VBP15: Preclinical characterization of a novel anti-inflammatory delta 9,11 steroid

Article abstract of DOI:10.1016/j.bmc.2013.02.009

Δ9,11 modifications of glucocorticoids (21-aminosteroids) have been developed as drugs for protection against cell damage (lipid peroxidation; lazaroids) and inhibition of neovascularization (anecortave). Part of the rationale for developing these compounds has been the loss of glucocorticoid receptor binding due to the Δ9,11 modification, thus avoiding many immunosuppressive activities and deleterious side effect profiles associated with binding to glucocorticoid and mineralocorticoid receptors. We recently demonstrated that anecortave acetate and its 21-hydroxy analog (VBP1) do, in fact, show glucocorticoid and mineralocorticoid receptor binding activities, with potent translocation of the glucocorticoid receptor to the cell nucleus. We concluded that Δ9,11 steroids showed novel anti-inflammatory properties, retaining NF-κB inhibition, but losing deleterious glucocorticoid side effect profiles. Evidence for this was developed in pre-clinical trials of chronic muscle inflammation. Here, we describe a drug development program aimed at optimizing the Δ9,11 chemistry. Twenty Δ9,11 derivatives were tested in in vitro screens for NF-κB inhibition and GR translocation to the nucleus, and low cell toxicity. VBP15 was selected as the lead compound due to potent NF-κB inhibition and GR translocation similar to prednisone and dexamethasone, lack of transactivation properties, and good bioavailability. Phamacokinetics were similar to traditional glucocorticoid drugs with terminal half-life of 0.35 h (mice), 0.58 h (rats), 5.42 h (dogs), and bioavailability of 74.5% (mice), and 53.2% (dogs). Metabolic stability showed ≥80% remaining at 1 h of VBP6 and VBP15 in human, dog, and monkey liver microsomes. Solubility, permeability and plasma protein binding were within acceptable limits. VBP15 moderately induced CYP3A4 across the three human hepatocyte donors (24-42%), similar to other steroids. VBP15 is currently under development for treatment of Duchenne muscular dystrophy.

Full text of DOI:10.1016/j.bmc.2013.02.009

Bioorganic & Medicinal Chemistry 21 (2013) 2241–2249
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
VBP15: Preclinical characterization of a novel anti-inflammatory
delta 9,11 steroid
Erica K. M. Reeves a, , Eric P. Hoffman a,b,d, Kanneboyina Nagaraju a,b,d, Jesse M. Damsker a, John M. McCall a,c
⇑
a ReveraGen BioPharma, Inc., 9700 Great Seneca Hwy Rockville, MD 20910, United States
b Center for Genetic Medicine Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC 20010, United States
c PharMac, LLC, P.O. Box 2253, Boca Grande, FL 33921 United States
d Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC 20052, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
D9,11 modifications of glucocorticoids (21-aminosteroids) have been developed as drugs for protection
Received 24 December 2012
Revised 3 February 2013
Accepted 11 February 2013
Available online 18 February 2013
against cell damage (lipid peroxidation; lazaroids) and inhibition of neovascularization (anecortave). Part
of the rationale for developing these compounds has been the loss of glucocorticoid receptor binding due
to the
D9,11 modification, thus avoiding many immunosuppressive activities and deleterious side effect
profiles associated with binding to glucocorticoid and mineralocorticoid receptors. We recently demon-
strated that anecortave acetate and its 21-hydroxy analog (VBP1) do, in fact, show glucocorticoid and
mineralocorticoid receptor binding activities, with potent translocation of the glucocorticoid receptor
Keywords:
Glucocorticoids
VBP15
Steroids
Anti-inflammatories
Neuromuscular disease
to the cell nucleus. We concluded that
ing NF- B inhibition, but losing deleterious glucocorticoid side effect profiles. Evidence for this was
developed in pre-clinical trials of chronic muscle inflammation. Here, we describe a drug development
program aimed at optimizing the 9,11 chemistry. Twenty 9,11 derivatives were tested in in vitro
screens for NF- B inhibition and GR translocation to the nucleus, and low cell toxicity. VBP15 was
selected as the lead compound due to potent NF- B inhibition and GR translocation similar to prednisone
D9,11 steroids showed novel anti-inflammatory properties, retain-
j
D
D
j
j
and dexamethasone, lack of transactivation properties, and good bioavailability. Phamacokinetics were
similar to traditional glucocorticoid drugs with terminal half-life of 0.35 h (mice), 0.58 h (rats), 5.42 h
(dogs), and bioavailability of 74.5% (mice), and 53.2% (dogs). Metabolic stability showed P80% remaining
at 1 h of VBP6 and VBP15 in human, dog, and monkey liver microsomes. Solubility, permeability and
plasma protein binding were within acceptable limits. VBP15 moderately induced CYP3A4 across the
three human hepatocyte donors (24–42%), similar to other steroids. VBP15 is currently under develop-
ment for treatment of Duchenne muscular dystrophy.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Anecortave was developed for inhibition of neo-vascularization
in age-related macular degeneration as a molecule that lacked
D
9,11 Steroids (21-aminosteroids) have been designed and
