Pharmacokinetics of hydroxymethylnitrofurazone, a promising new prodrug for chagas' disease treatment

Article abstract of DOI:10.1128/AAC.02522-12

Hydroxymethylnitrofurazone (NFOH) is a trypanocidal prodrug of nitrofurazone (NF), devoid of mutagenic toxicity. The purpose of this work was to study the chemical conversion of NFOH into NF in sodium acetate buffer (pH 1.2 and 7.4) and in human plasma and to determine preclinical pharmacokinetic parameters in rats. At pH 1.2, the NFOH was totally transformed into NF, the parent drug, after 48 h, while at pH 7.4, after the same period, the hydrolysis rate was 20%. In human plasma, 50% of NFOH was hydrolyzed after 24 h. In the investigation of kinetic disposition, the concentration of drug in serum versus time curve was used to calculate the pharmacokinetic parameters after a single-dose regimen. NFOH showed a time to maximum concentration of drug in serum (T_max) as 1 h, suggesting faster absorption than NF (4 h). The most important results observed were the volume of distribution (V) of NFOH through the tissues, which showed a rate that is 20-fold higher (337.5 liters/kg of body weight) than that of NF (17.64 liters/kg), and the concentration of NF obtained by in vivo metabolism of NFOH, which was about four times lower (maximum concentration of drug in serum [C_max]=0.83 μg/ml; area under the concentration-time curve from 0 to 12 h [AUC_0-12]=5.683 μg/ml · h) than observed for administered NF (C_max=2.78 μg/ml; AUC_0-12=54.49 μg/ml · h). These findings can explain the superior activity and lower toxicity of the prodrug NFOH in relation to its parent drug and confirm NFOH as a promising anti-Chagas' disease drug candidate. Copyright

Full text of DOI:10.1128/AAC.02522-12

Pharmacokinetics of Hydroxymethylnitrofurazone, a Promising New
Prodrug for Chagas’ Disease Treatment
Eliana Ometto Pavan Serafim,a,c Antonio Távora de Albuquerque e Silva,a Andréia de Haro Moreno,a Ednir de Oliveira Vizioli,a
Elizabeth Igne Ferreira,d Rosângela Gonçalves Peccinini,b Maria Lucia Ribeiro,c Man Chin Chunga
Lapdesf, Department of Drugs and Medicines, School of Pharmaceutical Sciences, University of São Paulo State, UNESP, Araraquara, SP, Brazila; Department of Natural
Drugs and Toxicology, School of Pharmaceutical Sciences, University of São Paulo State, UNESP, Araraquara, SP, Brazilb; Department of Organic Chemistry, Chemical
Institute, University of São Paulo State, UNESP, Araraquara, SP, Brazilc; Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo, USP, São
Paulo, SP, Brazild
Hydroxymethylnitrofurazone (NFOH) is a trypanocidal prodrug of nitrofurazone (NF), devoid of mutagenic toxicity. The pur-
pose of this work was to study the chemical conversion of NFOH into NF in sodium acetate buffer (pH 1.2 and 7.4) and in human
plasma and to determine preclinical pharmacokinetic parameters in rats. At pH 1.2, the NFOH was totally transformed into NF,
the parent drug, after 48 h, while at pH 7.4, after the same period, the hydrolysis rate was 20%. In human plasma, 50% of NFOH
was hydrolyzed after 24 h. In the investigation of kinetic disposition, the concentration of drug in serum versus time curve was
used to calculate the pharmacokinetic parameters after a single-dose regimen. NFOH showed a time to maximum concentration
of drug in serum (Tmax) as 1 h, suggesting faster absorption than NF (4 h). The most important results observed were the volume
of distribution (V) of NFOH through the tissues, which showed a rate that is 20-fold higher (337.5 liters/kg of body weight) than
that of NF (17.64 liters/kg), and the concentration of NF obtained by in vivo metabolism of NFOH, which was about four times
lower (maximum concentration of drug in serum [Cmax] ؍ 0.83 ␮g/ml; area under the concentration-time curve from 0 to 12 h
[AUC0–12] ؍ 5.683 ␮g/ml · h) than observed for administered NF (Cmax ؍ 2.78 ␮g/ml; AUC0–12 ؍ 54.49 ␮g/ml · h). These find-
ings can explain the superior activity and lower toxicity of the prodrug NFOH in relation to its parent drug and confirm NFOH
as a promising anti-Chagas’ disease drug candidate.
merican trypanosomiasis (Chagas’ disease) is an endemic tabolism, and elimination (ADME) barriers that restrict the appli-
A
parasitic disease caused by Trypanosoma cruzi, afflicting
cation of many chemical compounds as orally administered drugs
(14, 15).
much of Mexico, Central America, and South America, where an
estimated 100 million people are at risk and 8 to 11 million people
are infected, with 14,000 deaths per year. The acute cases are de-
tected in only 1% to 2% of those infected (1, 2). With the increase
in immigration to the First World, the disease is spreading to
developed countries such as the United States, Europe, and Japan,
which should be a concern if it is not controlled or has no treat-
ment available (3). Only two nitro-heterocyclic drugs are available
in the worldwide market for its treatment: benznidazole and
nifurtimox. Benznidazole is a nitroimidazole derivative active
against both the trypomastigote and amastigote forms of T. cruzi.
