International Journal of Pharmaceutics 587 (2020) 119692

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Development of a stable oral pediatric solution of hydrochlorothiazide by T

the combined use of cyclodextrins and hydrophilic polymers
Marzia Cirria, Francesca Maestrellia, Natascia Menninia, Lorenzo Di Cesare Mannellib,
⁎
Laura Michelib, Carla Ghelardinib, Paola Muraa,
a
Department of Chemistry, University of Florence, Florence, Italy
b
Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Pharmacology and Toxicology Section, University of Florence, Florence, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Hydrochlorothiazide (HCT) is widely used in pediatrics for hypertension management. Due to the lack of pe-
Hydrochlorothiazide diatric commercial forms, community or hospital pharmacies generally prepare HCT extemporaneous pediatric
Pediatric solution suspensions by dispersing in water a portion of a crushed tablet or the drug powder; however, any dose or
Hydroxypropyl-β-cyclodextrin stability control is usually done on these preparations. Obtaining stable HCT solutions is very challenging, due to
Sulfobutylether-β-cyclodextrin
its low water-solubility and pH-dependent degradation. The aim of this work was to develop a stable 2 mg/mL-
Hydrophilic polymers
HCT oral pediatric solution without using co-solvents. Combined use of cyclodextrins (CD) and hydrophilic
Stability
In vivo diuretic effect polymers was exploited to improve poor HCT solubility and stability. HPβCD and SBEβCD were selected, con-
sidering their safe toxicological profiles, while PVP resulted the best among the tested polymers. Low PVP
concentrations (0.2–1.0%) improved the solubilizing efficiency of both CDs, allowing to reach the prefixed HCT
concentration. Different CD-PVP concentrations were used to prepare several 2 mg/mL-HCT solutions in pH 5.5
buffer. The best stability was shown by solutions containing the highest SBEβCD concentration (25 mM), which
allowed a 3-months stability at 4 °C. In vivo studies on rats showed that such formulation allowed a more
pronounced and more reproducible diuretic effect than the corresponding HCT suspension.

1. Introduction
Most of the drug products available on the market have no in-
formation in the labeling for the use in pediatrics and there is a general
lack of medicines properly assessed and with approved indication for
the pediatric population, thus making the pharmacological treatment in
children a fundamental public health problem (Shah et al., 2007; Amer.
Acad. Pediatr., 2014). In fact, the marked differences in pharmacoki-
netic/pharmacodynamic of drugs between adult and pediatric patients,
would require specific studies for the pediatric population (Ivanovska
et al., 2014). Instead, clinical trials in this field are still very limited,
mainly due to shortage of available people and high complexity of the
research, joined to economic and ethical reasons. (Rose, 2008).
The need for increasing the availability of child-specific medicines
has been well recognized (Ward et al., 2017) and several efforts have
been made during the last decades for enhancing pediatric drug de-
velopment, by introducing suitable guidelines and policies and pro-
moting incentives to investments in new therapies for pediatric use
(WHO, 2011; EMA, 2013; Bucci-Rechtweg, 2017; Tomasi et al., 2017).
However, it will take some time before the effects of these legislations
and strategies can be seen and an adequate number of purposely for-
mulated drugs become actually available for children.
The lack of adequate pharmaceutical products authorized for pe-
diatric use led to a wide off-label use of dosage forms intended for
adults, by manipulation of tablets, capsules or concentrated suspensions
or solutions for obtaining the required proportion of drug dose to be
administered. However, these manipulations result in poor or un-
controlled dosing accuracy, especially harmful when very low dosages
are required. Dose calculations errors during manipulations can
happen, and they have even been recognized as the most frequent
medication mistake occurring in pediatric patients (Chua et al., 2010).
Moreover, the stability of extemporaneous formulations is limited and
uncertain, from both a physical, chemical and microbiological point of
view, and their bioavailability may be variable and/or unknown (Glass
and Haywood, 2006; Skwierczynski and Conroy, 2008). Also, the ex-
cipients commonly used in dosage forms for adults, such as pre-
servatives, sweeteners, fillers, solvents, coating and coloring agents, can
result harmful for children of various ages (Fabiano et al., 2011) and
the off-label use of drugs in pediatric patients leads to their exposure to
high risks of unanticipated adverse reactions (Bellis et al., 2013).