immunomodulatory glucocorticoid activity.10,11 The mechanism
of action is thought to be through inhibition of proliferation of vas-
cular endothelial cells, inhibition of uPA and matrix metallopro-
teinase 3, and stimulation of plasminogen activator inhibitor-1.11
Controlled clinical trials of posterior juxtascleral depot (delivered
at 6 month intervals) showed no safety issues, but marginal clinical
benefit.12,13 More recently, blinded controlled trials of anecortave
in steroid-induced elevated intraocular pressure (IOP) have been
carried out, with similar results of good safety profile.14 The best
dose of anecortave acetate showed a 31% reduction in IOP and this
developed to stably incorporate into cell membranes and inhibit li-
pid peroxidation without glucocorticoid or mineralocorticoid
activities, thus avoiding side effects associated with traditional cor-
ticosteroids.1 A few
D9,11 steroids brought to clinical trials were
Lazaroids and Anecortave. Lazaroids have extensive hydrophilic
head groups designed to optimize stability in membranes and lipid
peroxidation inhibitory activities (Fig. 1). Randomized controlled
trials of tirilazad mesylate in acute spinal cord injury,2,3 and in
stroke4–6 showed good safety profiles, but failed to show clear evi-
dence of efficacy. Pre-clinical work in additional indications con-
tinues, including multiple wound states and lung disease.7–9
benefit lasted
administration.15,16
Previous drug development programs pursuing lazaroids and
anecortave as prototype 9,11 glucocorticoids (21-aminosteroids)
have assumed that the 9,11 modification inhibited drug binding
to the glucocorticoid receptor (GR) and mineralocorticoid receptor
a mean duration of 56 days from a single
D
D
⇑
Corresponding author. Tel.: +1 (202) 476 1236; fax: +1 (240) 453 6208.
E-mail address: erica.reeves@reveragen.com (E.K.M. Reeves).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.009

2242
E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
(
)
Panel A
Panel B
OAc
O
O
OAc
O
OAc
TMS
H
H
H
OTMS
CH3
CH3
a, b
c
O
O
O
2
3TR (commercial)
3
d,e,f,g,h
O
O
OAc
OH
CH3
i
H
OH
H
OH
CH3
O
O
4
VBP 15
Figure 1. Structures and synthesis of
D
9,11 compounds. Panel A: Shown are two previously developed
D9,11 steroids (Anecortave, Tirilizad), and the lead compound
identified in the current study (VBP15). The
D
9,11 bond distinguishing these drugs from other steroidal compounds is shown by the black arrows. The lower panel shows the
chemical modifications to the chemical backbone. The R1, R2, and R3 residues shown are those that were chemically modified with alternative chemical structures (see red
box). Panel B: The synthesis of VBP15 that is pictured is typical for the synthesis of analogs in this series. (a) TMS imidazole, MeMgCl, THF; (b) CuAc2, H2O, DMPU, MeMgCl,
THF; (c) peracetic acid, toluene, ꢀ10 °C; (d) NaHSO3, TFA; (e) EtOAc, heptanes; (f) acetonitrile trituaration; (g) HBr, HC2Cl2, 40 °C; (h) MeOH crystallization; (i) K2CO3, MeOH,
followed by HCL and crystallization from MeOH/H2O.
(MR). This assumption was based largely on the lack of efficacy of
the drugs in inhibiting LPS-induced inflammation in pre-clinical
models—traditional glucocorticoids are impressively effective at
blocking lipopolysaccharide (LPS)-induced acute inflammation.10
However, we recently showed that both anecortave acetate and
its 21-hydroxy analog (VBP1) showed relatively high affinity bind-
by mRNA microarray, targeted RT-PCR, or luciferase reporter con-
structs.17 On the other hand, both drugs retained anti-inflamma-
tory activity via inhibition of NF-jB pathways, and showed
efficacy in inhibiting inflammation in vivo in two mouse models
of chronic inflammation in muscle (dystrophin-deficient mdx and
dysferlin-deficient SJL mice).17 The discrepancy between previous
findings of lack of anti-inflammatory activity, and our findings of
retention of anti-inflammatory activity may be explained by the
assays utilized. For example, McNatt et al. studied an acute model
of LPS-induced IL-1 induction, where anecortave failed to induce
IL-1, whereas glucocorticoids were effective in induction.10 Our
ing to both the GR and MR.17 The
D9,11 drugs induced GR translo-
cation from the cell cytoplasm to cell nucleus, shown by both
reporter and immunostaining assays, again suggesting effective
ligand/receptor interactions. Nuclear translocation of steroid hor-
mone receptors is a hallmark of classic glucocorticoid transcrip-
tional activity, and is necessary for downstream activation of
gene promoters via binding of ligand/receptor complexes to gluco-
studies focused on in vitro assays of NF-
jB inhibition and models
of chronic immunity not utilizing LPS.17
corticoid response elements (GREs). However, neither
D
9,11 drug
Here, we queried the chemical space around the
D9,11 steroid
showed significant GRE-mediated transcriptional activities, either
backbone, optimizing for anti-inflammatory properties (NF-
j
B

E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
2243
inhibition and GR nuclear translocation). The lead compound,
VBP15, shows excellent drug properties, and is in development
for Duchenne muscular dystrophy as the initial indication.
purity. Overall yield from 3TR is 43%. Purity is assigned by hplc/
ms. Structure was confirmed by proton and carbon NMR.
