Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

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Journal of Pharmaceutical and Biomedical Analysis

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Short communication

Ultra high performance liquid chromatography method for the

determination of pentamidine and analog in rat biological uids
a , M. Helvenstein a , L. Verdy a , Z. Kahvecioglu b , R. Conotte b ,
S. Hambye
J.-J. Vanden Eynde c , J.-M. Colet b , B. Blankert a,
a
Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons UMONS, Place du Parc 20, 7000 Mons, Belgium
b
Laboratory of Organic Chemistry, Faculty of Sciences, University of Mons - UMONS, Place du Parc 20, 7000 Mons, Belgium
c
Laboratory of Human Biology & Toxicology, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons
- UMONS, Place du Parc 20, 7000 Mons, Belgium

a r t i c l e

i n f o

Article history:
Received 30 October 2013
Received in revised form 31 January 2014
Accepted 3 February 2014
Available online 10 February 2014
Keywords:
Pentamidine
Accuracy proles
SPE
UPLC
Benzamidines

a b s t r a c t
Pentamidine isethionate (PTMD) is an antiprotozoal agent used in different parasitic diseases as Human
African Trypanosomiasis or Pneumocystis pneumonia. Given its side effects, numerous analogs are still
under development worldwide. PTMD has been recently described having a potential activity in myotonic
dystrophy (type 1). Here we present an UPLC method coupled to uo or PDA detection for PTMD and one
analog determination in rat plasma or urine. The chromatographic separation was achieved on a Acquity
UPLC HSS T3 analytical column using a mobile phase combining formic acid 0.1% (v/v) and acetonitrile
(ACN) at a constant ow rate of 0.4 mL/min. Preliminary, an innovative SPE (solid phase extraction)
procedure using Oasis WCX sorbent was processed and gave satisfying and reproducible results in
terms of extraction yields.
Additionally, the methods were successfully validated using the accuracy proles approach ( = 95%
and acceptance limits = 15%) over the ranges 2.88287.52 ng/mL and from 143.76 ng/mL to 1.72 g/mL
in rat plasma and urine for PTMD and for EBAB, from 4.23 to 423.39 ng/mL and from 211.69 ng/mL to
2.54 g/mL for plasma and urine, respectively.
The validated protocols were applied to a pharmacokinetic (PK) study on rats and permitted to point
out some relevant PK parameters on PTMD and its studied analog.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Pentamidine isethionate (PTMD), a member of the bisbenzamidines family, is an antiparasitic agent initially developed for the
treatment of Human African Trypanosomiasis in the 1930s [13].
In the following decades, the drug was used in other parasitic diseases such as leishmaniasis and more recently, with the emergence
of AIDS (Acquired ImmunoDeciency Syndrome), in Pneumocystis
pneumonia (a common opportunistic infection occurring in AIDS
patients). PTMD therefore remains a useful and essential active
compound in occidental countries [4].

Selected Paper presented at PBA 2013, Bologna, Italy, 30 June 3 July 2013.
Corresponding author. Tel.: +32 65373592.
E-mail address: bertrand.blankert@umons.ac.be (B. Blankert).
http://dx.doi.org/10.1016/j.jpba.2014.02.002
0731-7085/ 2014 Elsevier B.V. All rights reserved.

The PTMD mechanism of action is still not completely elucidated

but it could act as a minor groove binder of nucleic acids structures
[5]. The major disadvantage in the use of this therapeutic agent is
its toxicity, with side effects occurring in around 50% of the treated
patients. They range from local reactions such as necrosis at the
injection point to hypoglycaemia, hypotension or nephrotoxicity
[1].
This problematic has led to active research of less toxic analogs
(LTA) having at least the same therapeutic effect. Ideally, these new
compounds should also exhibit a better oral bioavailability, another
disadvantageous feature of PTMD [6]. The structural modications
of most LTA seek to optimize the central linker between the two
benzamidines groups, or to modify the grafted moieties on the amidine group [5,7,8]. The recent highlight of potential PTMD activity
in myotonic dystrophy (type 1) stimulates this thematic of research
even further [9].
Recently, the laboratory of Organic Chemistry (UMONS) has
synthesized piperazine-1,4-bisbenzamidines and analogs with a