⁎
Corresponding author at: Department of Chemistry, via Schiff 6, 50019 Sesto Fiorentino (FI), Italy.
E-mail address: paola.mura@unifi.it (P. Mura).

https://doi.org/10.1016/j.ijpharm.2020.119692
Received 31 May 2020; Received in revised form 16 July 2020; Accepted 21 July 2020
Available online 24 July 2020
0378-5173/ © 2020 Published by Elsevier B.V.
M. Cirri, et al.
Finally, poor palatability and/or taste of these extemporaneous pre-
parations can negatively influence the young patient compliance, and
consequently the relative clinical outcomes.
The European Medicinal Agency (EMA) reflection paper on pedia-
tric formulations indicates oral liquid dosage forms as the form of
choice due to the simplest applicability for administration to infants,
toddlers and children up to 5 years (EMA, 2006). Liquid formulations
offer the benefits of great dosing flexibility, swallowing ease, and low
choking risks. On the other hand, they present possible issues con-
cerning stability and palatability, acceptable dose volume (preferably
≤5 mL for children up to 5 years) and use of child-friendly excipients
(EMA, 2006). Among oral liquid formulations, solutions are preferred
to suspensions due to their better acceptance by pediatric patients, less
susceptibility to dosing errors arising from insufficient or non-homo-
geneous re-dispersion of the sediment, and greater ease of administra-
tion, also, if necessary, through enteral feeding tubes. Pediatric oral
solutions of drugs such as amlodipine (Van der Vossen et al., 2016) or
lorazepam (Van der Vossen et al., 2017) have been recently successfully
developed.
Hydrochlorothiazide (HCT) is a thiazide diuretic drug widely used
in pediatrics for the hypertension management, and it is part of the List
of Essential Medicines for Children set out by (WHO, 2013)). The re-
commended pediatric therapeutic HCT dosage is 1 or 2 mg/mL once
daily or two administrations every 12 h, depending on clinical situation
and patient’s response.
However, there are neither age-appropriate, nor liquid HCT for-
mulations available on the market. Moreover, the obtainment of HCT
solutions is made very challenging by its low aqueous solubility and its
pH-dependent hydrolytic degradation, with the lowest degradation rate
around pH 3.0–3.5 (Mollica et al., 1971); Tagliari et al., 2008; Mahajan
et al, 2012). Then, community or hospital pharmacies usually provide
HCT extemporaneous pediatric suspensions produced by dispersion of
the drug powder in sweetened solutions, or suspension of a portion of a
crushed tablet in suitable commercially available suspension bases, thus
running into all the disadvantages above illustrated (Glass and
Haywood, 2006; Chua et al., 2010; Skwierczynski and Conroy, 2008;
Fabiano et al., 2011).
Numerous efforts have been made to prepare stable HCT suspen-
sions. Tagliari et al. (2009) developed a pH 3.3 flocculated HCT sus-
pension containing 0.6% carboxymethylcellulose sodium, well re-dis-
persible after soft agitation; however, data about its stability on storage
are lacking. Santoveña et al. (2012) prepared four different oral pe-
diatric HCT suspensions, using glycerol as wetting agent, hydro-
xypropyl-methylcellulose or methylcellulose as stabilizer, and adjusting
pH at 3.0–3.5 with citric acid, to improve drug stability (Mollica et al.,
1971); the best result was obtained with the formulation containing
20% glycerol and 1% methylcellulose, which allowed a 3-weeks stabi-
lity when stored light-protected at 5 °C. Other authors proved that HCT-
spironolactone suspensions (5/5 mg/mL) obtained by commercial ta-
blets pulverization and then powder dispersion in an aqueous vehicle
containing an Ora-plus®/Ora-sweet® 1:1 mixture as suspending agent
(pH 4.2–4.4) were stable up to 60 days storage at 5 or 25 °C (Allen and
Erickson, 2011). Extemporaneous HCT aqueous suspensions prepared
using drug raw material showed a maximum 7-days shelf life when
stabilized by adjusting pH to 3.3 (Mendes et al., 2013). A 60 days-
stability was found for HCT suspensions (2 mg/mL) obtained by dis-
persing the drug in a 6.5% Syrspend®SF-PH4 aqueous vehicle (pH 4.0)
(Binson et al., 2019).