2.2. NF-jB screen
2. Materials and methods
2.1. Chemistry
C2C12 skeletal muscle cells stably transfected with a luciferase
reporter construct regulated under NF- B response element (Pa-
B inhibitors,
j
nomics, Fremont, CA) were used for screening NF-
j
as previously described.18 Myoblasts and myotubes grown in
growth and differentiating medium respectively were pretreated
All compounds were synthesized by Bridge Organics Co.
(Kalamazoo, Michigan). Systematic (IUPAC) names: Anecortave
([2-[(8R,10S,13S,14R,17R)-17-hydroxy-10,13-dimethyl-3-oxo-
2,6,7,8,12,14,15,16-octahydro-1H-cyclopenta[a]phenanthren-17-yl]-2-
with various concentrations (vehicle [DMSO]; 0.01–100
the drugs for a 24 h duration before stimulating with tumor necro-
sis factor- (TNF- ) (10 ng/ml) for another 24 h. After the comple-
lg/ml) of
a
a
oxo-ethyl] acetate). Tirilazad ((16a)-21-[4-(2,6-dipyrrolidin-1-yl-
tion of incubation, cells were washed once with PBS and lysed with
cell lysis buffer to measure luciferase activity (Promega Corp, Mad-
ison, WI) using Centro LB 960 luminometer (Berthold technologies,
GmbH & Co, Bad Wildbad, Germany) and IC50 values are calculated
for each compound.
pyrimidin-4-yl)piperazin-1-yl]-16-methylpregna-1,4,9(11)-triene-
3,20-dione). Additional chemical structures studied are described in
Table 1.
Traditional glucocorticoids have an 11-beta-hydroxy or an 11-
keto group whereas we used a double bond between carbons 9
and 11, leading to a
D9,11 C ring as the backbone, then developed
2.3. Cell viability
a series of steroids based on this scaffold (Fig. 1, Panel A; Table 1).
These scaffolds are not oxidatively metabolized to 11-beta hydroxy
compounds, and should have less off-target effects related to active
metabolites. We varied the D ring of the steroid, probing the effect
of different lipophilic groups at C16 and either hydroxy or hydro-
gen at C17. Twenty compounds were synthesized in the VBP series
Cell viability was determine by MTT assay (3-[4,5 dimethylthi-
azol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma, St., Louis,
Missouri) as per manufacturer’s protocols. Percent cell viability
was calculated relative to untreated cells, whereas relative lumi-
nescence units with TNF-
were considered as 100% percent. Data are represented as % inhibi-
tion relative to TNF- induced NF- B activation. Significance was
a stimulation in the absence of drugs
of
(R1, R2, R3) (Fig. 1; Table 1).
The synthesis of the 9,11 steroid begins with commercially
D9,11 compounds varying in structure at the three positions
a
j
D
calculated using a one-way repeated measure ANOVA to determine
if there is an effect of drug concentration on cell viability.
available steroid 3TR (Fig. 1; Panel B). Copper catalyzed Michael
addition of MeMgCl with TMSCl through a 1,4 addition introduced
a 16-alpha methyl group to give silyl enol ether 2 with 93% purity
by hplc/ms and in nearly quantitative yield. The product was iso-
lated as a toluene slurry which was oxidized with 32% by weight per-
acetic acid in acetic acid to yield intermediate 3 which was not
isolated but was converted to compound 4 as a powder that precip-
itated from EtOAc/heptane trituration in 60% yield from 3TR and
with 86% purity. In order to remove a small amount of the delta
9,11 epoxide, compound 4 was treated with HBr in methylene chlo-
ride. Yield from the HBr treatment after MeOH/water crystallization
was 90% with 97% purity and an overall yield from 3TR of 54%. Final-
ly, the acetate of compound 4 is removed by K2CO3 hydrolysis. Meth-
anol/water crystallization of VBP15 gave a 79% yield with 98.3%
2.4. Nuclear translocation assays
Translocation assays were performed by DiscoveRx (Fremont,
CA) using GR Nuclear Translocation PathHunter cells (DiscoveRx;
Fremont, CA). This assay is based on the detection of protein–pro-
tein interactions between the GR and a nuclear fusion protein con-
taining steroid receptor co-activator peptide. The receptor is
tagged with the ProLink component of enzyme fragment comple-
mentation assay system and the steroid receptor co-activator pep-
tide (SRCP) is fused to the enzyme acceptor component. When the
receptor is bound by ligand it translocates to the nucleus where it
