Influence of Vehicles Used for Oral Dosing of Test Molecules on the
Progression of Mycobacterium tuberculosis Infection in Mice
Shubhra Singh, Richa Dwivedi, and Vinita Chaturvedi
Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow, India
Preclinical evaluation of drug-like molecules requires their oral administration to experimental animals using suitable vehicles.
We studied the effect of oral dosing with corn oil, carboxymethyl cellulose, dimethyl sulfoxide, and polysorbate-80 on the pro-
gression of Mycobacterium tuberculosis infection in mice. Infection was monitored by physical (survival time and body weight)
and bacteriological (viable counts in lungs) parameters. Compared with water, corn oil significantly improved both sets of pa-
rameters, whereas the other vehicles affected only physical parameters.
E very year, progressive infection with Mycobacterium tubercu-
losis leads to approximately 8 million new tuberculosis (TB)
cases and 3 million deaths worldwide. Furthermore, the spread of
multidrug-resistant (MDR) TB has become a major threat to
global TB control programs. This alarming situation emphasizes
the need for new drugs against TB (28, 31).
Evaluation of the in vivo efficacy of a new drug candidate in
a suitable animal model is a critical step in determining
whether it will enter the preclinical and clinical development
phases (10). When infected with a high number of CFU of M.
tuberculosis by the intravenous route, the lung of a mouse har-
bors a bacillary population that is similar in number and in
metabolic state to that present in the lungs of TB patients (10).
Thus, the mouse model can provide information on lead mol-
ecule activity that can be extrapolated to humans (8, 22). Out-
bred mice, particularly Swiss mice, are used for in vivo assays
for anti-TB activity because of their heterogeneity, which is
akin to that of the human population (3).
For in vivo evaluations, a lead molecule is preferably adminis-
tered as a solution or suspension by the oral route (1), which is an
important requirement for its successful development as a new
drug. Water is the universally accepted “ideal” vehicle for oral
dosing, having no chemical, biochemical, immunological, or
pharmacological effects on the test molecule, the host, or the
pathogen (2). However, if the molecule is highly hydrophobic,
then edible oils (such as corn or peanut oil) are used as vehicles
(21). For moderately hydrophobic molecules, certain solubilizing
agents, such as carboxymethyl cellulose (CMC), dimethyl sulfox-
ide (DMSO), and polysorbate-80 (Tween 80), can be added to the
aqueous dosing vehicle (23, 24).
The influence of dosing vehicles on the progression of disease
in animal models, if any, is an important consideration for in vivo
evaluation of test molecules. There is hardly any information
available on whether these vehicles themselves could modulate the
disease process, thereby influencing the outcome of the evaluation
of the drug candidates. This is particularly important in cases of
chronic infections, such as TB, where treatments are given to the
animals for long periods of time (6, 12, 19). We therefore studied
the effect that some of the vehicles themselves could exert on the
progression of M. tuberculosis infection in mice. Five vehicles—
water, corn oil, CMC, DMSO, and Tween 80 —were compara-
tively studied. Study parameters were physical, i.e., mean survival
6026 aac.asm.org Antimicrobial Agents and Chemotherapy
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time (MST) and body weight, as well as bacteriological, i.e., CFU
recovered from the lung.
Corn oil was administered undiluted, whereas dilutions of
CMC (0.5%), Tween 80 (0.05%) and DMSO (10%) were made in
sterile water. Female outbred Swiss mice (16 to 18 g), obtained
from the Laboratory Animal Division of the Central Drug Re-
search Institute, Lucknow, India, were infected intravenously
(2 ⫻ 107 bacilli/mouse) with M. tuberculosis H37Rv (ATCC
27294) and caged in groups of 8 to 10 animals. Each group was
given water or the other vehicles separately (0.2 ml/mouse, by oral
gavage, once daily, for 30 days). The progress of infection was
monitored in the control (untreated) mice by determining
numbers of viable bacilli (CFU) on day 1 postinfection and then at
weekly intervals (Fig. 1). For this, serial dilutions of lung homoge-
nates (1 g tissue/ml) in physiological saline were spread on Middle-
brook 7H11 agar medium (containing OADC [oleic acid-albumin-
dextrose-catalase] supplement). The plates were incubated at 37°C,
and CFU were counted after 3 to 4 weeks (4). Numbers of CFU in the
lungs of mice from each experimental (vehicle-treated) group were
determined on day 30.
The corn oil-fed mice showed far less decline in body weight
(weight on day 0 ⫺ weight on day 21) than the water-fed animals
(0.50 ⫾ 1.00 g and 4.67 ⫾ 3.06 g, respectively [Table 1]). This
difference was statistically significant (P ⫽ 0.047 [Table 2]). More
importantly, oil-fed animals also exhibited a ⬎1-log reduction in
lung CFU compared with the water-fed animals (Table 1). This dif-
ference was also statistically significant (P ⫽ 0.0016) (Table 2). How-
ever, a significant difference was not observed between MST (mean
survival time of all animals in a group) of the water-fed (24.75 ⫾ 4.50
days) and oil-fed (25.25 ⫾ 3.69 days) animals (Table 1).
Among the three aqueous vehicles, administration of DMSO
resulted in “healthier” physical parameters. The MST with DMSO
was ⬎30 days (no deaths up to day 30) compared with 28.00 ⫾
2.31 days with CMC and 28.75 ⫾ 2.50 days with Tween 80 (Table
1). All three MSTs were higher than those observed with water and
Received 17 August 2012 Accepted 18 August 2012
Published ahead of print 27 August 2012
Address correspondence to Vinita Chaturvedi, Vinita_chaturvedi@cdri.res.in.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01702-12
p. 6026 – 6028 November 2012 Volume 56 Number 11
 Influence of Vehicles on M. tuberculosis Infection