The only study in literature on HCT solutions development regarded
a 0.5 mg/mL solution (Li, 2013), that was the maximum drug con-
centration giving a clear solution, according to its saturation solubility
(≈0.57 mg/mL, Baka et al., 2008; Altamimi et al., 2018). The mono-
graphy of this HCT 0.5 mg/mL oral solution is reported on the new on-
line European Paediatric Formulary (2019), and consists in a sucrose
solution (200 mg/mL) buffered at pH 3.2, to reduce drug hydrolysis,
added with propylene glycol (2.75 mg/mL) as co-solvent and methyl-
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International Journal of Pharmaceutics 587 (2020) 119692
parahydroxybenzoate (0.77 mg/mL) as preservative, and has a 6-
months shelf-life (Eur. Paed. Formul., 2019). However, products con-
taining high propylene glycol concentrations should not be adminis-
tered to infants below 4 years, due to possible accumulation problems,
owing to their limited metabolic pathway, with consequent risks of
toxic effects (EMA, 2006). Moreover, the pediatric use of sucrose as
sweetener should be generally limited, since it is considered potentially
cariogenic, and it must be completely avoided in therapy of diabetic
patients (EMA, 2013).
Oral liquid HCT pediatric formulations based on solid lipid nano-
particles (SLN) and nanostructured lipid carriers (NLC), were pre-
viously successfully developed (Cirri et al., 2017; Cirri et al., 2018a;
Cirri et al., 2018b). However, in spite of their safety in use, due to the
absence of potentially harmful excipients, higher bioavailability than
simple HCT suspensions, and good stability on storage (up to 3 months
at 4 °C), such formulations were not suitable for a quick setting-up in
hospital pharmacies, due to the elaborated preparation procedure, re-
quiring a series of steps with the use of specific rather complex appa-
ratus.
Therefore, based on all these premises, the aim of this work was to
develop a stable oral pediatric aqueous solution of HCT at a target
concentration of 2 mg/mL, easy and fast to prepare also in a pharmacy,
without using potentially harmful co-solvents, with a well-tolerable pH
value, endowed with an adequate shelf-life, supported by stability
studies, and thus able to assure right administration dose, efficiency of
the therapeutic treatment, and stability during storage.
Our approach, aimed to overcome the problems of poor solubility
and stability of HCT, was based on the combined use of cyclodextrin
(CD) complexation and hydrophilic polymers. CDs are cyclic torus-
shaped oligosaccharides consisting of six to eight glucose units, named
α -, β -, and γ-CDs, respectively. CDs, characterized by a hydrophilic
outer surface and a hydrophobic central cavity, are known for their
ability to form inclusion complexes with a variety of lipophilic drugs,
thus improving their solubility and/or chemical or physical stability
(Kurkov and Loftsson, 2011; Jansook et al., 2018). Several studies re-
ported the positive effect of water-soluble polymers in improving CD
complexing, solubilizing and stabilizing efficacy, (Loftsson and
Fridriksdottir, 1998; Mura et al., 2001; Ammar et al., 2006; Cirri et al.,
2006; Taupitz et al., 2013; Wang et al., 2013), due to the synergistic
effect of polymer and CD on the formation of drug:CD:water-soluble
polymer ternary systems (Loftsson et al., 1994; Ribeiro et al., 2003;
Loftsson and Másson, 2004).
Some studies concerning the characterization of HCT interactions in
solution with βCD are reported in literature (Denadai et al., 2006;
Onnainty et al., 2013). The HCT-βCD complex prepared by fluid bed
showed an increase in HCT intrinsic dissolution rate and diuretic effect
(Pires et al., 2011). Complexation with βCD increased HCT solubility,
protected it from hydrolysis up to 30 days and enhanced its in vivo
bioavailability (Mendes et al., 2016). However, these authors reported a
water solubility value for HCT of 0.037 mg/mL, and obtained a max-
imum solubility increase by βCD complexation at pH 3.3 up to
0.080 mg/mL. Altamimi et al. (2018) showed that βCD complexation
was more effective than the use of surfactants such as sodium lauryl
sulfate or Tween 20 in improving HCT solubility (reaching a maximum
solubility value of about 1.4 mg/mL) and in preventing its degradation
up to 60 days at room temperature.