Table 1
Chemical structures of compounds tested
VBP#
A
16
17
21
NF-
jB inhibition EC50 (M) Rank NF-
jB
GR nuclear translocation (%) Rank GR translocation Sum rank
Pred/Dex
6
15
7
1
16
2
3
41
17
11
5
13
10
4
1.67E-08
5.64E-08
6.59E-08
5.41E-08
6.29E-08
6.23E-08
1.29E-07
2.12E-07
8.95E-08
7.23E-07
1.74E-06
9.36E-08
4.35E-07
1.85E-06
4.95E-07
9.88E-07
5.24E-06
1
3
6
2
5
4
9
10
7
100.00
54.20
80.40
48.80
49.20
44.50
57.60
51.50
27.00
37.70
28.70
15.30
21.80
24.70
16.70
19.90
22.70
23.50
14.70
8.50
1
4
2
7
6
8
3
5
2
7
8
9
Delta 1,4 CH3
Delta 1,4 CH3
Delta 1,4 CH3
H
OH
OH OH
OAc
OH OH
Delta 1,4 Beta CH3 OH OH
H
Delta 4
H
11
12
12
15
18
22
25
26
26
28
29
30
31
31
40
41
42
Delta 1,4
Delta 4
H
H
OH OH
OH Oac
H
H
OH Morpholino
ene OAc
OH Dipropylamino
OH Pyrrolidine
ene OH
Delta 1,4 Ethyl
OH
11
9
Delta 4
Delta 4
CH3
H
Dipropylamino
13
15
8
10
18
15
12
17
16
14
13
19
20
21
Delta 1,4 ene
Delta 4
Delta 4
H
H
11
16
12
14
17
18
21
21
21
Delta 1,4 ene
18
14
9
12
59
58
Delta 4
Delta 4
Delta 4
Delta 4
Delta 4
CH3
CH3
H
Me
nButyl
H
H
Pyrrolidine
Morpholino
OH N-Me Piperazine 5.78E-06
H
H
H
N-Me Piperazine >1e-5
OH
OH
>1e-5
>1e-5
Delta 1,4 nButyl
6.90

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E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
recruits the SRCP and complementation occurs producing a chemi-
luminescent signal. A ten point agonist dose curve, ranging from
1 ꢁ 10ꢀ5 to 1.69 ꢁ 10ꢀ10 M was performed on all VBP compounds.
Dexamethasone was performed in parallel as the standard refer-
ence control. Percentage activity is calculated using the following
formula: % Activity = 100% ꢁ (Mean Relative Light Units [RLU] of
test sample—mean RLU of vehicle control)/(mean MAX RLU control
ligand—mean RLU of vehicle control)). EC50 is calculated for all the
compounds.
compound or positive control (Verapamil) was added to micro-
somal solutions at a final concentration of 2 M VBP compound,
l
0.5 mg/mL microsomes, 5 mM MgCl2 and 5 mM PBS. The reaction
was started with the addition NADPH solution at a final concentra-
tion of 1 mM and carried out at 37 °C. H2O was used instead of
NADPH solutions in the negative control. Aliquots were taken from
the reaction solution at 0 and 60 min. The reaction was stopped by
the addition of 3 volume of cold methanol. Aliquots of the superna-
tant were used for LC/MS/MS analysis for metabolite analysis and
identification was performed as described above.
2.5. Transactivation assays
2.8. CYP induction
HEK-293 cells stably transfected with a luciferase reporter con-
struct regulated under glucocorticoid response element (GRE) (Pa-
nomics, Fremont, CA) were grown according to manufacturer’s
instructions. Cells were treated with various concentrations (0.01
CYP1A2 and CYP3A4 induction studies were conducted by Phar-
maron. Cryopreserved human liver microsomes from 3 donors
(separate incubations) were obtained commercially from CellzDir-
ect (Invitrogen). Hepatocytes were cultured on a collagen substra-
tum for three days prior to study initiation according to
manufacturer’s instructions. Hepatocyte cultures were treated dai-
to 100 lg/ml) of the drug for 6 h. Cells were washed once with
PBS and lysed with cell lysis buffer to measure luciferase activity
(Promega Corp, Madison, WI) using Centro LB 960 luminometer
(Berthold technologies, GmbH & Co, Bad Wildbad, Germany).
ly with fresh media containing 1-, 10-, and 100 lM VBP15, vehicle
(negative control) or appropriate positive control (rifampin for
CYP3A4 and omeprazole for CYP1A2). 24-h after the final treat-
ment, CYP3A4 and CYP1A2 activity was determined using the
FDA recommended probe substrates testosterone and phenacetin
respectively. Assay analysis was conducted via LC/MS/MS. The per-
centage inductions relative to positive control were calculated. A
greater than 40% of positive control in any one of the three donors
for a CYP was considered a potential inducer of that CYP.
2.6. Pharmacokinetics
Pharmacokinetic studies were carried out at Pharmaron (US
headquarters 6 Venture, Suite 250, Irvine, CA 92618; work per-
formed in Pharmaron-Beijing,
100176). Mice were purchased from Huafukang Bioscience, Beijing,
China. Dogs were purchased from Marshall Inc., Beijing, China.