TABLE 2 Comparison of changes brought in the assessment parameters

with experimental vehicles
P value fora:
Net decline in Bacterial load
Comparison avg body wt in lungs
Distilled water vs oil 0.0471* 0.0016**
CMC vs distilled water NS NS
CMC vs oil 0.017* 0.0007#

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FIG 1 Progression of M. tuberculosis infection in Swiss mice infected with 2 ⫻
107 bacilli/mouse without any oral dosing. Each bar represent log10 CFU
(mean ⫾ SD) in lungs of three mice each on day 1 and at weekly intervals up to
4 weeks (W4) postinfection. Statistical significance is indicated by numbers in
boxes: 1, P ⫽ 0.073; 2, P ⫽ 0.0006; 3, P ⫽ 0.0001; 4, P ⫽ 0.035.
oil. As none of the mice in DMSO group died, it was not possible
to calculate statistical significance of the differences in MST. The
decline in body weight with all three vehicles was also less than that
with water (Table 1), though the differences were not significant
(P ⬎ 0.05). With respect to oil, only CMC showed a significantly
greater decline in the body weight (3.00 ⫾ 1.15 g, P ⫽ 0.0176)
(Table 2). Finally, the numbers of CFU in lungs with all aqueous
vehicles (Table 1) were close to that obtained with water (P ⬎
0.05) but significantly higher than that obtained with oil (P ⫽
0.0007 versus CMC, 0.0021 versus DMSO, and 0.0060 versus
Tween 80) (Table 2).
The apparent beneficial effects of corn oil could be due to the
presence of 86% unsaturated fatty acids, of which two-thirds is
linoleic acid and one-third is oleic acid (15). Linoleic acid has been
found to inhibit mycobacteria in vitro (15, 29); thus, it could also
produce the same effect in vivo. The three aqueous vehicles
(DMSO, CMC, and Tween 80) caused some apparent, though not
significant, modulation of only the physical parameters, particu-
larly MST. Among the three, administration of DMSO resulted in
much improved physical parameters. This may influence the gen-
eral body physiology and/or metabolism, which may in turn mod-
ulate the handling of the pathogen by the infected host (5, 7, 9, 11,
13, 14, 17, 18, 25–27, 30).
Measurement of survival time or body weight of animals is an
appropriate, cost- and time-effective criterion to assess the pro-
gression of infection and to evaluate new drugs against M. tuber-
culosis in vivo (10, 16, 20). Our results, however, show that weight
loss and MST may not be reliable criteria and that determination
TABLE 1 Survival, average body weight, and bacterial load in lungs of
M. tuberculosis-infected mice treated with different vehicles
Net decline (from Bacterial load in
MST on day day 0 to 21) in lung (log10
Vehicle 30 avg body wt (g)a CFU/g tissue)a
Distilled water 24.75 ⫾ 4.50 4.67 ⫾ 3.06 11.44 ⫾ 0.212
Corn oil 25.25 ⫾ 3.69 0.50 ⫾ 1.00 10.36 ⫾ 0.122
CMC 28.00 ⫾ 2.31 3.00 ⫾ 1.15 11.84 ⫾ 0.242
DMSO ⬎30 1.50 ⫾ 1.00 11.75 ⫾ 0.316
Tween 80 28.75 ⫾ 2.50 2.00 ⫾ 1.63 11.80 ⫾ 0.451
a
Values are means ⫾ standard deviations.
DMSO vs distilled water NS NS
DMSO vs oil NS 0.0021**
Tween 80 vs distilled water NS NS
Tween 80 vs oil NS 0.0060**
a
NS, not significant; *, statistically significant; **, very significant; #, extremely
significant.
of bacterial load in the infected organs is a true measure of infec-
tion. Moreover, in a previous screening for TB-induced weight
loss, some active compounds or drugs possessing anabolic prop-
erties could have affected the results (20). In conclusion, the vehi-
cles used for oral dosing of test molecules can influence physical as
well as bacteriological parameters in mice infected with M. tuber-
culosis.
ACKNOWLEDGMENT
We are grateful to Director, Central Drug Research Institute, Lucknow,
India, for providing necessary facilities and support to carry out the work.
This paper has CDRI communication number 8299.
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Antimicrobial Agents and Chemotherapy
ERRATUM
Influence of Vehicles Used for Oral Dosing of Test Molecules on the
Progression of Mycobacterium tuberculosis Infection in Mice
Shubhra Singh,a,b Richa Dwivedi,a,c Vinita Chaturvedia
Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow, Indiaa; IFTM University, Moradabad, Indiab; Academy of Scientific &
Innovative Research (AcSIR), New Delhi, Indiac

Volume 56, no. 11, p 6026 – 6028, 2012. Page 6026: The author affiliation line should appear as shown above.
Page 6027: The Acknowledgments section should include the following statement, “Shubhra Singh and Richa Dwivedi are senior
research fellows of ICMR New Delhi and would like to thank IFTM University, Moradabad, and AcSIR, New Delhi, respectively, for
registering them for the Ph.D. degree.”

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.02911-14

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