However, it is well known that safety in use and complexing/solu-
bilizing power of βCD are limited by its rather low water solubility,
which led to the search for more soluble βCD-derivatives (Brewster and
Loftsson, 2007). Moreover, some of these highly-soluble derivatives,
such as in particular hydroxypropyl-β-cyclodextrin (HPβCD) and sul-
fobutylether-β-cyclodextrin (SBEβCD), in addition to the higher com-
plexing and solubilizing power, further possess improved toxicological
profiles compared to native βCD (Brewster and Loftsson, 2007), thus
making them more suitable for pediatric use.
Therefore, in the present study HPβCD and SBEβCD were selected as
M. Cirri, et al.
complexing partners for HCT, while hydrophilic polymers poly-
vinylpyrrolidone, hydroxypropylmethylcellulose and sodium carbox-
ymethylcellulose were tested for their effectiveness as ternary compo-
nents. In addition, a maltitol-syrup (Lycasin®80/55) was added to the
developed formulations, in order to enhance their taste, making them
more palatable and also suitable for children with diabetic diseases. The
developed formulations were tested for physical, chemical and micro-
bial stability on storage and for in vivo diuretic activity in rats.
2. Materials and methods
2.1. Materials
Hydrochlorothiazide (HCT) was a gift from Menarini (Italy).
Hydroxypropyl-β-Cyclodextrin (Kleptose®HP, HPβCD) with an average
molar substitution (MS) of 0.85 (average molecular weight 1443 g/mol)
and Lycasin®80/55, a maltitol syrup (55% maltitol on dry basis) with
an average viscosity of 3000 mPa*s at 20 °C, were kindly donated from
Roquette (Lestrem, France), while sulfobutylether-β-Cyclodextrin
(Captisol®, SBEβCD) from Ligand Pharmaceuticals, Inc. (San Diego,
CA). Polyvinylpyrrolidone K30 (average molecular weight 40000,
PVP), hydroxypropyl-methylcellulose (HPMC), Sodium carbox-
ymethylcellulose (NaCMC) and sodium benzoate were supplied by
Sigma Aldrich Co. (St. Louis, USA). Purified water was obtained by
reverse osmosis (Elix® Millipore, MD, USA). All other chemicals were of
analytical grade.
2.2. Phase-solubility studies
An excess amount of HCT was added to pH 5.5 phosphate buffer
aqueous solutions containing increasing concentrations of HPβCD or
SBEβCD (0–25 mM) in the presence or the absence of a fixed amount of
hydrophilic polymer (PVP, HPMC or CMC). The suspensions were kept
at constant temperature (25 °C) under magnetic stirring at 500 rpm up
to equilibrium (48 h), and then an aliquot was filtered (0.45 µm pore
size) and UV assayed according to the procedure described below
(section 2.3). No interferences on the spectrophotometric assay of HCT
due to the presence of CD and/or polymer were found. Each test was
performed in triplicate.
The apparent stability constant (K1:1) of the HCT-CD complexes
were calculated from the slope of the phase-solubility diagrams and the
drug solubility in the medium (Higuchi and Connors, 1965).
The solubilizing efficiency (SE) was obtained by the ratio of drug
solubility in the presence of the highest CD concentration used and that
of the plain drug.
2.3. HCT spectrophotometric assay
Drug concentration in phase-solubility and storage stability studies
was spectrometrically determined at 272.2 nm (Shimadzu UV/vis 1601,
Tokyo, Japan). The analytical method was validated according to the
ICH Q2(R1) guidelines (2005). Five standard solutions of the drug were
prepared in the concentration range from 3.0 to 20 mg/L. The equation
of the regression line was y = 0.0623x + 0.003, r2 = 0.9991. The LOQ
(Limit of Quantitation) and LOD (Limit of Detection) values were
0.5 mg/L and 0.15 mg/L, respectively. The precision of the method (as
repeatability), determined by a six times analysis of a same drug
sample, was 0.55%. The accuracy of the method, expressed as % re-
covery of a known added drug amount, was 100.5 ± 1.5%.
2.4. Preparation of HCT liquid formulations
Two different series of liquid HCT (2 mg/mL) formulations were
prepared, using, respectively, a simple pH 5.5 phosphate buffer or a 80/
20 v/v phosphate buffer (pH 5.5):Lycasin®80/55 mixture, (with or
without 0.05% w/v sodium benzoate as preservative) as solvent.
