Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00657

Diacylglycerol lipase-beta (DAGLβ) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLβ inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLβ inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLβ-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of ～5 μg of liposomal KT109 was sufficient to achieve ～80% inactivation of DAGLβ in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLβ inhibitors to macrophages.

Full text of DOI:10.1021/acs.molpharmaceut.7b00657

Article
pubs.acs.org/molecularpharmaceutics
Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to
Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo
Myungsun Shin,† Helena W. Snyder, Giulia Donvito, Lesley D. Schurman, Todd E. Fox,
,#
∥,#
⊥
⊥
‡,§
⊥
‡,§
,†,‡
Aron H. Lichtman, Mark Kester, and Ku-Lung Hsu*
†
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, United States
University of Virginia Cancer Center, University of Virginia, Charlottesville, Virginia 22903, United States
‡
§
∥
Department of Materials Science and Engineering, University of Virginia, Charlottesville, Virginia 22904, United States
⊥
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298 United States
S Supporting Information
*
ABSTRACT: Diacylglycerol lipase-beta (DAGLβ) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce
bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling.
Previously, we demonstrated that DAGLβ inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory
signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we
could exploit the phagocytic capacity of macrophages to localize delivery of DAGLβ inhibitors to these cells in vivo using
liposome encapsulated KT109. We used DAGLβ-tailored activity-based probes and chemical proteomic methods to measure
potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of ∼5 μg of
liposomal KT109 was sufficient to achieve ∼80% inactivation of DAGLβ in macrophages with no apparent activity in other
tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with
free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by
targeted in vivo delivery of DAGLβ inhibitors to macrophages.
KEYWORDS: endocannabinoid, 2-arachidonoylglycerol, diacylglycerol lipase, macrophage, inflammation,
activity based protein profiling, chemical proteomics, diacylglycerol, prostaglandin, eicosanoid, lipid signaling, chronic pain,
neuropathic pain, inflammation, nonsteroidal anti-inflammatory drugs, analgesic, liposome, drug delivery, pain
4
,9−15
INTRODUCTION
inflammation using DAGL-selective inhibitors.
Two
■
1
−4
mammalian isoforms, DAGLα and DAGLβ, have been
Diacylglycerol lipases
(DAGLs) are principal biosynthetic
4
,15
enzymes for the endocannabinoid 2-arachidonoylglycerol (2-
identified, and development of chemical proteomic
and
5
,6
7
2
,3
AG ), a lipid agonist of the GPCRs CB1 and CB2. DAGLs
are transmembrane serine hydrolases that hydrolyze arach-
idonate-esterified diacylglycerols (DAGs) to biosynthesize 2-
AG, which can be further cleaved by downstream enzymes to
release arachidonic acid (AA) for cyclooxygenase (COX)-
mediated eicosanoid production (e.g., PGE2, Figure 1). The
regulation of both 2-AG and AA by DAGLs not only establishes
important cross-talk between endocannabinoid and eicosanoid
genetic tools have enabled investigation of isoform-specific
functions that are separated anatomically and even by cell type
Special Issue: Pharmacology by Chemical Biology
Received: July 31, 2017
Revised: August 21, 2017
Accepted: August 30, 2017
4
,8
signaling but also offers new opportunities for blocking
©
XXXX American Chemical Society
A
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Figure 1. DAGLβ hydrolyzes DAGs to regulate lipid precursors important for inflammation. DAGLβ hydrolyzes diacylglycerol (DAG) to produce 2-
arachidonoylglycerol (2-AG). DAG can signal by activating protein kinase pathways and 2-AG is the predominant lipid messenger for cannabinoid
receptors. 2-AG can be further hydrolyzed by a downstream serine hydrolase (2-AG hydrolase) to produce arachidonic acid (AA), which is a
substrate for cyclooxygenase 1 and 2 enzymes (COX1/2). COX1/2 along with PG synthases biosynthesize eicosanoids including PGE2, which signal
through prostaglandin receptors. Nonsteroidal anti-inflammatory drugs (NSAIDs) block COX1/2 activity.