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

exible ethylenediamine linker instead of the central rigid piperazine. Assessed in vitro on Pneumocystis cultures and in vivo on
immunocompromised rats presenting Pneumocystis carinii pneumonia, two LTA showed an in vitro activity similar to pentamidine
and one of the synthetized analog (EBAB, namely compound 6 in
[10]) was also very efcient in vivo [10].
Additionally, EBAB has also shown promising antioxidant, neuroprotective and anticonvulsant properties as described by Vamecq
et al. [11].
In a previous work, we developed and validated a HPLCUV
method for the determination of PTMD and the two in vitro active
cited LTA, in rat plasma [12]. Although our method permitted the
elaboration of preliminary pharmacokinetic proles (at a dose of
20 mg/kg) and suggested a likely explanation concerning the difference of in vivo activity between the two LTA (the second analog
being rapidly eliminated by the organism), the method presented
some limitations. The method was time consuming; unsatisfying
regarding its limit of quantication (LOQ) when working at a lower
dosage (4 mg/kg) and the extraction step developed in plasma did
not operate on rat urine.
The goal of the present work is to develop, optimize and validate (using accuracy proles) a new generic analytical method
(for plasma or urine samples) adapted for both analysis of PTMD
and the selected LTA (EBAB). The second LTA mentioned above
was no further studied due to its lack of in vivo activity [10]. A
UPLC approach will be for the rst time performed in the frame
of PTMD quantication in biouids. Coupled to uorescence (or
to a Photo Diode Array detector), it could help us to ameliorate
the LOQ within a shorter analysis run time. To fully achieve these
objectives, an alternative extraction procedure will be also investigated.
To demonstrate that the new method is well suited for
its purpose, we performed a pharmacokinetic study that
allowed us to complete our knowledge of the behavior of the
two drugs and to determine some pharmacokinetic parameters.

2. Materials and methods

2.1. Reagents
Pentamidine
[PTMD,
{4,4 -(pentane-1,5-diylbis(oxy)]bibis(2-hydroxyethanesulfonate))}]
sbenzenecarboximidamide
was purchased from SigmaAldrich (St. Louis, MO, USA). Hexamidine
[HXMD,
4,4 -(hexamethylenedioxy)dibenzamidine
bis(2-hydroxyethanesulfonate)], was bought as a commercial
antiseptic solution named Hexomedine (Melisana, Switzerland).
LTA, EBAB and N-methyl-EBAB, were synthesized in the Laboratory of Organic Chemistry, Faculty of Sciences, University of Mons
(Belgium). EBAB, for 1,2-ethane bis-1-amino-4-benzamidine is also
named {4,4 -(1,2-ethanediyldiimino)bisbenzenecarboximidamide
dihydrochloride salt}.
is
also
named
{4,4 -(N-methylN-methyl-EBAB
1,2-ethanediyldiimino)bis(benzenecarboximidamide)
dihydrochloride salt}. HXMD and N-methyl-EBAB were used
as internal standard (IS) for PTMD and EBAB, respectively. Their
synthesis pathways and elemental analyses were previously
described in the literature [11,12].
Phosphoric acid, ammonium hydroxide, methanol (HPLC grade)
came from ChemLab (Zedelgem, Belgium). Dimethylsulfoxide
(DMSO) was bought from Baker (Mallinckrodt Baker, Deventer,
Netherlands). Formic acid and acetonitrile (UPLC grade) came
from Biosolve (Valkenswaard, The Netherland). Ultrapure water
(18.2 M cm) was obtained with a Reference A+ Milli Q water
purication system (Millipore, Brussels, Belgium).