3
International Journal of Pharmaceutics 587 (2020) 119692
The series of HCT simple solutions were prepared by adding the
exactly weighed drug amount (200 mg) in 100 mL of pH 5.5 phosphate
buffer, and keeping under magnetic stirring at ambient temperature, in
the presence of different suitable concentrations of SBEβCD or HPβCD
and hydrophilic polymer (as indicated by phase-solubility studies), al-
lowing to obtain complete drug solubilization and achieve the target
drug solubility (2 mg/mL).
The same procedure was followed for preparation of the series of
HCT syrups, where a 20% of the total volume of phosphate buffer was
replaced by Lycasin®80/55.
2.5. Stability studies under storage
Chemical, physical and microbiological stability studies of the dif-
ferent formulations were performed during 3 months on samples stored
at room temperatures and at 4 °C, in light-protected glass vials.
Chemical stability studies were performed by biweekly monitoring
the residual drug concentration by spectrophotometric assay (UV–Vis
1601 Shimadzu spectrometer, Kyoto, Japan) at λ = 272.2 nm (see
Section 2.3). In the light of the high accuracy and precision demon-
strated by the spectrophotometric technique, and the absence of in-
terferences in the drug assay, it was considered suitable also for stabi-
lity studies. In fact, these were merely aimed at determining the
residual drug concentration present in solution, and not at separating
and quantifying its eventual degradation products, already known and
characterized (Gumieniczek et al., 2018). HCT formulations were con-
sidered stable when drug concentration remained above 90% of the
initial concentration, without showing variations in physical properties.
Physical stability was checked weekly monitoring, by visual in-
spection, any eventual modification in organoleptic properties such as
odour, clarity and colour, appearance of precipitation. Eventual pH
changes of the solutions were controlled by pH measurements, per-
formed in triplicate using a Basic 20 pH-meter (Crison Instruments,
Spain).
Microbiological stability was checked by biweekly monitoring, by
visual inspection, of possible cloudiness phenomena or possible mold
formation.
2.6. In vivo studies
In vivo studies were carried out using male Sprague-Dawley rats
(Envigo, Varese, Italy), weighing around 200–250 g at the beginning of
the experimental procedure. Rats, housed in CeSAL (Centro
Stabulazione Animali da Laboratorio, University of Florence, Italy)
were used not earlier than seven days after they arrived. Rats were
housed in 26x41 cm cages (4 in each one), kept at room temperature
(23 ± 1 °C) with a 12 h light/dark cycle (light at 7 a.m), and nourished
according to a standard laboratory diet, providing water ad libitum. All
animal treatments were performed in agreement with the Directive
2010/63/EU of the European Parliament and of the European Union
Council (22 September 2010) on protection of animals used for scien-
tific purposes. The Florence University ethical policy is in compliance
with the Guide for Care and Use of Laboratory Animals of US National
Institutes of Health (NIH Publication No. 85–23, revised 1996;
University of Florence assurance number: A5278-01). Formal approval
to perform the experiments was obtained from the Italian Ministry of
Health (No. 54/2014-B) and from the Animal Subjects Review Board of
Florence University. Experiments involving animals have been reported
in agreement with ARRIVE guidelines (McGrath and Lilley, 2015).
Every effort was undertaken to lower the number of animals utilised
and minimize their suffering.
The HCT diuretic effect was evaluated following the same procedure
of a previous study of the Authors (Cirri et al. (2017), in accordance
with the method reported by Compaore et al. (2011). Rats were sub-
divided into four groups (n = 5) for the acute (single oral dose) study,
and they had free access to both water and food. Prior to treatment, an
M. Cirri, et al.
oral dose of 2.5 mL/100 g body weight of physiological saline (0.9%
NaCl) was administered to all rats (Wiebelhaus et al., 1965), so that to
impose a uniform water and salt load. An hour later, rats received the
following oral treatments: group I (control), 0.9% saline; group II, HCT
suspension 10 mg kg−1; group III, HCT- SBEβCD solution, 10 mg kg−1;
group IV, HCT- SBEβCD syrup, 10 mg kg−1. Immediately after ad-
ministration, the rats were put in metabolic cages (one per cage),
purposely designed to separate fecal material from urine and the cu-
mulative urine and feces output at various times (1, 2, 4, 6 and 24 h)
was measured (Asif et al., 2014). The diuretic activity was expressed in
mL/100 g of body weight (Asif et al., 2014), while the diuretic index
was calculated by the ratio between the diuresis of rats treated with the
test formulation and that of the control group (Danamma et al., 2011).