9
within the same tissue. From these studies, DAGLβ has
emerged as the isoform with a more specialized role in
inflammation because of its enriched expression and activity in
Here, we set out to test whether we could use liposomes to
localize delivery of DAGLβ inhibitors to macrophages in vivo.
We hypothesized that our approach would not only support
macrophages as the site of action but also establish a new
strategy for development of targeted anti-inflammatory agents.
We developed an encapsulation strategy compatible with the
hydrophobic nature of KT109 to produce homogeneous
liposomal formulations suitable for in vivo testing. We used
DAGLβ-tailored activity-based probes and chemical proteomic
methods to evaluate potency and selectivity of liposomal
KT109 across cells and tissues from treated mice. Finally, we
tested whether enhanced delivery of liposomal KT109 to
macrophages in vivo was sufficient to reverse nociceptive
behavior in the mouse LPS inflammatory pain model.
4
9
macrophages and microglia, and its role in regulating
proinflammatory lipid (PGE2) and cytokine (TNF-α) path-
4
ways.
Discovery of first generation DAGLβ inhibitors was enabled
by development of tailored activity-based protein profiling
ABPP) methods to overcome challenges with detection and
(
4
,15
quantitation of native DAGL activity.
In addition to being
highly sensitive, ABPP methods allowed rapid evaluation of
DAGLβ inhibitor potency and selectivity directly in complex
lysates. Using chemical proteomics to guide inhibitor
optimization, the 1,2,3-triazole urea inhibitor KT109 emerged
as the first in vivo-active DAGLβ inhibitor suitable for cell and
animal physiology studies.
adopted this strategy to expand development of covalent as well
as reversible DAGL inhibitors.
using DAGLβ inhibitors showed potent reductions in PGE2 as
well as TNF-α signaling in lipopolysaccharide (LPS)-stimulated
4
,11
Subsequent studies have since
EXPERIMENTAL SECTION
■
Laboratory Animals. Subjects consisted of male C57BL/6J
mice obtained from either Jackson Laboratories (Bar Harbor,
Maine) or breeding pairs in the Virginia Commonwealth
University vivarium for use in LPS pain studies. For selectivity
studies, C57BL/6J mice were obtained from breeding pairs in
the University of Virginia vivarium. Animal experiments were
conducted in accordance with the guidelines of the Institutional
Animal Care and Use Committee of each respective institution.
Materials. KT109, HT-01, and fluorophosphonate-rhod-
10,13,14,16
Cell biology studies
4
macrophages treated with KT109. Treatment of mice with
KT109 produced antinociceptive activity in the lipopolysac-
charide (LPS) inflammatory pain model as well as in nerve
11
injury and paclitaxel neuropathic pain models. These results
are consistent with established roles for PGE2 and TNF-α
inflammatory signaling in sensitization of peripheral sensory
1
amine (FP-Rh) were synthesized and purity confirmed by H-
NMR and HPLC analysis as previously described.
1
7,18
4,15,19
neurons in chronic pain
and the effectiveness of KT109 to
11
block these pathways in vivo. Results from the mouse pain
models also showed that local injection of KT109 in the LPS-
treated inflamed paw reversed allodynic pain responses,
presumably through disruption of DAGLβ function in
Isoflurane (Isothesia, Henry Schein) was purchased from the
Center for Comparative Medicine at University of Virginia.
Brewer thioglycollate medium was purchased from Fluka
Analytical. The following lipids were purchased from Avanti
polar lipids as 25 mg/mL stock solutions in chloroform: 1,2-
17,18
macrophages that accumulate at inflammatory sites.