This drug is the first-line drug treatment, with a half-life of 12 h, its
elimination is predominantly renal, and it is the only drug on the
market in Brazil. Nifurtimox is a nitrofuran compound; its half-
life is about 3 h because it is extensively metabolized by the liver. It
also presents activity against trypomastigotes and amastigotes.
The mechanism of its action is not totally understood (4); how-
ever, several derivatives showing potential anti-Chagas’ disease
activities have been prepared (5, 6, 7).
Among the studied compounds, hydroxymethylnitrofurazone
(NFOH), a prodrug of nitrofurazone (NF) (Fig. 1), was effective
when tested in LLC-MK2 cell cultures infected with trypomastigote
forms of T. cruzi (10). The results showed higher trypanocidal
activity in all stages of development forms of the parasite with low
toxicity. Trossini and colleagues (16) demonstrated that NFOH
was also able to inhibit 60% of cruzipain activity, compared to
30% inhibition by NF.
In preclinical genotoxicity studies (Ames mutagenic test using
TA102 and TA98 strains of Salmonella enterica serovar Typhimu-
rium in the presence, or not, of S9), NFOH was shown to be four
times less toxic than NF (17). These results were confirmed by
micronucleus assays that showed NFOH activity similar to that of
control (water) as well as by acute-toxicity tests that showed 50%
lethal dose (LD50) values higher than 2,000 mg/kg of body weight
(rats, per os) versus 556 mg/kg for NF, the parent drug (P. L.
Bosquesi, M. Mello, J. L. Santos, and M. C. Chung, unpublished
data).
Both drugs possess negative characteristics such as severe and
frequent side effects (vomiting, anorexia, peripheral neuropathy,
allergic dermatopathy, mental disturbance, and mutagenicity)
and selective sensitivity for different strains of T. cruzi; and the
long periods of treatment (8, 9) and lack of active drugs for the
treatment of the chronic form of the disease increase the impor-
tance of the discovery of new drugs.
New therapeutic drugs have been obtained by using the prod-
rug design strategy (4, 6, 7, 9, 10, 11, 12, 13). The prodrugs have
been an effective way of overcoming absorption, distribution, me-
Received 5 March 2013 Returned for modification 1 June 2013
Accepted 6 September 2013
Published ahead of print 30 September 2013
Address correspondence to Man Chin Chung, chungmc@fcfar.unesp.br, or
Rosângela Gonçalves Peccinini, peccinin@fcfar.unesp.br.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02522-12
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Antimicrobial Agents and Chemotherapy p. 6106–6109
December 2013 Volume 57 Number 12

Pharmacokinetics of NFOH for Chagas’ Disease
FIG 1 Structures of nitrofurazone (NF) (left) and hydroxymethylnitrofura-
zone (NFOH) (right).
In 2010, NFOH was tested against T. cruzi in a murine model of
Chagas’ disease and was found to be as effective as benznidazole in
keeping direct parasitemia at undetectable levels after 60 days of
treatment, and negative PCR results without histopathological le-
sions after 180 days of finishing the treatments showed that levels
of anti-T. cruzi antibodies were very low in mice treated either
with NFOH or with benznidazole (11).
FIG 2 Conversion profile of NFOH to NF* in sodium acetate buffer (pH 1.2)
at 37°C.
The development of prodrugs relies significantly on the knowl-
edge of their pharmacokinetic profile, and at this phase, the phar-
macokinetic data from animal studies provide information about
their ability to release the drug in biological systems and provide
guidance on the level of doses that can be used in humans in future
clinical trials (18, 19).
Hence, the present work was conducted with the objectives to
studytheinvitrohydrolysisoftheprodrugNFOHtoNF(considering
chemical and enzymatic hydrolysis) and to evaluate its pharmacoki-
netic profile in rats in order to determine the first pharmacokinetic
parameters for this new compound. For the accomplishment of these
studies, a bioanalytical method for the determination of NF and
NFOH in serum was also developed and validated.