The gastrointestinal motility was evaluated measuring the number of
faecal pellets produced by the animals during the entire experiment, at
the same times considered for the diuretic activity.
Each value represents the mean ± SEM of 5 rats per group, per-
formed in two different experimental sets. The analysis of variance was
performed by ANOVA. A Bonferroni’s significant difference procedure
was used as post-hoc comparison. P values of < 0.05 were considered
significant. Data were analysed using the ‘Origin 9′ software
(OriginLab, Northampton, MA, USA).
3. Results and discussion
The objective of this study was the development of a stable oral
liquid HCT pediatric formulations with a target concentration of 2 mg/
mL, without the use of potentially harmful co-solvents, and with a well-
tolerable pH value, higher than the pH 3–4 values commonly used to
improve its stability against hydrolysis (Mollica et al., 1971; Tagliari
et al., 2008; Mahajan et al, 2012) in the until now developed aqueous
drug suspensions (Mendes et al., 2013, Binson et al., 2019), as well in
the existing 0.5 mg/mL HCT solution (Eur. Paed. Formul., 2019),
CD complexation was exploited to reach the prefixed goal, in the
attempt to improve at the same time drug solubility and stability in
aqueous solution. Our previous studies already proved the solubilizing
ability of different βCD-derivatives towards HCT (Maestrelli et al.,
2020). Among the examined derivatives, SBEβCD and HPβCD were
selected for the present study, due to their safer toxicological profiles
than both native βCD or methylated-βCDs (Brewster and Loftsson,
2007), which make them suitable for parenteral and pediatric use.
With a view to further improve the drug solubility, and reach the
desired target value of 2/mg/mL, the possible favorable effect of the
presence of hydrophilic polymers, such as NaCMC, PVP, or HPMC, in
enhancing the CD solubilizing and stabilizing ability towards HCT has
been evaluated, based on the positive results reported in literature
about the effectiveness of this combined approach (Loftsson and
Fridriksdottir, 1998; Mura et al., 2001; Ammar et al., 2006; Cirri et al.,
2006; Taupitz et al., 2013; Wang et al., 2013). These polymers were
selected also considering their wide and consolidated use as safe
pharmaceutical excipients (Schwarz, 1990: Rowe et al., 2009; Franco
and De Marco, 2020).
A pH 5.5 phosphate buffer was selected as aqueous medium to
prepare the final HCT solutions, as a good compromise between drug
permeability, stability, and tolerability. In fact, based on its pKa value
(7.9, according to O'Neil, 2013), HCT at pH 5.5 was still predominantly
present in the unionized form, which is more permeable than the io-
nized one through the biological membranes. Moreover, even though
lower pH values, around 3.0–3.5, should provide a higher drug stability
(Mollica et al., 1971; Tagliari et al., 2008; Mahajan et al, 2012), pH 5.5
should assure a better tolerability for pediatric patients, considering
they have a gastric pH around 4.5 (Nguyen et al., 2015).
3.1. Phase-solubility studies
An initial screening was conducted by performing experiments with
4
International Journal of Pharmaceutics 587 (2020) 119692
Table 1
Stability constant (K1:1) and solubilizing efficiency (SE) of HCT complexes with
HPβCD or SBEβCD in the presence of 0.2% w/v of hydrophilic polymer at 25 °C
in pH 5.5 phosphate buffer.
CD K 1:1 (M−1) SEa
HPβCD 106 ± 3 3.1
HPβCd + PVP 101 ± 5 3.5
HPβCd + HPMC 107 ± 4 3.2
HPβCd + NaCMC 128 ± 5 3.2
SBEβCD 213 ± 4 4.7
SBEβCd + PVP 173 ± 4 4.9
SBEβCd + HPMC 214 ± 4 4.7
SBEβCd + NaCMC 253 ± 3 4.7
a
ratio between drug water solubility in presence of 25 mM CD used and
alone.