B
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dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-dis-
tearoyl-sn-glycero-3-phosphocholine (DSPC); and 1,2-dioleoyl-
sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (ammonium salt) (PEG2000 PE). The liposome
extrusion kit and polycarbonate membranes were also
purchased through Avanti polar lipids. Sepharose beads
Gel-Based Competitive Activity Based Protein Profil-
ing (ABPP). Proteomes (1 mg/mL) were treated with either
HT-01 or FP-Rh at 1 μM final concentration for 30 min at 37
°C. The reaction was quenched using SDS-PAGE loading
buffer. After separation by SDS-PAGE (10% acrylamide),
samples were visualized by in-gel fluorescence scanning using a
Chemidoc MP imaging system.
(Sepharose CL-4B, 20% ethanol) were purchased from GE
Evaluation of Liposomal KT109 in LPS Model of
Inflammatory Pain. Mice were given an injection of 2.5 μg of
LPS from Escherichia coli (026:B6, Sigma), in 20 μL of
physiological sterile saline (Hospira Inc., Lake Forest, IL, USA)
into the plantar surface of the right hind paw. As previously
reported, this dose of LPS elicits mechanical allodynia without
producing measurable increases in paw thickness. Mice were
returned to their home cages after the LPS injection. Twenty-
two hours following LPS administration, each mouse was given
an intraperitoneal (i.p.) injection of ghost liposome or
liposomal KT109 (8, 4, or 1 μg) and tested for mechanical
allodynia at 24 h.
Health Care, Inc.
Preparation and Characterization of Liposomal
KT109. Liposomal KT109 and matching “ghost” liposomes
(containing the same lipid components but no compound)
were prepared as described in detail in the methods section of
the Supporting Information. Ghost liposomes were used as
controls for in vitro and in vivo experiments. All liposomal
KT109 stocks were made up as 1 mL sample sizes, limited by
the 1 mL total volume of the extrusion syringes. However,
multiple sets of liposomes were often made simultaneously to
deliver larger quantities of liposomes for experimental use.
Liposomal KT109 size and homogeneity were characterized via
dynamic light scattering (DLS, Malvern Zetasizer, Nano Series)
and nanoparticle tracking analysis (NTA, Malvern NanoSight
LM10). KT109 concentrations in liposomes were determined
by LC−MS as described below. Liposomes were kept
refrigerated and used within a month.
Quantification of KT109 in Liposomal KT109 Stocks
by LC−MS. LC−MS was performed on an I-class Acquity
coupled to a TQ-S mass spectrometer (Waters Corporation,
Milford, MA). Samples were analyzed on a 2.1 mm ID × 5 cm
C8 Kinetex column (Phenomenex) with the column oven set
to 50 °C. Mobile phases consisted of water with 0.1% formic
acid (A) and methanol with 0.1% formic acid (B). A gradient of
20
Behavioral Assessment of Mechanical Allodynia.
Baseline responses to light mechanical touch were assessed
using von Frey filaments following habituation to the testing
21
environment, as described elsewhere. In brief, mice were
acclimated to the testing conditions in which they were given a
daily 30 min habituation session for 4 days. They were placed
under an inverted wire mesh basket, which allowed unrestricted
air flow, that was on top of a wire mesh screen, with spaces 0.5
mm apart. During acclimation and testing, each mouse was
unrestrained and singly housed. The von Frey test utilizes a
series of calibrated monofilaments, (2.83−4.31 log stimulus
intensity; North Coast Medical, Morgan Hills, CA, USA)
applied randomly to the left and right plantar surfaces of the
hind paw for 3 s. Lifting, licking, or shaking the paw was
considered a response.
50% B to 99% B over 1 min was held for 0.5 min before re-
equilibration at 0.5 mL/min. KT109 was detected by multiple
reaction monitoring (MRM) analysis using the 423.3 > 202.1
transition with a collision energy of 10 using argon as the
collision gas. Concentrations of KT109 in liposomal KT109
stocks were estimated to be ∼20 μg/mL by LC−MS (Figure
S1).