Monkeys were purchased from Hainan, Inc., Jinggang, China and
Guangxi Inc., Guidong China.
6 Taihe Road, BDA Beijing,
3. Results
2.6.1. Analysis
3.1. In vitro screening of VBP compounds for NF-
transrepression and GR nuclear translocation
jB
All analyses were conducted on a Shimadzu liquid chromato-
graph separation system equipped with degasser DGU-20A3, sol-
vent delivery unit LC-20AD, system controller CBM-20A, column
oven CTO-10ASVP and CTC Analytics HTC PAL System. . Samples
We screened the 20 compounds for their ability to inhibit NF-
B using a luciferase reporter construct stably transfected into
j
were loaded onto a Phenomenex Luna 5
l
C18 (2) (2.0 ꢁ 50 mm)
C2C12 myogenic cells.18 Both undifferentiated myoblasts, and dif-
ferentiated, multi-nucleated myotubes were studied. VBP com-
coupled with a preguard column with a mobile phase of 0.1% formic
acid in acetonitrile (A) and 0.1% formic acid in water. Mass spectro-
metric analysis was performed using an API 4000 instrument from
AB Inc (Canada) with an ESI interfaceThe data acquisition and con-
trol system were created using Analyst 1.5 software from ABI Inc.
pounds were able to inhibit TNF-alpha induced NF-
degrees resulting in reduced percent NF- B activity. Figure 2 dis-
plays the top 5 NF- B inhibitors in myoblasts and myotubes. These
results indicate that several of the VBP compounds are able to in-
jB to varying
j
j
hibit NF-
cells.
jB at potency similar to prednisolone in vitro in muscle
2.6.2. Mouse
CD1 mice (n = 3/drug) were injected via tail vein with a solution
of 10 mg/kg VBP6 (10% ethanol and 40% PEG400; pH 7.0) or 10 mg/
kg VBP15 (10% ethanol, 10% DMSO, and 30% PEG400; pH 7.0). Oral
administration (PO) was conducted by feeding CD1 mice (n = 3/
drug) an aqueous emulsion of either VBP6 or VBP15 in 30% Labrafil.
Blood samples were taken at 0-, 5-, 15-, 30 min and 1-, 2-, 4-, 8-,
and 24 h.
Cell viability was assayed on C2C12 cells grown and treated con-
currently to those measuring NF- B activity. We discovered that a
C16-methyl group showed strong inhibition of NF- B and the low-
est cell toxicity, while larger alkyl groups (ethyl and butyl) either
decreased activity or enhanced toxicity. We now favor a 1,4 in
the A ring because of improved metabolic stability, a 9,11 in
j
j
D
D
the C ring, a methyl at C16, and either hydroxyl or hydrogen at C17.
Compounds were tested for potency in causing translocation of
the GR from the cytoplasm to nucleus using a reporter assay in
both myoblasts and myotubes.18 EC50 for GR Translocation [LogM]
2.6.3. Dog and rat
Sprague–Dawley rats (n = 3) were intravenously injected with a
solution of 10 mg/kg VBP15 (10% ethanol, 10% DMSO and 30%
PEG400; pH 7.0). Beagle dogs (n = 3) were intravenously injected
with a solution of 10 mg/kg VBP15 (8% ethanol, 8% DMSO, 50%
PEG400 and 34% HP-b-CD (20%W/V in water). Oral administration
for SD rats (n = 3) and beagles (n = 3) was conducted by feeding an
emulsion of VBP15 in 30% Labrafil. Blood samples were taken at 0-
5-, 15-, 30 min and 1-, 2-, 4-, 8-, and 24 h.
was then plotted against the IC50 for NF-jB inhibition (Figure 3).
VBP6, VBP7 and VBP15 clustered with prednisone and dexametha-
sone, showing high activities for both assays. VBP6 and VBP15 are
both 21-hydroxyls with VBP7 being the acetate (prodrug) of VBP6.
3.2. VBP compounds lack transactivation activity
To determine if VBP compounds dissociate transrepression from
2.7. Metabolic stability
transactivation, we screened the top NF-
for their ability to induce GRE-dependent gene transcription using
GRE-luciferase reporter construct as we have previously
jB inhibiting compounds
Stability studies were conducted by Pharmaron using liver
microsomes from human, monkey, dog, rat and mice. Either VBP
a

E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
2245
Myoblasts
A
B
Myotubes
Prednisolone
VBP2
100
100
Prednisolone
VBP2
VBP3
VBP6
VBP7
VBP15
VBP5
VBP6
90
80
70
60
50
40
30
90
80
70
60
50
40
30
VBP7
VBP15
-10.0 -9.5
-9.0
-8.5
-8.0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
-10.0
-9.5
-9.0
-8.5
-8.0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
Log [Drug] M
log [Drug] M
Figure 2. NF-jB inhibition in myogenic cells. (A) Dose response of the top NF-
jB inhibiting VBP compounds in C2C12 myoblasts. (B) Dose response of the top NF-jB inhibiting
VBP compounds in C2C12 differentiated myotubes. Standard deviations are shown.