0 to 24 h (AUC0-24) was calculated by the trapezoidal method, and the area
under the curve from time zero extrapolated to infinity (AUC0-ϱ) was
calculated by the equation AUC0-ϱ ϭ AUC0-24 ϩ Cpn/kel (where Cpn is
the last concentration in serum determined and kel is the elimination
constant). The AUC0–ϱ was used for the calculation of the total clear-
ance (CLTF ϭ dose/AUC0-ϱ), where F is bioavailability; the apparent
distribution volume (V/F) was calculated using the equation V/F ϭ
CLT/␤).
(iv) Statistical analysis. Statistical analyses were performed using the
software GraphPad Instat, followed by the Sigma-Stat software. The data
were analyzed by one-way analysis of variance (ANOVA).
RESULTS
MATERIALS AND METHODS
The study of the kinetic disposition of drugs requires analytical
methods with compatible sensitivity and selectivity for determi-
nation of their concentration in serum after administration of
single or multiple doses. The HPLC method for the determination
of NFOH and NF in serum was developed, validated, and applied
in the studies of hydrolysis and to evaluate the pharmacokinetic
profile of compounds.
Figures 2 and 3 show the results obtained for the in vitro con-
version of NFOH to NF at pH 1.2 and 7.4, respectively. Figure 4
shows the results for the in vitro conversion of NFOH to NF* in
human plasma at 37°C. NF* represents the NF obtained from
NFOH.
In the investigation of kinetic disposition in rats that received
NFOH (Fig. 5) and NF (Fig. 6) in a single-dose regimen, the con-
centration of serum versus time curves obtained were used to cal-
culate the pharmacokinetic parameters.
Figure 7 clearly shows that, after reaching Cmax, the NFOH
hydrolysis profile in human plasma (ex vivo) differs from the data
In vitro hydrolysis of prodrug NFOH to NF. The decomposition of
NFOH was studied at 37°C in aqueous sodium acetate buffer solutions
(pH 1.2 and 7.4) at a concentration of 15 ␮g · mlϪ1. The solution was kept
at constant temperature in a water bath, and at appropriate time intervals,
samples were analyzed by high-pressure liquid chromatography (HPLC).
The analyses were performed on 3 aliquots at each time point. The results
were expressed as the average peak area. The same procedure was carried
out with human plasma (ex vivo hydrolysis), and the times of the analyses
were 0.0, 0.25, 0.50, 1, 2, 4, 8, 12, 24, 48, 72, and 120 h.
Pharmacokinetic profile of NFOH and NF in rats. (i) Animals. Male
Wistar rats (weighing around 250 g) were housed at constant temperature
(23 Ϯ 1°C), humidity (55% Ϯ 5%), and light cycle (12/12 h) with food
and water ad libitum. The animals were deprived of food 12 h before oral
administration of compounds. The experiments were conducted during
the light phase.
(ii) Experimental protocol. Pharmacokinetic studies of NF and
NFOH were carried out at an equivalent oral dose of 200 mg/kg in male
Wistar rats. This dose was based on recent studies of toxicity in rats (data
not published), and the sample size (n ϭ 40 per group) was based on the
method published by Liu and Chow (18). NF and NFOH solutions were
prepared on the day of administration by dissolving appropriate amounts
of drugs directly in dimethyl sulfoxide (DMSO); group I received NF, and
group II received NFOH, both by gavage. The animals were killed by
decapitation at 0.25, 0.5, 1, 2, 4, 8,12, and 24 h after administration. Five
animals were used for each point. The blood samples were centrifuged and
submitted to HPLC analysis. All experimental procedures were reviewed
and approved by the Research Ethics Committee of the School of Phar-
maceutical Science, UNESP, Araraquara, Brazil.
(iii) Pharmacokinetic analysis. The NFOH and NF kinetic disposi-
tions were evaluated after a single oral administration in rats. The phar-
macokinetic parameters were calculated based on the concentration in
serum versus time curves. The half-life of elimination (t1/2␤) was calcu-
lated by the graphic method, and the constant of elimination (␤) was
FIG 3 Conversion profile of NFOH to NF* in sodium acetate buffer (pH 7.4)
calculated by the equation ␤ ϭ 0.693/t1/2␤. The area under the curve from at 37°C.
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Serafim et al.
FIG 6 Time-dependent concentration of NF in plasma obtained after NF
administration to male Wistar rats. Data are expressed as means and SEM
(n ϭ 50).
FIG 4 Conversion of NFOH to NF* in human plasma samples (pH 7.4) at
37°C.
NFOH was about 20-fold higher than the parent drug NF V, an
impressive value suggesting that the new compound can cross the
tissues to reach the target, while NF remains in the bloodstream.