both the selected CDs in the presence of a fixed amount (0.2% w/v) of
three different hydrophilic polymers, i.e. PVP, NaCMC and HPMC, in
order to select the most effective ternary component. Phase-solubility
studies were performed in pH 5.5 buffer solution, selected, as explained
above, as the medium for the preparation of the final oral HCT for-
mulations. AL-type phase-solubility diagrams were obtained for both
CDs in the presence of all the tested polymers, indicating the formation
of soluble inclusion complexes (Higuchi and Connors, 1965). The ob-
tained values of apparent stability constants (K1:1) and Solubilizing
Efficiency (SE) are collected in Table 1, together with the corresponding
data previously obtained for HCT:HPβCD and HCT:SBEβCD inclusion
complexes in the absence of polymers (Maestrelli et al., 2020), for
comparison purposes.
As known, the affinity of ionizable drugs, such as HCT, for the in-
clusion into the hydrophobic cavity of CDs is inversely related to the
ionization degree of the host molecule. In particular, in the case of HCT,
considering its pKa value (7.9, according to O'Neil, 2013), a decrease of
the complex stability constant is expected with increasing the pH value,
due to a progressive decrease in drug hydrophobicity with increasing its
ionized fraction in solution. However, at pH 5.5 the drug is still largely
present in the unionized form, and previous studies performed on the
complex of HCT with native βCD showed that there was not significant
difference between the K1:1 values determined at pH 3.0 and pH 5.5
(Onnainty et al., 2013).
The obtained K1:1 values indicated that only the addition of NaCMC
led to an increase of the complex stability with both CDs, but it did not
enhance the solubilizing power of HPβCD and SBEβCD toward the
drug, while HPMC affected neither complexing nor solubilizing prop-
erties of both CDs.
On the contrary, PVP was the only hydrophilic polymer able to in-
crease both the intrinsic HCT solubility (S0) (0.77 mg/mL vs 0.62 mg/
mL), and the CD solubilizing efficiency, even though a slight decrease of
the complex stability constant was observed, due to the increased value
of S0.
Therefore, based on these findings, PVP was selected for further
phase-solubility studies, to evaluate the effect of the increase of its
concentration (up to 1% w/v) on the CD solubilizing power, and in-
vestigate a possible synergistic effect between polymer and CD in en-
hancing HCT solubility. The results of these studies are shown in Fig. 1,
and summarized in Table 2 in terms of K1:1, SE and highest HCT con-
centration achieved in solution (Csmax). The reduction of the stability
constant values of HCT complexes with both HPβCD and SBEβCD be-
came more evident with increasing the PVP concentration in the
medium, due to the almost constant values of the slopes of the related
phase-solubility diagrams (see Fig. 1) joined to the concomitant in-
crease in drug intrinsic solubility S0. Interestingly, a progressive in-
crease in CD solubilizing efficiency was observed, even though there
was not a true synergistic effect between polymer and CD, since the
total increase in drug solubility was almost equal to the sum of the
M. Cirri, et al. International Journal of Pharmaceutics 587 (2020) 119692

Fig. 1. Phase-solubility diagrams in pH 5.5 buffered water of hydrochlorothiazide (HCT) with HPβCD (A) or SBEβCD (B), alone (●), or in the presence of PVP at 0.2%
(□), 0.5% (▴) or 1% w/v (○).

Table 2 recommended only in parenteral dosage forms in neonates (EMA,

Stability constant (K1:1) and Solubilizing Efficiency (SE) of complexes of hy- 2006).
drochlorothiazide (HCT) with the tested CDs in the presence of different PVP
concentrations (0.2, 0.5 and 1% w/v) at 25 °C in pH 5.5 phosphate buffer, and
HCT concentration achieved (Cs max) in the presence of 25 mM CD. 3.3. Stability studies of HCT liquid formulations
−1 a
CD % PVP K1:1 (M ) SE Cs max (mg/mL)
All the developed HCT solutions were stored both at room tem-
HPβCD — 106 ± 3 3.1 1.9 perature and at 4 °C, protected from light, and drug concentration,
HPβCD 0.2 101 ± 5 3.5 2.1 possible pH changes, or organoleptic properties modifications or mi-
HPβCD 0.5 84 ± 3 3.7 2.2 crobial contaminations (in terms of mold formation) were biweekly
HPβCD 1.0 78 ± 2 3.8 2.3 monitored. The stability was considered acceptable as long as the
SBEβCD — 213 ± 4 4.7 2.9
SBEβCD 0.2 173 ± 4 4.9 3.0
percentage of HCT remained above 90% of the initial value, and no
SBEβCD 0.5 164 ± 5 5.0 3.1 organoleptic modifications or microbiological contamination occurred.