A
B
Figure 3. Correlation of NF-jB and GR nuclear translocation potency in (A) myoblasts and (B) myotubes. IC50 values for NF-kB inhibition activity (X axis) and GR nuclear
translocation activity (Y axis) were plotted for all VBP series compounds, relative to prednisone and dexamethasone.
described.18 Results show that there was a significant dose-depen-
dent response in prednisolone treated cells (p <0.0001) but there
was no increase in luciferase transcription at even the highest
doses tested for any of the VBP compounds (Fig. 4). These data sug-
gest we are achieving our desired SAR goals of dissociating the
gene transcriptional (classic glucocorticoid) properties from the
was calculated by comparing the dose-corrected area under curve
(AUC) non-intravenous divided by AUC intravenous. IV AUC was
considered 100%. We had different formulations for the oral versus
IV which improved solubility, and hence a larger AUC, for the oral
formulation compared to the IV formulation leading to F% >100%.
PK results for VBP15 indicate that it had low-medium clearance
18.8 ml/min/kg. The terminal half-life was 0.35 h. Volume of distri-
bution was 0.75 L/kg indicating that it also did not have high tissue
accumulation. In the PO arm, Cmax = 6787 ng/ml at 2 h, and bio-
availability (F%) was 74.5% (Table 2; Fig. 5).
anti-inflammatory (NF-jB) properties.
3.3. Pharmacokinetics in CD1 mice following intravenous and
oral administration
The two top candidates from the in vitro assays (VBP6 and
VBP15) were compared for bioavailability through PK studies.
The relatively poor solubility of VBP15 required formulation in
8% DMSO + 8% Ethanol + 50% PEG400 + 34% HP-b-CD (20% W/V).
PK results for VBP6 indicate that it had a relatively high
clearance of 48.2 ml/min/kg, which was about 50% of the mouse
blood hepatic flow. The terminal half-life was 0.67 h. Volume of
distribution (Vss) was 0.87 L/kg, indicating that it did not have high
tissue accumulation. In the PO arm, Cmax = 10203 ng/ml and
percentage bioavailability (F%) was 128% (Table 2, Fig. 5). The F%
3.4. Metabolic stability across multiple species
The in vitro metabolic stability of VBP6 and VBP15 was studied
in different species of liver microsomes. Results show P80%
remaining at 1 h of VBP6 and VBP15 in human, dog, and monkey
microsomes (Fig. 6). VBP6 had poor stability in mouse liver micro-
somes (34%) compared to VBP15 (88%). Both compounds showed
poor stability in rat, presumably due to the high first pass clearance
traditionally seen with glucocorticoids in rats.19

2246
E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
120000
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
UnTx
Prednisolone
VBP3
VBP6
VBP7
VBP15
-10.0
-9.5
-9.0
-8.5
-8.0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
Concentration [Log M]
Figure 4. VBP compounds do not induce GRE-dependent gene transcription. A reporter construct with luciferase driven by glucocorticoid response elements (GREs) was
studied, as previously described.18 Prednisone induced luciferase expression, while none of the VBP series did. Standard deviations are shown.
Table 2
PK analysis of VBP6 and VBP15 following IV and PO administration in CD1 mice
Drug
Route Dose
(mg/kg)
Formulation
Clobs (mL/
min/kg)
T1/2
(h)
Tmax
(h)
Cmax (ng/ AUClast
mL) (h ng/mL)
AUCInf
(h ng/mL)
MRT
(h)
AUC/D
(h ng/mL)
Vssobs
(L/kg)
F
(%)
VBP6
IV
10
10% EtOH + 40%
PEG400
30%
48.2
NA
0.667 0.0833 8.637
0.659 1.00 10.203
0.354 0.0833 11.167
3.447
22.023
8.838
3.457
22.031
8.842
0.289 345
NA 440
0.667 884
0.874
NA
NA
128
NA
PO
50
10
Labrafil
VBP15 IV
PO
10% EtOH
10% DMSO
40% PEG400
30%
18.8
0.757
50
NA
0.678 2.00
6.787
32.912
32.932
NA
658
NA
74.5
Labrafil
A
B
VBP6 PK Mean Profile
100000
IV
10000
1000
100
10
PO
1
0
2
4
6
8
10
Time (h)
Figure 5. PK mean profiles for (A) VBP6 and (B) VBP15 in mice. Error bars indicate standard deviation.
3.5. Lead compound selection
3.6. Absorption, distribution, metabolism and excretion (ADME)
and pharmacokinetics of VBP15
Potential lead compounds were narrowed to VBP6 and VBP15
based on superior activities (high NF-
j
B inhibition, low cytotoxic-
We performed a series of ADME and pharmacokinetic studies to
characterize our lead compound. Table 3 summarizes the results of
these ADME experiments. PK results for VBP15 in rats indicate that it
had a clearance of 20.2 mL/min/kg. The terminal half-life was 0.58 h.