These findings can explain the superiority of the activity of NFOH
over that of NF (9, 10). In addition, the active NF* metabolite
obtained by in vivo hydrolysis of NFOH showed that the concen-
tration of NF* is about four times lower (Cmax ϭ 0.83 ␮g/ml;
AUC0–12 ϭ 5,683 ␮g · h/ml) than that of the NF administered
orally (Cmax ϭ 2.78 ␮g/ml; AUC0–12 ϭ 54.49 ␮g · h/ml). This
result can explain the 4-fold-lower toxicity observed by Guido and
coworkers (17).
We observed that the simple inclusion of a methylhydroxy
group in a molecule of NF may promote the improvement of its
ADME profile in vivo. The literature showed that the inhibition of
the trypanothione reductase was a possible mechanism of action
of nitrofurazone (22, 23, 24); Trossini and coworkers (16) showed
cruzipain inhibition and an increase of 30% in affinity for the
enzyme. In addition, the most interesting and important point is
the simplicity of the NFOH synthesis and its high yield (10), sug-
gesting a very cheap compound for chemical scale-up. All these
findings strongly suggested NFOH as a promising anti-Chagas’
disease candidate drug.
Conclusions. Hydrolysis studies carried out at pH 1.2 and 7.4
indicated that NFOH is stable in vitro and is not totally converted
in human plasma by plasma enzymes. The in vivo experiments
showed different hydrolysis profiles from those obtained in vitro
in surprising results. The pharmacokinetics of NFOH showed that
this compound is 20-fold more distributed than the parent drug
NF. These findings may explain the superior activity against all
stages of T. cruzi forms and lower toxicity of NFOH in relation to
NF, and unlike what is observed with hydroxymethyl compounds,
which are usually unstable, the results showed that NFOH is
obtained in vivo, suggesting the participation of some other
systems in the metabolism of NFOH when administered to the
animal.
The pharmacokinetic parameters (Table 1) were calculated as
values required for the subsequent evaluation and study of dosage.
NFOH reaches Tmax in 1 h compared with 4 h for NF, suggesting
faster absorption than NF. The values of Cmax showed that the
maximum concentration of NFOH in plasma is 0.99 ␮g/ml com-
pared to 2.78 ␮g/ml of NF, but the distribution volume (V) of
NFOH is 337.5 liters/kg while that of NF is 17.64 liters/kg.
DISCUSSION
According to the literature, N-hydroxymethyl derivatives are hy-
drolyzed by chemical action and not by enzymatic attack (14, 20,
21). The experiments in this study were performed to verify if
NFOH was stable at the pH of stomach and/or blood. The results
showed that at pH 1.2, NFOH was totally converted into NF* after
48 h, while at pH 7.4, after the same period, the conversion rate
was only 20%, showing chemical stability at pH 7.4. However,
when the NFOH experiment was performed in human plasma, it
was 50% hydrolyzed into NF* after 24 h; this result could be ex-
plained by the action of plasmatic enzymes since in a buffered
medium (pH 7.4), 50% degradation occurs only after 120 h, sug-
gesting that the molecule of NFOH has a different behavior from
that of other hydroxymethyl compounds.
The effect of NFOH administered in rats showed that the plas-
matic concentrations of NF* observed after the administration of
NFOH were significantly lower than the concentrations obtained
with direct administration of NF. After calculation, the V of
FIG 5 Time-dependent concentrations of NFOH and NF* in plasma, ob-
tained after NFOH administration to male Wistar rats. Data are expressed as FIG 7 Comparative study of the hydrolysis of NFOH after reaching Cmax in
means and standard errors of the means (SEM) (n ϭ 50).
human serum and in vivo.
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Pharmacokinetics of NFOH for Chagas’ Disease
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Parameter (unit)
max (␮g/ml)
max (h)
el (hϪ1
NFOH
NF*
NF
C
T
k
0.99
1.00
0.099
7.00
33.41
337.5
5.683
5.986
10.102
0.83
4.00
0.231
3.00
2.78
4.00
0.178
3.9
3.140
17.64
54.49
63.70
5.618
)
t1/2␤ (h)
CL/F (liters/h · kg)
V/F (liters/kg)
AUC0–12 (␮g/ml · h)
AUC0-ϱ (␮g/ml · h)
MRT
13.36
14.23
a Abbreviations: NF, nitrofurazone administered by gavage; NF*, NF obtained by
hydrolysis of administered NFOH; Cmax, maximum concentration; Tmax, maximum
time; kel, elimination constant; t1/2, half-life; CL/F, clearance/oral bioavailability; V/F,
volume of distribution/oral bioavailability; AUC, area under the curve; MRT, mean
residence time.
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