In Vivo Studies Using Liposomal KT109. C57BL/6J mice
were injected with thioglycollate solution (4% w/v) in the
peritoneal cavity 4 days prior to compound treatment in order
to recruit sufficient macrophages for analyses. Mice were
treated with ghost or liposomal KT109 (2.5 or 5 μg;
intraperitoneal, i.p.; 4 h), anesthetized with isoflurane,
sacrificed, and thioglycollate-elicited macrophages harvested as
Statistical Analysis. Enzyme activity remaining was
determined by comparing integrated band intensities of
inhibitor with vehicle-treated samples for gel-based ABPP
assays. Linear regression analysis was used to generate KT109
standard curves from LC−MS studies. In the LPS inflammatory
pain study, Holm−Sidak’s multiple comparisons test was used
for post hoc analysis following a significant one-way ANOVA.
The antinociceptive ED50 value 95% confidence limit of
liposomal KT109 was calculated using least-squares linear
regression analysis. The potency ratio with 95% confidence
limit of KT109 between its formulation in liposomes (present
study) and free compound in a standard vehicle (previous
4
11
22
previously described. For selectivity profiling, other tissues
study ) was calculated using linear regression. Data are
shown as mean ± SEM. For analysis of DAGLβ inhibition in
paw, significance was determined by unpaired Student’s t test.
All statistical analyses were performed using GraphPad Prism or
Excel.
were harvested at the same time as macrophages. Macrophages
and tissues were either used immediately or flash frozen in
liquid nitrogen and stored at −80 °C until use.
Preparation of Tissue Proteomes. Tissues were washed
twice with ice cold lysis buffer (0.25 M sucrose, 20 mM
HEPES, and 2 mM DTT in ddH O). Tissues were processed
using dounce homogenization and placed in ice for 15 min.
Tissue homogenates were centrifuged at 800 × g for 5 min at 4
RESULTS
2
■
Encapsulation of KT109 in Liposomes. As KT109 is
insoluble in aqueous media, typical passive loading mechanisms
for liposomal encapsulation were ineffective. We therefore
explored whether KT109 could be trapped within the lipid
bilayer during the dry-down step of the liposome formulation
process. In brief, KT109 was dissolved in chloroform along with
a cocktail of natural and unnatural phospholipids. Next, the
KT109/lipid mixture was dried down under a nitrogen stream.
Addition of aqueous buffer (PBS) followed by shaking and
vortexing produced liposomal KT109 (see Methods section in
°
C. The resulting supernatant was isolated to remove debris.
Supernatants were centrifuged at 100,000 × g for 45 min at 4
C. The supernatant was removed and remaining pellet
°
resolubilized in assay buffer (20 mM HEPES in ddH O) by
2
passing through a 26-gauge syringe multiple times, and referred
to as soluble and membrane fractions, respectively. Protein
concentrations were determined using a Bio-Rad DC protein
assay.
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Figure 2. Characterization of liposomal KT109 by DLS, NTA, and LC−MS. (A) Graph showing the average size distribution by intensity of
liposomal KT109 as determined by dynamic light scattering (DLS). Liposomal KT109 was diluted 1:100 for this analysis. The peak average is 101 ±
2
nm, which corresponds to a Z-average of 89 ± 0.3 nm. (B) Liposomal KT109 size and homogeneity as characterized by nanoparticle tracking
analysis (NTA). Representative scatter graph of four replicate analyses of liposomal KT109 intensity (absorbance unit, a.u.) as a function of size
8
(
nm). (C) Finite track length adjustment (FTLA) analysis of scatter graphs is used to calculate average particle concentration (9 ± 0.5 × 10
particles/mL) and mean particle size (91 ± 2 nm). (D) Representative extracted ion chromatogram from MRM analysis by LC−MS of free KT109
standard compared with KT109 in liposomes. The equivalent retention times confirm the identity of KT109 encapsulated in liposomes. MRM
analysis was performed using a 423 to 202 transition for KT109.