55

2.2. Instrumental conditions

Chromatographic analyses were performed on a Waters Acquity
H-Class UPLC System (MA, USA) equipped with an Acquity UPLC
HSS T3 (1.8 m; 2.1 mm 100 mm) as analytical column, maintained at 45 C. An optimized gradient elution was carried out at
a constant ow rate of 0.4 mL/min. The mobile phase combines
formic acid 0.1% (v/v) and acetonitrile (ACN). The percentage of
ACN was initially set at 5% and regularly increased up to 15% within
1 min. Thereafter it was gradually increased to 30% until 2.5 min,
kept constant until 3.8 min and quickly raised to 95% at 4.5 min.
The total run time was 4.6 min and the injection volume was xed
at 10 L.
For PTMD, a uorescence detection is performed ( excitation:
270 nm;  emission: 345 nm). For EBAB, the PDA detector, set at
320 nm was chosen.
All data acquisition and chromatograms analyses were performed using Empower Software 3.0 (Waters).
2.3. Stock and standard solutions
Stock solution (1 mg/mL) was made in puried water for PTMD
and LTA. For LTA, DMSO was added to improve the compounds
solubility (nal concentration 7% (v/v)). HXMD was purchased in a
1 mg/mL commercial solution form.
For each analyte (PTMD and EBAB), standard solutions were
prepared in ultrapure water in an adequate range regarding the
two biological matrixes, plasma (0.1, 0.5, 2, 5, 7 and 10 g/mL) and
urine (5, 10, 20, 30, 40 and 60 g/mL). HXMD standard solutions
in water of 3 and 20 g/mL were prepared for plasma and urine,
respectively. N-methyl-EBAB standard solutions in water of 5 and
20 g/mL were prepared for plasma and urine, respectively.
2.4. Sample pre-treatment
For the development and validation steps, plasma (collected
with K3 EDTA as anticoagulant agent) and urine from healthy Wistar
male rats were purchased from Janvier Labs (Le Genest, France).
Calibration samples were prepared by spiking 100 L of plasma (or
200 L of urine) with the adequate volume of stock solution of the
target analyte and internal standard and diluted with 200 L (or
300 L for urine) of phosphoric acid 4% (v/v) in water.
2.5. Solid phase extraction (SPE)
Oasis WCX Elution Plates (30 M, Waters) were used with
an extraction plate manifold (Waters, Milford, MA, USA). As recommended by the manufacturer, each well of the plate was
conditioned three times with 200 L of methanol and equilibrated
with 200 L of ultrapure water. The samples (prepared as explained
in Section 2.4) were loaded in each well. Ammonium hydroxide
(5%, v/v in water) and methanol washes were successively performed. Elution was done in collection plates with 2 L 25 L of
formic acid 2% (v/v) in methanol, and quickly diluted with 50 L
(for plasma) or 150 L (for urine) of ammonium hydroxide 2% (v/v)
in water.
During the validation step, recoveries at all the levels of concentration were calculated on three consecutive days. Areas of the
peaks after WCX extraction were compared to those of standard
solutions.
2.6. Validation methodology
The developed UPLC method was validated using accuracy proles strategy; extensive details are reported in [12]. Briey, for

56

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

Fig. 1. Structures and typical chromatograms at LLOQ of pentamidine (1; 3.15 min) and hexamidine (2; 3.4 min) in rat plasma (a) and urine (b).

each analyte, 6 calibration standards (CS) and 6 validation standards (VS) are realized in each biological matrix. The concentration
range for CS and VS for PTMD and EBAB in plasma raised from
2.88 to 287.52 ng/mL and from 4.23 to 423.39 ng/mL, respectively (expressed as free drug). The concentration range for CS
and VS for PTMD and EBAB in urine raised from 143.76 ng/mL
to 1.72 g/mL and from 211.16 ng/mL to 2.54 g/mL, respectively
(expressed as free drug). The concentrations of IS HXMD were 87.3
and 582.07 ng/mL for plasma and urine, respectively. The concentrations of IS N-methyl-EBAB were 208.50 and 834.0 ng/mL for
plasma and urine, respectively.
Each CS and VS were prepared on three consecutive days and
analyzed each day in triplicate for CS and four times for VS. Linear,
quadratic, linear after square root transformation and linear after
logarithm (base 10) transformation regressions were tested and the
best model was selected. -Expectation tolerance intervals were
computed for each analyte and matrix using set at 95% [1315].
All data were computed with Excel software (Microsoft, USA). Accuracy proles were drawn using acceptance limits at 20% at the
LLOQ (lower limit of quantication) and at 15% for the remaining
concentration range.

