Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor salicyl-AMS in mice

Article abstract of DOI:10.1128/AAC.00918-13

Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence. The mycobactin biosynthesis inhibitor salicyl-AMS [5=-O-(N-salicylsulfamoyl)adenosine] inhibitsM. tuberculosis growth in vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of salicyl- AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibitedM. tuberculosis growth in the mouse lung, providing the first in vivo proof of concept for this novel antibacterial strategy. Copyright

Full text of DOI:10.1128/AAC.00918-13

Pharmacokinetic and In Vivo Efficacy Studies of the Mycobactin
Biosynthesis Inhibitor Salicyl-AMS in Mice
Shichun Lun,a Haidan Guo,a John Adamson,b Justin S. Cisar,c,d Tony D. Davis,c,e Sivagami Sundaram Chavadi,f J. David Warren,g
Luis E. N. Quadri,f Derek S. Tan,c,d,e William R. Bishaia,b,h
Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAa; KwaZulu-Natal Research Institute for Tuberculosis and HIV,
Durban, South Africab; Molecular Pharmacology & Chemistry Program and Tri-Institutional Research Program, Memorial Sloan-Kettering Cancer Center, New York, New
York, USAc; Tri-Institutional Training Program in Chemical Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, USAd; Pharmacology Program, Weill
Cornell Graduate School of Medical Sciences, New York, New York, USAe; Department of Biology, Brooklyn College, City University of New York, Brooklyn, New York, USAf;
Department of Biochemistry, Weill Cornell Medical College, New York, New York, USAg; Howard Hughes Medical Institute, Chevy Chase, Maryland, USAh
Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence.
The mycobactin biosynthesis inhibitor salicyl-AMS [5=-O-(N-salicylsulfamoyl)adenosine] inhibits M. tuberculosis growth in
vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of sali-
cyl-AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration
did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibited M. tuberculosis growth in the mouse lung, providing
the first in vivo proof of concept for this novel antibacterial strategy.
iderophore production and iron acquisition by Mycobacte- vivo proof of concept for the use of siderophore biosynthesis in-
S
rium tuberculosis are important for growth under iron-limited
hibitors as novel antibacterials.
conditions and within human macrophages (1). They are also
essential for the virulence of M. tuberculosis during infection (2).
Importantly, gene expression profiling indicates that free iron is
limited in human lungs (3). Hence, M. tuberculosis must compete
for iron that is retained by high-affinity host proteins such as
transferrin and lactoferrin. The siderophore-dependent iron ac-
quisition system of M. tuberculosis consists of two major sidero-
phore variants, cell wall-associated mycobactin and secreted sol-
uble carboxymycobactin, both of which bind iron with high
affinity (4, 5). It is believed that carboxymycobactin binds iron in
the extracellular aqueous environment and transfers the iron to
the cell wall-associated mycobactin that then transports the iron
across the cell wall into the cytoplasmic space (5, 6). Biosynthesis
of mycobactins is carried out by multiple enzymes, most of which
are encoded by a cluster of genes designated mtbA to mtbJ (7).
Systematic mutational studies of nine of the mbt genes (mbtA to
mtbH and mbtT) in the Mycobacterium smegmatis model indicate
that eight of the genes (all but mbtH) are required for mycobactin
production (8). MbtA, a bifunctional enzyme encoded by the
mbtA gene (Rv2384 in M. tuberculosis), catalyzes the initiation of
mycobactin biosynthesis through a two-step reaction involving
ATP-dependent adenylation of salicylate to generate a salicyl-
AMP intermediate, followed by salicylation of an aroyl carrier
protein domain of the nonribosomal peptide synthetase MbtB (7,
9). The importance of the mycobacterial siderophore biosynthesis
pathway has led to the development of a designed small-molecule
inhibitor, salicyl-AMS [5=-O-(N-salicylsulfamoyl)adenosine] (see
Fig. S1 in the supplemental material), that specifically targets
MbtA by mimicking the salicyl-AMP intermediate (9–11). In vitro
studies have confirmed that salicyl-AMS inhibits MbtA activity
and also inhibits M. tuberculosis growth in iron-depleted media (9,
12), with the MIC for M. tuberculosis H37Rv found to be 0.5 ␮g/ml
in a study with an alamarBlue assay (13). In this study, we have
characterized the pharmacokinetics of salicyl-AMS and its thera-
peutic efficacy in a murine infection model, leading to the first in
Compound bioavailability is essential for in vivo efficacy. In
order to investigate the bioavailability of salicyl-AMS, we con-
ducted single-dose pharmacokinetic studies with mice via two
routes, oral gavage and intraperitoneal (i.p.) injection. Salicyl-
AMS sodium salt was synthesized on a large scale via an optimized
route (see the supplemental material for the synthesis procedure
used) and dissolved in 1ϫ phosphate-buffered saline (PBS) at a
concentration of 5 or 20 mg/ml. Twenty-gram female BALB/c
mice (Charles River Laboratories, Wilmington, MA) were given a
single dose of either 50 or 200 mg/kg, either orally or by i.p. injec-
tion, in a volume of 0.2 ml. At 7, 15, 30, 60, and 120 min after
compound administration, animals were euthanized and cardiac
blood (ϳ0.7 ml) and lungs were collected. Plasma was separated
by centrifugation at 12,000 rpm (benchtop microcentrifuge) for
20 min at 4°C. Mouse lungs were homogenized in 0.5 ml of liquid
chromatography (LC)-mass spectrometry (MS) grade water. Sali-
cyl-AMS concentrations in plasma and lung homogenate super-
natants were analyzed by LC-tandem MS (LC-MS/MS; AB SCIEX
QTRAP 5500 system) with, as an internal standard, stable-iso-
tope-labeled salicyl-AMS synthesized from uniformly 13C10- and
15N5-labeled adenosine (Cambridge Isotope Labs; see the supple-
mental material for the synthesis procedure used) with detection
of mass transitions 467.0/135.8 and 482.1/146.1. Data analysis was
Received 1 May 2013 Returned for modification 15 June 2013
Accepted 5 July 2013
Published ahead of print 15 July 2013
Address correspondence to William R. Bishai, wbishai1@jhmi.edu, Derek S. Tan,
tand@mskcc.org, or Luis E. N. Quadri, LQuadri@brooklyn.cuny.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.00918-13.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00918-13