Figure 3. Chemical proteomic evaluation of liposomal KT109 potency and selectivity in vivo. (A) General scheme of competitive gel-based activity
based protein profiling (ABPP). Proteomes were pretreated with inhibitors (30 min at 37 °C) followed by labeling with activity based probe (HT-01
or FP-Rh, 1 μM, 30 min at 37 °C). Probe-labeled proteomes were separated by SDS-PAGE and analyzed by in-gel fluorescence scanning. (B)
Thioglycollate-elicited macrophages were isolated after 4 h treatment with ghost liposomes or liposomal KT109 at the indicated doses. Cells were
lysed and membrane proteomes subjected to gel-based ABPP analysis using HT-01. The degree of inactivation (% inhibition) was quantified using
integrated band intensities from fluorescence gel analyses. (C) Activity and selectivity of liposomal KT109 against serine hydrolases detected in
tissues from treated mice using gel-based ABPP with FP-Rh. Treatment with liposomal KT109 resulted in negligible activity across measured tissues.
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Figure 4. Efficacy and selectivity of liposomal KT109 in the LPS inflammatory pain model. (A) Intraperitoneal (i.p.) administration of liposomal
KT109 reverses LPS-induced allodynia in C57BL/6J mice. *P < 0.05 vs LPS + ghost liposome-treated paw. Data reflect mean ± SEM, n = 13
(
(
control paw), 10 (LPS + ghost liposome), 6 (LPS + liposomal KT109 (1 μg)), 6 (LPS + liposomal KT109 (4 μg)), and 3 (LPS + liposomal KT109
8 μg)). (B) Native DAGLβ activity in LPS-treated and contralateral hind paws (control) from representative mice was measured by gel-based ABPP
using HT-01. The identity of the DAGLβ band detected in paws was confirmed by in vitro treatment of paw proteomes using free KT109 (DAGLβ
4
specific inhibitor) compared with the DAGLβ-inactive control inhibitor KT195 (left panel). Native DAGLβ activity was quantified in paws of mice
treated with ghost liposome or liposomal KT109 under basal and inflammatory states (LPS-injected paw). Intraperitoneal administration of
liposomal KT109 (4 μg) resulted in ∼20% inhibition of total DAGLβ activity in the LPS-treated paw (estimated by ratio of fluorescent signals of
DAGLβ band from paws of liposomal KT109- compared with ghost liposome-treated mice). Liposomal KT109 (4 μg) did not inhibit DAGLβ
activity in contralateral paw (no LPS treatment). *P < 0.05 for DAGLβ activity remaining in LPS-injected versus contralateral paws. Liposomal
KT109 (4 μg, i.p.) showed negligible activity against serine hydrolases in brain, spinal cord, liver, and spleen of treated mice (under inflammatory
states) as measured by gel-based ABPP using HT-01 (C) and FP-Rh (D).
the Supporting Information for more details). Although this
method provided us a route for liposomal KT109 formulation,
the method proved to be very inefficient with a large portion of
compound remaining suspended within the PBS solution rather
than entrapped within the lipid bilayer during spontaneous
liposome formation upon rehydration. To counter this issue,
we used a higher starting concentration of KT109 (5 mg/mL)
to overcome limitations resulting from encapsulation ineffi-
ciency. More than 99% of the nonencapsulated KT109 is
separated from liposomal KT109 during the liposome extrusion
step, remaining trapped on one side of the polycarbonate
membrane. The remaining free drug is separated from the
liposome portion during size-exclusion chromatography to
yield pure liposomal KT109.