times during 48 h (before administration, 10, 30, 60 min and 2, 4, 6,

8, 21, 26, 48 h after administration). The obtained blood was centrifuged 15 min at 3000 g and the resulting plasma gathered and
stored at 80 C until analysis. Urine was collected each 6 h using
metabolic cages and stored at 80 C until analysis.
Urinary creatinine levels were measured by Jaffe Method
(Roche/Hitachi Modular P analyser, Roche Diagnostics) to ensure
that no major renal insufciency was present in tested rats [16].
Urine and plasma samples were analyzed following the hereby
described method. Plasma concentrations versus time curves were
plotted. Concentrations of each unchanged (not metabolized)
compound excreted in urine were determined for 24 h on two consecutive days.
Based on these results, main pharmacokinetic data were extrapolated: maximal concentration (Cmax ), time to the Cmax (Tmax ) and
24 h excretion percentage. Area under curve (AUC) was calculated
by the trapezoidal method for the area from time 0 (t0 ) to the
time of the last measured concentration. Half-time was determined
using the equation T1/2 = 0.693/Ke , where Ke (elimination rate constant) is the slope obtained by drawing a plot of ln C (C = plasmatic
concentration) vs time.

2.7. Pharmacokinetic study

3. Results and discussion

To compare in vivo behavior of the two studied compounds, a

preliminary pharmacokinetic (PK) study of each compound was
carried out. Experiments were performed on 8 Wistar male rats
divided into 2 groups. One group received PTMD and the other one
EBAB consisting of a subcutaneous dose of 4 mg (salt)/kg. One animal in each group served as control. Blood was collected from the
tail vein in the presence of EDTA as anticoagulant agent, at dened

3.1. Developing the SPE and UPLC method

Different gradient mobile phase compositions have been tested
in order to obtain the best separation parameters for both biological matrixes. The nal composition chosen, a mixture of ACN
and formic acid 0.1% is common for both compounds in both
matrixes. The chromatographic separation performed under these

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

57

Fig. 2. Structures and typical chromatograms at LLOQ of EBAB (3; 2.2 min) and N-methyl-EBAB (4; 2.4 min) in rat plasma (a) and urine (b).

conditions is efcient and gives rise to well-shaped peaks with a

rapid time analysis shorter than 5 min. The chromatograms registered for PTMD and EBAB, in each matrix at the lower limit of
quantication (LLOQ), show sharped and symmetrical peaks; the
targeted compounds are separated with a high resolution and selectivity, free from any interferences (Figs. 1 and 2).
The selected SPE stationary phase (Oasis WCX) is an ion
exchange mode, specially designed for very basic compounds.
Thanks to its selectivity, it allowed a very good clean up for both
matrixes. Classical SPE extraction in large cartridges includes an
evaporation step after elution, before resolubilization and injection
in LC. Performing this protocol leads to the degradation of EBAB,
illustrated by the apparition of a new peak in the chromatogram
(data not shown). This new peak only seems to appear after the
evaporation step. Its presence is likely due to the instability of the
studied compound in more drastic acidic conditions resulting from
solvent evaporation step. To overcome this issue, a new extraction
procedure was achieved using Elution SPE plates. They require no
evaporation step (molecule degradation is avoided), limit samples
losses and contribute to accentuate time saving.
Mean recoveries, with the WCX extraction, provided satisfying yields, although somewhat lower in urine than in plasma (see
Table 1).
3.2. Validation
Based on experimental validation data, we computed trueness
(expressed in terms of relative bias (%)), precision (intermediate
precision and repeatability) and accuracy. Four different response
functions were t for each method and matrix, tolerance intervals limits were calculated ( = 0.95%) and accuracy proles were