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Antimicrobial Agents and Chemotherapy p. 5138–5140
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Salicyl-AMS for Tuberculosis Chemotherapy
TABLE 1 Single-dose pharmacokinetics of salicyl-AMS in mouse
C
max (␮g/ml)a
T
max (min)a
AUC (␮g · min/ml)a
Sample and dose
(mg/kg)
Oral
I.p.
Oral
I.p.
Oral
I.p.
F
rel (%)b
Plasma
50
200
0.3 Ϯ 0.05
1.2 Ϯ 0.96
64.6 Ϯ 1.76
329.7 Ϯ 109.0
11.0 Ϯ 5.66
7.0 Ϯ 0.00
7.0 Ϯ 0.00
7.0 Ϯ 0.00
15.9 Ϯ 0.59
58.6 Ϯ 12.60
1,280.0 Ϯ 261.6
9,185.0 Ϯ 1,105.0
1.24
0.6
Lungc
50
0.4 Ϯ 0.11
1.9 Ϯ 1.80
29.2 Ϯ 7.29
356.2 Ϯ 72.10
7 Ϯ 0.00
7 Ϯ 0.00
7 Ϯ 0.00
7 Ϯ 0.00
12.3 Ϯ 0.72
56.4 Ϯ 10.67
671.7 Ϯ 163.8
6,020 Ϯ 369.1
1.83
0.94
200
a Data are shown as means Ϯ standard deviations of three mice.
b Relative bioavailability was calculated as follows, where D is the dose: Frel ϭ 100 ϫ [(AUCoral ϫ Di.p.)/(AUCi.p. ϫ Doral)].
c Data for lungs were normalized to ␮g/g for Cmax and ␮g · min/g for AUC.
done with GraphPad Prism 4. Assays detected free salicyl-AMS in tiple-comparison test was conducted. These data demonstrated
the plasma and lungs after both oral and i.p. administrations, in- that the mycobactin biosynthesis inhibitor salicyl-AMS signifi-
dicating that this compound is bioavailable in mice. The lung-to- cantly inhibited the growth of M. tuberculosis in mouse lungs, as
plasma exposure ratio (lung area under the concentration-time shown by 0.87- and 1.10-log10 lower CFU burdens in low-dose-
curve [AUClung]/plasma AUC [AUCplasma]) was 0.52 at 50 mg/kg and high-dose-treated mice, respectively, compared with those in
and 0.66 at 200 mg/kg. Similar kinetic patterns were observed after untreated controls after 2 weeks of treatment (P Ͻ 0.01, Table 2).
single-dose administration of salicyl-AMS between the two sam- The CFU data also showed a dose dependency at this time point.
ple types, plasma and lung, and more interestingly, between the After 4 weeks of treatment, although the lung CFU burden in the
two administration routes, oral gavage and i.p. injection (see Fig. low-dose group was still lower than that in the untreated group,
S2 and S3 in the supplemental material; Table 1). Although salicyl- the difference was not statistically significant.
AMS is bioavailable after oral administration, the concentrations
Our study also provides insights into the toxicity profile of
achieved in plasma and lung samples after oral gavage are mar- salicyl-AMS. Previous studies have shown that salicyl-AMS is
ginal compared to those obtained after i.p. injection, as indicated nontoxic to the murine leukemia cell line P388 at concentrations
by the dose-normalized relative bioavailability (Table 1). In gen- of Ͼ200 ␮M (10, 11). We have also confirmed this lack of toxicity
eral, there are two in vivo working patterns for antimycobacterial for Chinese hamster ovary cells at concentrations of up to 500 ␮M
agents, time over MIC dependent and maximum drug concentra- (data not shown). Notably, however, all of the mice treated with
tion in serum (Cmax) dependent (14). The findings in this study the higher dose (16.7 mg/kg) of salicyl-AMS died before the week
are important because these data, i.e., Cmax ϭ 1.2 ␮g/ml and AUC ϭ 4 time point (Table 2). This drug toxicity could also contribute to
58.6 ␮g · min/ml (Table 1), clearly suggest that oral administration the decreased efficacy observed at week 4 in the low-dose group.
of this inhibitor is less likely to achieve an effective systemic level, Since MbtA has no mammalian homologues, this toxicity is likely
regardless of the in vivo mode of action of salicyl-AMS. This also to be the result of off-target effects and/or brought about by a drug
highlights the need to optimize the bioavailability of this lead metabolite(s) generated in vivo. Further lead optimization of sali-
compound after oral administration in future work, because oral cyl-AMS through medicinal chemistry may address the animal
administration is preferable for tuberculosis drugs.
toxicity. The less-than-ideal in vivo efficacy of salicyl-AMS could
Next, we examined the in vivo efficacy of salicyl-AMS in a also be attributable to its rapid clearance, as the calculated lung
mouse aerosol infection model. Thirty-five 6-week-old female half-lives were only 13.3 and 19.3 min for 50 and 200 mg/kg,
BALB/c mice (Charles River Laboratories, Wilmington, MA) were respectively (based on 30-, 60-, and 120-min data points). Despite
aerosol infected with 10 ml of a late-log-phase M. tuberculosis these rapid clearance pharmacokinetic parameter values, salicyl-
H37Rv culture by an inhalation exposure system (Glas-Col Inc.). AMS still demonstrated in vivo efficacy, suggesting that MbtA in-
Five mice were sacrificed at day 1 for implantation determination, hibition is a promising antimycobacterial strategy.
which was found to be 3.31 log10 per mouse lung. Mice were ran-
domly grouped to low-dose (5.6 mg/kg), high-dose (16.7 mg/kg),
and untreated groups. From day 1 onward, mice were treated by
i.p. injection of 0.2 ml of a PBS solution containing salicyl-AMS
sodium salt with the corresponding dose, daily (5 days per week).
At 2 and 4 weeks after treatment initiation, five mice in each group
were sacrificed and their lungs were removed. All of the animal
procedures used in this study were approved by the Institutional
Animal Care and Use Committee of the Johns Hopkins University
School of Medicine. The lungs were homogenized by bead beat-
ing, diluted with 1ϫ PBS, and plated on Middlebrook 7H11 selec-
tive agar plates (Becton Dickinson & Co., Franklin Lakes, NJ).
CFU per lung were enumerated after incubation for 4 weeks. Data
are shown as means and standard deviations (Table 2). One-way
In conclusion, we have characterized the pharmacokinetics
TABLE 2 In vivo efficacy as represented by lung CFU burden
Mean lung CFU (log10) Ϯ SDa
Salicyl-AMS at:
Untreated
Time point
control
5.6 mg/kg
16.7 mg/kg
Wk 0
Wk 2
Wk 4
3.31 Ϯ 0.022
7.92 Ϯ 0.105
8.40 Ϯ 0.669
3.31 Ϯ 0.022
7.05b Ϯ 0.084
7.72 Ϯ 0.049
3.31 Ϯ 0.022
6.82b Ϯ 0.316
NAc
a Mean and SD from four mice.
b P Ͻ 0.01 compared with untreated group as analyzed with one-way ANOVA and
Tukey’s multiple comparison test.
analysis of variance (ANOVA) was carried out, and Tukey’s mul- c NA, not available.
October 2013 Volume 57 Number 10
aac.asm.org 5139

