrich concentration in membrane
structures including the endoplasmic reticulum, the presumed
intracellular site of action for DGAT1 inhibition. Meanwhile,
the low portal vein concentrations highlight that the unique
absorption characteristics of 7, rather than first-pass metabo-
lism, underly the vanishingly low plasma concentrations of 7.
Compounds 4−7 all show clearance from plasma well below
hepatic blood flow following intravenous administration (Clp
for 7 = 10 mL/min/kg in rats; Clp for 4−6 all <19 mL/min/kg
in rats), which argues against hepatic extraction contributing to
the low circulating levels of 7 following oral dosing.
Encouraged by the efficacy profile of 7 in rodents, the
pharmacodynamic effect of compound 7 was further evaluated
in a dog model of hypertriglyceridemia. In this model, beagle
dogs were given a high-fat challenge 30 min after receiving an
oral 1 mg/kg dose of compound 7. The plasma triglyceride
level at baseline was 35.4 4.7 mg/dL in four dogs treated with
vehicle, and a marked increase in triglycerides ensued for at
least 6 h following caloric challenge (Figure 3). Oral
Figure 2. Plasma triglyceride levels 2 h following Intralipid challenge
for compounds 4−6 (5 mg/kg). *p < 0.05 for compound 6.
expressing enterocytes. This unique recycling allows for
effective blunting of intracellular triglyceride synthesis despite
the intrinsic low permeability of 6 and weaker cellular potency
relative to nontransported inhibitors such as 5.
During the course of our optimization efforts, we identified
the closely related analogue 7, which was 17-fold more potent
than 6 in inhibiting cellular triglyceride synthesis (DGAT1 IC50
= 39 nM; cell IC50 = 390 nM; see Table 2). Consistent with our
Table 2. Rat Enterocyte and Plasma Concentrations of
Compound 7 Following a 5 mg/kg Oral Dose
a
time (h) duodenum (nM) jejunum (nM) p. vein (nM) plasma (nM)
2
6
500
630
610
930
2020
1400
22
8
16
7
b
17
5
BDL
a
b
Portal vein. BDL = below detection limit (<3 nM).
Figure 3. Plasma triglycerides in dogs administered compound 7 (1
mg/kg, 0.5% carboxymethylcellulose suspension, solid line) or vehicle
(dotted line) followed by lipid challenge. Plasma concentrations of 7
shown in red. Intrinsic biochemical potency of 7 (39 nM) indicated
with dashed red line.
earlier findings, the propanoic acid derivative 7 also exhibited
strong efflux potential in Caco-2 cells, and this disposition was
independently confirmed in a MDR1-MDCK assay where the
P-glycoprotein transporter is specifically overexpressed
(MDCK efflux ratio = 47).22 We cannot rule out the
participation of other transporters such as MRP2 and BCRP,
which are known to also function on the apical membrane of
enterocytes.23
preadministration of compound 7 (0.5% carboxymethylcellu-
lose suspension vehicle) robustly suppressed the plasma TG
excursion compared to vehicle alone, leading to a mean
To better understand the potential role of transporter-
mediated efflux on local enterocyte concentrations in vivo, we
measured enterocyte and portal vein concentrations of 7
following a 5 mg/kg oral dose to rats (0.5% methyl cellulose/
0.1% Tween-80 suspension vehicle). Tissue samples were
extensively washed to minimize the contribution of unabsorbed
compound to enterocyte concentration measurements. Two
hours after an Intralipid challenge, the concentration of 7 (500
nM) in duodenal enterocytes was well above the intrinsic
potency of 7 (39 nM) and also 23-fold higher than the level
detected in portal vein and plasma (Table 2). At this time
point, intracellular triglyceride levels were significantly lower in
rats dosed with 7 (67.8% reduction compared to vehicle),
confirming robust inhibition of DGAT1 at this time point. The
AUC0−6h reduction of 75
6% (p < 0.005, paired t test).
Consistent with our observations for 7 in rats, robust
pharmacodynamic effects were seen up to 6 h in dogs despite
plasma levels of 7 that were below the intrinsic biochemical
potency (39 nM) and cellular potency (390 nM) of 7
throughout the postprandial period. Despite the complete
blunting of postprandial triglycerides in plasma, no visible
steatorrhea was observed in the any of the studies described
above.
A potential advantage of our gut-targeting approach is to
reduce risk of on- or off-target effects that may be driven by
systemic exposure. We studied the toxicological effects of 7
over 2 weeks in rats at doses exceeding the pharmacologically
active range (10, 30, and 100 mg/kg; 5% hydroxypropyl β-
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cyclodextrin solution vehicle). At the 100 mg/kg dose, which is
at least 20-fold higher than the dose required for a
pharmacodynamic effect in rats, no adverse effects were
observed following in-life observation, hematology, and clinical
chemistry. Thus, intestinally targeted DGAT1 inhibitors have
the potential to favorably impact postprandial metabolism with
a large therapeutic margin. Interestingly, appreciable plasma
levels of 7 were observed at the highest dose level examined
(AUC0−24h = 9800 nM·h), suggesting that the mechanism(s)
which limits the systemic exposure of 7 can be saturated at
superpharmacological doses.
We have thus demonstrated that intestinally restricted
DGAT1 inhibitors can exert robust effects in rodent and
nonrodent preclinical models of hypertriglyceridemia. While
this single inhibitor cannot definitively establish the contribu-
tion of each DGAT1-expressing tissue to overall energy
metabolism, the profound postprandial effects of an intestinally
restricted inhibitor certainly underscore the importance of
intestinal DGAT1 in modulating nutrient handling. A better
understanding of the role of nongut tissues expressing DGAT1
awaits the development of inhibitors with diverse pharmaco-
kinetic signatures to potentially modulate DGAT1 activity in a
tissue-specific fashion.
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization data for all new compounds and biological
assay information. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (617) 871-7523. E-mail: michael.serrano-wu@
novartis.com.
■
Author Contributions
All authors contributed to the writing of the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Brian Jones and Jason Elliott for helpful suggestions
to this manuscript, as well as members of the MAP-ADME
group for profiling support.
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