drawn (data not shown). For each one, linear regression model
(the simplest model to use), allowed the validation of each analytical quantication method on the whole range of concentration
(data of interest summarized in Table 1). Fig. 3 shows accuracy
proles for each compound in each matrix, considered with linear regression model. As all tolerance intervals are comprised
within the acceptance limits, the validation tool permits to conclude to the validation of the developed quantication methods on
the whole tested concentration ranges. It means for PTMD from
2.88 to 287.52 ng/mL and from 143.76 ng/mL to 1.72 g/mL in rat
plasma and urine, respectively. For EBAB, it ranges from 4.23 to
423.39 ng/mL and from 211.69 ng/mL to 2.54 g/mL for plasma and
urine, respectively. The plasma assay presents a LLOQ of 2.88 ng/mL
and 4.23 ng/mL for PTMD and EBAB, respectively while urine assay
shows a LLOQ of 143.76 ng/mL and 211.69 ng/mL for PTMD and
EBAB, respectively.
3.3. Pharmacokinetic proles
Samples collected from in vivo study were determined with
the validated reversed-phase UPLC method. PTMD and EBAB are
measurable in plasma at the rst collecting time until 21 h for
PTMD (48 h for EBAB) suggesting they are rapidly reabsorbed
and distributed. Plasma results permit the construction of pharmacokinetic proles as shown in Fig. 4. Cmax and Tmax for
PTMD are located at 85.14 ng/mL and 62 min and for EBAB at
755.8 ng/mL and 13 min, respectively. AUC for PTMD and EBAB
were 18,689 and 97,855 ng min/mL, respectively. T1/2 were calculated at 271 and 138 min for PTMD and EBAB, respectively.
These data show the difference of plasmatic behavior of the two
compounds.

58

Table 1
Validation data of linear regressions of PTMD and EBAB in rat plasma and urine.
Analyte

PTMD

EBAB

Plasma

Urine

Plasma

Regression
model

VS

Concentration
(ng/mL)

Trueness

Precision

Accuracy

Extraction efciency

Relative bias (%)

Repeatability/intermediate
precision (RSD, %)

-Expectation lower and upper

tolerance limits of the relative error (%)

Average recovery (%)

Linear

1
2
3
4
5
6

2.88
14.38
57.50
143.76
201.27
287.52

7.41
5.51
1.39
1.14
1.57
2.68

2.58/4.77
1.65/2.00
0.83/2.89
1.04/1.65
0.48/0.46
1.87/3.91

[4.39; 19.20]
[10.26; 0.77]
[8.69; 5.90]
[2.88; 5.16]
[2.62; 0.53]
[12.40; 7.03]

92 8%

Linear

1
2
3
4
5
6

143.76
287.52
575.04
862.56
1150.09
1725.13

3.54
0.89
2.81
0.12
6.42
2.23

0.94/3.25
0.97/2.80
0.54/3.36
0.46/3.69
0.67/3.10
0.84/2.92

[4.65; 11.72]
[7.92; 6.13]
[5.72; 11.34]
[9.49; 9.25]
[1.41; 14.25]
[5.13; 9.58]

75 2%

Linear

1
2
3
4
5
6

4.23
21.17
84.68
211.69
296.37
423.39

4.43
1.89
0.40
0.24
0.20
1.01

6.00/5.71
2.31/5.15
1.99/2.14
0.84/3.65
0.62/0.74
0.55/1.71

[8.48; 17.35]
[10.96; 14.73]
[4.57; 5.37]
[9.47; 8.99]
[1.95; 1.55]
[5.31; 3.28]

84 10%

Linear

1
2
3
4
5
6

211.69
423.39
846.77
1270.16
1693.55
2540.32

0.38
1.29
2.70
3.53
1.88
0.46

1.88/2.83
1.11/3.6
0.39/3.77
0.58/4.29
0.67/1.9
0.31/2.34

[6.50; 7.26]
[10.37; 7.79]
[12.27; 6.86]
[14.42; 7.36]
[2.88; 6.64]
[5.48; 6.39]

55 4%

EBAB

Urine

HXMD

Plasma
Urine

87.3
582.07

Internal Standard (PTMD)

103 9%
74 5%

N-methyl EBAB

Plasma
Urine

208.5
834

Internal Standard (EBAB)

93 6%
61 5%

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

PTMD

Matrix

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

59

Fig. 3. Accuracy proles representations (using linear regressions) for PTMD in rat plasma (a) and urine (b) and EBAB in rat plasma (c) and urine (d); = 95%,  = 15% except
at LLOQ = 20%.