Lun et al.
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geting of MbtA with salicyl-AMS slowed the growth of M. tuber-
culosis in mouse lungs. Although mouse infection models do not
exactly reproduce human tuberculosis pathologies such as granu-
loma structures and cavities, they are the most convenient and
cost-effective animal models with which to study the pharmaco-
kinetics and efficacy of new tuberculosis therapies, and optimized
leads can be advanced to nonhuman primate models for further
assessment in the future. Lead optimization efforts to achieve
lower drug toxicity, a longer half-life, higher efficacy, and better
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ACKNOWLEDGMENTS
This work was supported by NIH grants AI079590, AI036973, and
AI037856 to W.R.B.; AI068038 and GM100477 to D.S.T.; and AI075092
to L.E.N.Q. and by the Howard Hughes Medical Institute (W.R.B.).
L.E.N.Q. acknowledges the endowment support from Carol and Larry
Zicklin.
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molecule inhibition of siderophore biosynthesis in Mycobacterium tuber-
culosis and Yersinia pestis. Nat. Chem. Biol. 1:29–32.
We thank Laurene Cheung for technical assistance and Elisa De
Stanchina (MSKCC) and Francis Sirotnak (MSKCC) for helpful discus-
sions.
D.S.T., L.E.N.Q., and J.S.C. hold U.S. Patent 8,461,128 on salicyl-AMS
and analogues. We have no conflicts of interests to declare.
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