± 2 nm in diameter, which corresponds to a Z-average value of
89 ± 0.3 nm in diameter. Like DLS, NTA measures the
Brownian motion of particles in a suspension and their speed
related to the Stokes−Einstein equation. However, unlike DLS,
which can only measure time-dependent fluctuations in
scattering intensity, NTA can measure individual particle
motion by video. NTA, therefore, is able to provide a more
quantitative analysis as well as enhanced size resolution. For
NTA, a laser beam is passed through the liposomal suspension.
As the liposomes scatter light, they may be visualized by
microscope. A representative distribution plot of liposome
intensity as a function of size as measured by NTA is shown in
Figure 2B. As shown in Figure 2C, we used NTA finite track
length adjustment (FTLA) analysis to calculate the average
Characterization of Liposomal KT109. To determine the
size and homogeneity of liposomal KT109, we measured these
parameters by both dynamic light scattering (DLS) and
nanoparticle tracking analysis (NTA). As shown in Figure 2A,
the DLS profile revealed a single peak that centers around 101
8
particle concentration (9 ± 0.5 × 10 particles/mL) and a mean
particle size (91 ± 2 nm) for a typical liposomal KT109 sample.
In summary, characterization of liposomal KT109 using
complementary DLS and NTA techniques confirm that stable,
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homogeneous liposomes of desired diameter are routinely and
reproducibly fabricated.
treatment with 4 and 8 μg of liposomal KT109 significantly
attenuated LPS-induced allodynia compared with mice
receiving LPS and ghost liposomes. At 8 μg of KT109, we
observed full reversal of LPS-induced allodynia (Figure 4A).
Using these data, the calculated ED50 value for liposomal
KT109 was 11 μg/kg (9−13 μg/kg; 95% confidence limits),
which represents a >100-fold enhancement in potency
To determine the concentration of KT109 encapsulated in
liposome, we developed a targeted multiple-reaction monitor-
ing (MRM) LC−MS method to quantify KT109 concen-
trations in liposome samples (423 to 202 MRM transition,
Figure 2D). We first generated a standard curve of free KT109
and showed high correlation between KT109 concentrations
11
compared with treatments using free KT109.
2
and MRM signal intensities from 0.0025 to 0.25 μg/mL (R =
We also confirmed that liposomal KT109 was active in the
LPS-injected paw by directly measuring native DAGLβ activity
at this site by gel-based ABPP using HT-01 (Figure 4B). First,
we confirmed that the ∼70 kDa band detected in paw
proteomes represented endogenous DAGLβ by demonstrating
that pretreatment with KT109 (DAGLβ inhibitor) but not
KT195 (DAGLβ-inactive control inhibitor) in vitro blocked
HT-01 probe labeling (left panel, Figure 4B). Next, we
measured DAGLβ activity in contralateral (control) and LPS-
injected paws of mice treated with ghost liposome or liposomal
KT109. We observed a modest but significant decrease in
endogenous DAGLβ activity (∼20% inhibition, P = 0.037) in
the LPS-injected but not contralateral control paw (right
panels, Figure 4B). Future studies are needed to determine
whether increased accumulation of macrophages at inflamma-
tory sites is a likely mechanism explaining why liposomal
KT109 blocks DAGLβ activity only in the LPS-injected paw. In
contrast to activity in the LPS-injected paw, we observed no
apparent inhibition of other SHs in central (brain and spinal
cord) and peripheral (liver and spleen) tissues from liposomal
KT109-treated mice as measured by gel-based ABPP (Figure
0
.99, Figure S1). Using our standard curve, we were able to
estimate the concentration of encapsulated KT109 as ∼20 μg/
mL (Figure S1). We calculated encapsulation efficiency based
on the ratio of total encapsulated (20 μg) versus starting (5
mg) KT109 amounts, which results in an estimated efficiency of
∼
0.4%. Future studies will be required to improve the
formulation process and increase the amount of KT109 that
is effectively incorporated into liposomes.
Chemical Proteomic Analysis of Liposomal KT109.