Volume of distribution (Vss) was 0.77 L/kg, indicating that it did not
ity, high GR binding and GR translocation). PK data showed that
VBP15 had a longer Tmax and lower Cmax across species, leading
to a greater and preferred AUC compared to VBP6. Thus, VBP15
was selected as the lead compound for all further studies.

E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
2247
CYP1A2. VBP15 moderately induced CYP3A4 across the three do-
nors (24–42%). This indicates that VBP15 is a potential inducer of
CYP3A4, similar to other steroidal compounds.
4. Discussion
We have recently shown that an existing
D9,11 steroidal com-
pound (anecortave acetate) was able to bind and translocate the
glucocorticoid receptor (GR) and inhibit anti-inflammatory
pathways (NF-j
B).17 Based on these data we synthesized 20
new compounds not previously known to the literature, with
the goal of carrying out a lead optimization program using the
D
9,11 backbone, in vitro screening assays included potent inhibi-
tion of NF- B, potent translocation of the GR to the nucleus, loss
of GRE-mediated transcriptional activity, and low cytotoxicity.
We found that a C16-methyl group was important for NF-
j
Figure 6. Metabolic stability of VBP6 and VBP15 in liver microsomes across
multiple species. VBP15 shows better stability in rat and mouse microsomes
compared to VBP6. HLM = human liver microsomes, MRLM = male rat liver micro-
somes, MMLM = male mouse liver microsomes, MDLM = male dog liver micro-
somes, MKLM = male monkey liver microsomes.
jB
inhibitory activity, while larger alkyl groups (ethyl and butyl)
either decreased activity or enhanced cytotoxicity of myogenic
cells. Other optimizations of chemistry included a
D1,4 in the A
ring (improved metabolic stability), a 9,11 in the C ring, a
D
have high tissue accumulation. In the PO arm, Cmax = 2543 ng/mL
and percentage bioavailability (F%) was 47.8% (Table 4).
VBP15 in beagle dogs had medium-high clearance of 24.7 mL/
min/kg. The terminal half-life was 5.42 h. Volume of distribution
was 1.93 L/kg indicating that it had certain tissue distribution. In
the PO arm, Cmax = 814 ng/mL, and bioavailability (F%) was 53.2%
(Table 4).
methyl at C16, and either hydroxyl or hydrogen at C17. VBP15
was selected as the lead compound based on these findings.
VBP15 also showed desirable ADME and PK properties. Like many
steroids, VBP15 shows oral absorption in a target range accept-
able for oral delivery. The drug can be formulated for PK and
other studies in vehicles that are typical for related steroids. PK
and metabolite work suggest that the primate and mouse will
probably be the preferred species for PK.
3.7. Metabolite identification
Synthetic glucocorticoids are among the most commonly pre-
scribed drugs due to their potent anti-inflammatory properties,
and remain standard of care in many conditions such as arthritis,
dermatitis, asthma, muscular dystrophy, and auto-immune
disorders.20–22 However, glucocorticoids have many off-target
effects that together contribute to a relatively broad side effect
profile. These include but are not limited to disruption of glucose
metabolism, immune suppression, adrenal suppression, thymocyte
apoptosis, osteoporosis, erythroblast proliferation, elevation of
intraocular pressure, cataract, and mood changes. Many of these
side effect profiles are associated with GRE-mediated transcrip-
tional activities of steroids (classic glucocorticoid activity). We
have shown that VBP15 lacks GRE-mediated transcriptional
Human, monkey, dog, rat and mouse hepatocytes were incu-
bated at a final concentration of 10 lM VBP15 for 2 and 4 h. Four
metabolites were identified in the human and monkey hepato-
cytes, 3 were identified in the dog (peak 4 is the major metabolite),
5 metabolites were identified in rat (peak 3 is the major metabo-
lite) and 2 in the mouse (Table 5).
3.8. CYP induction
There was no induction of CYP1A2 by VBP15 seen across the
three donors therefore VBP15 is not considered an inducer of
Table 3
ADME properties of VBP15
In vitro properties
Units
M)
Value & class
187.18
Target range
Solubility (pH, media)
Stability—microsomes
(
l
l
M
>60
>30
t1/2 (min)
Human = 82%
Rat = 35.4%
Mouse = 88.8%
Dog = 92.5%
Monkey = 100%
88.06%
1.9%, 7.9%, 15.3%
>50 lM for all
Stability—plasma (species)
CYP450 Inhibition (2C9,2D6,3A4)
% Remaining at 1 h
% Inhibition at 10 mM
IC50 (mM)
>80%
<40%
>10
>10
<2
Permeability—Caco-2
Papp (a–b, 10ꢀ6 cm/s)
Efflux ratio
11.2
0.61
hERG— (method)
Free Cmax—plasma
Ames test
IC50 (mM)
Total Cmax (mM) Fu,
Positive/negative
Positive/negative
20
0.12
Negative
Negative
0.25 = 0.648
0.5 = 0.620
1.0 = 0.614
2.0 = 0.593
4.0 = 0.583
6.0 = 0.581
8.0 = 0.433
l
M
>10
⁄
plasma
Negative
Negative
Micronucleus test
Blood Brain Barrier (rats)a
Ratio (brain/plasma) (h)
a
The brain concentration was not corrected for vascular content.