Creatininuria was not critically different in tested animals as

compared to matching controls.
The Cmax for EBAB is surprisingly high compared to our previous results [12]. This led to a maximal concentration higher than
the validated range of concentrations. Assessing the quality of these
data, linearity was checked successfully in triplicate on an enlarged
range (4.231270.2 ng/mL) of samples undergoing the whole
analytical procedure.

During the rst 24 h, unmetabolized PTMD urinary excretion

was evaluated at 0.3 0.1% of the administered drug. During the
next day, excretion percentage was stable at 0.31 0.07%. Previous informations available in literature on urinary elimination of
PTMD are old and scarce, but are conrmed by currently generated data. It reinforces the idea that PTMD urinary excretion is
low and time delayed likely due to a large distribution in tissues
[17,18].

Fig. 4. Pharmacokinetic proles (rat plasmatic concentrations vs time) of PTMD (a) and EBAB (b).

60

S. Hamb
ye et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 5460

For EBAB, urine samples were diluted ten times to t in the calibration range. During the rst 24 h, EBAB urinary excretion was
estimated at 7 3% of the administered drug. During the next day,
excretion percentage was lower at 2.77 0.08%.
Based on these preliminary results, the two studied compounds
exhibit a distinct in vivo behavior. PTMD, as expected, gives a
low maximal plasmatic concentration, and is slowly and delayed
excreted in urine. EBAB seems to reach rapidly a higher maximal
plasmatic concentration (Fig. 4). The EBAB urinary excretion takes
place in a faster and more ample manner, likely due to a lower
distribution in tissues than PTMD.
4. Conclusion
We developed an original and simple generic UPLC method coupled to DAD or uorescence detection for accurate quantication of
PTMD and a LTA namely EBAB in rat plasma and urine. To the best
of our knowledge, this sensitive method is the rst developed and
successfully validated (by accuracy proles strategy) with UPLC for
PTMD and EBAB quantication.
An Oasis WCX 96-well elution protocol was rstly applied
to the studied compounds. It allows an efcient and time saving extraction for both compounds from two different biological
matrixes, plasma and urine. The recovery for EBAB is signicantly
improved (by 30%) in comparison with classical acetonitrile protein
precipitation [12].
Compared to our previously described HPLC method [12], the
validated concentration ranges permit to determine samples with
lower concentrations. The method gains in sensitivity with a LLOQ
in plasma of 2.88 ng/mL for PTMD and of 4.23 ng/mL for EBAB. It
means this parameter is lowered by a factor ten for PTMD and more
than fteen for EBAB. Furthermore, the time of analysis is cut by a
factor 5 as the total time run is lower than 5 min.
This method is a suitable and valuable tool in the investigation
of pharmacokinetic data. It has been applied for both compounds
and allowed their determination in plasma over 24 h after a single
subcutaneous injection (4 mg/kg). Urinary levels were measured
for 48 h after the single dose injection. PK data of PTMD and EBAB
in urine and plasma highlight differences in the behavior of both
compounds. PTMD presents a low maximal plasmatic concentration and is eliminated via a low and delayed urinary excretion. At
the opposite, EBAB reaches a maximum plasmatic concentration
faster but with a more important urinary excretion.
The described method should easily be transferable to the analysis of other similar analogs as the clean-up of biomatrixes is really
relevant. Furthermore, it would also be easily transferable to human
plasma and urine analysis with the advantage that large samples
volumes are foreseeable and put in perspective to obtain even lower
LLOQ.
Acknowledgments
This research work was supported by grants from ABMM
(Association Belge pour les Maladies Neuromusculaires), AFM

(Association Francaise pour les Myotonies), FNRS (Fonds de la

Recherche Scientique (projet FRSM No 3.4614.11) and FRMH
(Fond pour la recherche mdicale en Hainaut).
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