Using our first generation liposomal KT109 formulation, we
asked how liposome delivery would impact KT109 potency and
selectivity in vivo. For these analyses, we evaluated native
DAGLβ and other serine hydrolase (SH) activities by gel-based
4
competitive ABPP using DAGL-tailored (HT-01 ) and broad
2
3
spectrum (FP-Rh ) SH probes (Figure 3A). ABPP analysis
provides a molecular snapshot of liposomal KT109 bioavail-
ability and selectivity in vivo because covalent enzyme
inactivation by KT109 during treatment is not affected by
tissue lysis and processing. Mice were treated with ghost
liposome or liposomal KT109 (2.5 or 5 μg) for 4 h, animals
sacrificed, and tissues processed and analyzed by gel-based
ABPP assay (Figure 3A). Remarkably, we observed dose-
dependent inhibition of DAGLβ in peritoneal macrophages
despite these low amounts of administered KT109 with ∼80%
inhibition observed at the 5 μg dose (Figure 3B). Compared
with administration of free KT109 (5 mg kg dose ),
liposomal delivery reduced the amount of total KT109 required
for comparable DAGLβ inactivation in peritoneal macrophages
by >20-fold (∼120 vs 5 μg of KT109 in free- compared with
liposomal-delivery, respectively).
4
C,D). Collectively, our results help support that liposomal
delivery of KT109 dramatically enhances in vivo efficacy and
selectivity and that the LPS-injected paw is the likely site of
action for reversal of LPS-induced allodynia.
−
1
4
DISCUSSION
■
4
DAGLβ expression and activity is enriched in macrophages,
providing an opportunity to exploit the phagocytic capacity of
25
these immune cells for targeted delivery of inhibitors. We
explored liposomal delivery of KT109 with the goal of lowering
the total amount of compound required for DAGLβ
inactivation in macrophages in vivo. The effective increase in
potency using liposomal KT109 would allow for more localized
blockade of PGE2 and TNF-α signaling at inflammatory sites
We observed excellent selectivity for liposomal KT109 across
other tissues profiled (Figures 3C and S2−S5). We measured
tissues where DAGLβ is known to be active (liver, brain ) as
2
,4
9
well as tissues that are potential liabilities for compound toxicity
(kidney, heart). In all tissues profiled, we observed negligible
17,18
cross-reactivity with the exception of a minor off-target activity
against a ∼35 kDa serine hydrolase in kidney (Figure S2) and
heart (Figure S3) proteomes that is likely ABHD6 based on
molecular weight and reactivity with HT-01 probe. ABHD6 is
a known off-target for several mechanism-based inhibitors
where macrophages are known to accumulate.
We discovered that systemic delivery of ∼5 μg of KT109 in
liposomes was sufficient to achieve ∼80% inactivation of
DAGLβ in macrophages. Enhanced delivery of liposomal
KT109 to macrophages in vivo was sufficient to reverse
nociceptive behavior in the mouse LPS inflammatory pain
model. The calculated ED50 value for liposomal KT109 was 11
μg/kg (9−13 μg/kg; 95% confidence limits), which represents
a >100-fold enhancement in potency compared with treatments
24
4
including triazole ureas like KT109. Gel-based ABPP analysis
of brain and liver proteomes from liposomal KT109-treated
mice did not reveal any overt off-target activity (Figures 3C and
S4−S5). In summary, we discovered that liposomal delivery of
KT109 dramatically enhances potency against DAGLβ in
macrophages while minimizing cross-reactivity in other tissues
where DAGLβ is active.
Liposomal KT109 Exhibits High Potency in Reversing
LPS-Induced Mechanical Allodynia. Next, we examined
whether liposomal KT109 would reverse LPS-induced
mechanical allodynia with increased potency compared with
11
using free KT109. While active in macrophages, the use of
liposomal delivery resulted in negligible activity of KT109 in
central as well as peripheral tissues under basal (Figures 3C and
S2−S5) and inflammatory states (Figure 4C,D). The dramatic
increase in potency supports our hypothesis that systemic
administration of liposomal KT109 results in accumulation and
targeted disruption of DAGLβ activity in macrophage in vivo.