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E. K. M. Reeves et al. / Bioorg. Med. Chem. 21 (2013) 2241–2249
Table 4
PK properties of VBP15 across species
PK properties
Units
h
Mouse 10 mpk IV 50 mpk PO
Rat 10 mpk IV 50 mpk PO
Dog 10 mpk IV 30 mpk PO
Target range
>3
t1/2
0.354 (IV)
0.678 (PO)
8842 (IV)
32932 (PO)
18.8 (IV)
11167 (IV)
6787 (PO)
0.083 (IV)
2.0 (PO)
0.58 (IV)
5.42 (IV)
2.25 (PO)
6791 (IV)
10844 (PO)
24.7 (IV)
814 (PO)
2.29 (PO)
8339 (IV)
19937 (PO)
20.2 (IV)
AUC0–1,
,
h ng/mL
>500 (PO)
<25% HBF
total unbound
CL
mL/min/kg
ng/mL (nM)
Cmax,
,
2543 (PO)
total unbound
Tmax
h
4.00 (PO)
6.00 (PO)
Vss
F
L/kg
%
0.757 (IV)
74.5
0.770 (IV)
47.8
1.93 (IV)
53.2
>20%
Table 5
VBP15 metabolites identified across species
Peak
no.
Rt
Expected
Mass
shift
Biotransformation
Metabolites in hepatocytes
(min)
m/z
Human
Monkey
Dog
Rat
Mouse
1
2
3
4
5
6
5.52
6.37
7.78
9.69
10.42
12.86
371.2
373.2
371.2
369.2
355.2
357.2
16
18
16
14
0
Oxidation
Oxidation + hydrogenation
Oxidation
Methylation
Parent drug
+
+
+
+
+
+
+
+++
+
+
+
ꢀ
ꢀ
+++
+
ꢀ
ꢀ
+
+++
ꢀ
ꢀ
ꢀ
+
+
+++
+
+++
+
2
Hydrogenation
+
+
activity, although it inhibits NF-jB and causes translocation of the
References and notes
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complexes with NF-
B promoter elements (transrepression properties). VBP15 appears
to disassociate these two subactivities, with GR translocation likely
due to NF- B transcriptional complexes. Thus, we expect that pre-
jB transcriptional complexes that target NF-
j
j
clinical studies will show superior side effect profiles of VBP15
compared to other steroid-based anti-inflammatories, as we have
previously shown for anecortave acetate.17
The extensive effect profiles of pharmacological glucocorti-
coids often limit prescription despite proven efficacy,
particularly in children. For example, in Duchenne muscular dys-
trophy, daily prednisone is considered standard of care in some
countries, but the efficacy if offset by increased bone fragility,
mood changes, weight gain, and muscle catabolic pathways.23–
26
Thus, there is non-adoption of this standard in some coun-
tries, and relatively poor adherence even in those countries
adopting daily glucocorticoids.27 The VBP15 described here is
being developed for Duchenne muscular dystrophy as the initial
indication, in collaboration with the Muscular Dystrophy Associ-
ation Venture Philanthropy, and National Institutes of Health
Therapeutics for Rare and Neglected Disease program. It is our
hope that VBP15 not only provides a superior side effect profile
relative to prednisone, but will also improve efficacy by opening
the therapeutic window (allowing increased dosing relative to
prednisone).
17. Baudy, A. R.; Reeves, E. K.; Damsker, J. M.; Heier, C.; Garvin, L. M.; Dillingham, B.
C.; McCall, J.; Rayavarapu, S.; Wang, Z.; Vandermeulen, J. H.; Sali, A.; Jahnke, V.;
Duguez, S.; DuBois, D.; Rose, M. C.; Nagaraju, K.; Hoffman, E. P. J. Pharmacol.
Exp. Ther. 2012, 343, 225–232.
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Immunopharmacol. 2009, 9, 1209–1214.
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Acknowledgments
Supported in part by Grants from the Congressionally Directed
Medical Research Program of the US Department of Defense
(W81XWH-09-1-0218; W81XWH-11-1-0754), Muscular Dystro-
phy Association Venture Philanthropy (Phase 1 and Phase 2), Foun-
dation to Eradicate Duchenne, NIH Wellstone Muscular Dystrophy
Research Center (1U54HD053177-01A1), and NIH Therapeutics for
Rare and Neglected Disease (TRND) program.
23. Sandri, M.; Sandri, C.; Gilbert, A.; Skurk, C.; Calabria, E.; Picard, A.; Walsh, K.;
Schiaffino, S.; Lecker, S. H.; Goldberg, A. L. Cell 2004, 117, 399–412.

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