As a result, we can reduce the total amount of KT109 required
for efficacy to minimize cross-reactivity in other tissues and
improve overall selectivity in vivo.
11
free compound from our recent study. As shown in Figure
A, KT109 significantly reversed LPS-induced allodynia
F(4,33) = 32, p < 0.0001]. Post hoc testing revealed that
4
[
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We also demonstrated that chemical proteomic methods
were critical for evaluating the cells and tissues where liposomal
KT109 is active. Importantly, the use of HT-01-based ABPP
analysis allowed us to confirm potent inactivation of DAGLβ in
macrophages as well as to measure general off-target activity
against other detected SHs in central and peripheral tissues. An
interesting finding from our chemical proteomics analyses was
detection of DAGLβ inhibition by liposomal KT109 only in the
LPS-injected paw (Figure 4B). While we cannot fully explain
why DAGLβ activity was unaffected in control paws, our
current findings support macrophages as the likely site of action
for the antinociceptive activity of liposomal KT109. Future
studies will focus on isolation of macrophages from paws of
treated mice to further clarify the specificity of liposomal
KT109 for targeting macrophages versus other cell types at
inflammatory sites. We are also cognizant that DAGLβ
inactivation at other sites in addition to the paw may contribute
to the observed efficacy in vivo. Future studies are needed to
investigate whether changes in macrophage trafficking and/or
phagocytic capacity affects liposomal KT109 access and
clearance during inflammation, and whether this could
represent a general strategy to target compounds to
inflammatory sites. Incorporation of targeting agents, e.g.,
ACKNOWLEDGMENTS
■
We thank all members of the Hsu Lab and colleagues at the
University of Virginia for helpful discussions and review of the
manuscript. This work was supported by the University of
Virginia (start-up funds to K.-L.H.), VCU School of Pharmacy
(
start-up funds to A.H.L.), NanoSTAR (to H.S.), and National
Institutes of Health (DA035864 to K.-L.H.; 1F31NS095628 to
L.D.S.; T32DA007027, P30DA033934, and R01NS093990 to
A.H.L.).
ABBREVIATIONS
■
ABPP, activity based protein profiling; DAGLβ, diacylglycerol
lipase-beta; DAGLα, diacylglycerol lipase-alpha; AA, arach-
idonic acid; COX, cyclooxygenase; DAG, diacylglycerol;
GPCR, G-protein-coupled receptor; LPS, lipopolysaccharide;
FP-Rh, fluorophosphonate-rhodamine; DOPE, 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine; DSPC, 1,2-distearoyl-sn-glyc-
ero-3-phosphocholine; PEG2000 PE, 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy(polyethylene glycol)-
2
000]; i.p., intraperitoneal; DLS, dynamic light scattering;
NTA, nanoparticle tracking analysis; FTLA, finite track length
adjustment; MRM, multiple-reaction monitoring; LC−MS,
liquid chromatography−mass spectrometry; NSAIDs, non-
steroidal anti-inflammatory drugs; TNF-α, tumor necrosis
factor-alpha; PGE2, prostaglandin E2; PLGA, poly(lactide-co-
glycolide); PLA/PEO, poly(lactic acid)−poly(ethylene oxide)
26
antibodies against the macrophage-specific marker F4/80, in
future studies should further enhance the utility of this
approach to target specific macrophage subsets.
While our preliminary studies using liposomal KT109 were
successful, we recognize the low encapsulation efficiency of our
current formulation process. Currently the encapsulation
efficiency of KT109 within the formulated liposomes is
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ORCID
Ku-Lung Hsu: 0000-0001-5620-3972
Author Contributions
These authors contributed equally.
#
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.molpharmaceut.7b00657
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