SBEβCD 1.0 155 ± 4 5.1 3.2 The results of chemical stability studies, in terms of residual HCT
concentration, of the series of pH 5.5 aqueous solutions are depicted in
a
ratio between drug water solubility in presence of 25 mM CD concentration Fig. 2 (A, B, C). Samples stored at room temperature in the absence of
and alone.
0.05 %w/v Na Benzoate showed a faster drug degradation than the
corresponding refrigerated samples, reaching the minimum acceptable
solubility increases given by each carrier separately. value of residual concentration after only 4 weeks (Fig. 2A). Moreover,
However, our first goal was achieved: in fact the joined presence of also mold formation was observed at this time.
PVP and HPβCD allowed to achieve the desired HCT concentration Storage at 4 °C enabled a slowing down of HCT degradation rate: all
(2 mg/mL), not attainable with HPβCD alone, even at the highest the samples showed higher values of HCT residual concentration during
concentration used (25 mM); on the other hand, addition of PVP al- 4 weeks compared to those stored at room temperature (Fig. 2B).
lowed to reduce the amount of SBEβCD to reach the target HCT solu- However, also in these storage conditions, mold formation was detected
bility (2 mg/mL) from 25 mM up to 13 mM. in all the samples after 4 weeks. The only exception was the solution
containing 25 mM SBEβCD, that showed the highest stability, main-
3.2. Preparations of HCT liquid formulations taining a suitable residual concentration up to 8 weeks, without dis-
playing microbiological contamination. Solutions 2 containing 0.05%
Based on the data obtained by phase-solubility studies, four dif- w/v Na Benzoate, stored at 4 °C, showed a longer stability period for all
ferent 2 mg/mL HCT solutions in pH 5.5 phosphate buffer were then set the examined formulations, maintaining an acceptable residual HCT
up, containing, respectively, 25 mM SBEβCD alone, 15 mM SBEβCD concentration during 8 weeks, without showing any mold formation.
with 0.2% PVP, 25 mM HPβCD with 0.2% PVP and 20 mM HPβCD with Among them, the solutions containing the highest CD concentrations
1% PVP were the best, and, in particular, the solution containing 25 mM
A second series of analogous formulations was also prepared where SBEβCD was the only one stable up to three months (Fig. 2C). The
a 20% of the pH 5.5 phosphate buffer solution was replaced by a higher improvement in drug chemical stability observed in the presence
maltitol syrup (Lycasin®80/55), with the aim to exploit its flavouring of SBEβCD than HPβCD can be attributed to the higher stability of its
and sweetening properties for improving taste and palatability of the complex with the drug (see Table 2), considering that the stronger the
formulations. Moreover, the low-glycemic and insulinemic indexes of complex, the lesser the amount of free drug subjected to degradation.
Lycasin®80/55 make it ideal for the preparation of sugar-free syrups. In Complexation with native βCD provided a protective effect up to
addition, the composition of this maltitol syrup has been specifically 30 days against HCT hydrolytic degradation in unbuffered aqueous
developed to generate very little acid production when in contact with solution (Mendes et al., 2016).
the plaque bacteria, so that it is considered tooth-friendly. Moreover, 25 mM SBEβCD also exhibited a preservative effect
The two series of formulations, as solutions or syrups, were pre- against mold formation, previously observed by other Authors and at-
pared both in absence and in presence of Na Benzoate (0.05% w/v) as tributed to its high ability to reduce the water activity of the formula-
preservative, since it is considered suitable for daily use in pediatric tion, which is considered one of the most important factors affecting the
formulations (Embrechts et al., 2010). In fact, its use is not growth of microorganisms (Mosher et al., 2005).

5
M. Cirri, et al.
Fig. 2. Stability of the different hydrochlorothiazide (HCT) pH 5.5 buffered
solutions stored at room temperature (A) or at 4 °C in light-protected glass vials,
in the absence (B) or in the presence (C) of Na Benzoate as preservative.
The obtained results are very satisfactory, considering that the sta-
bility of the different HCT suspensions previously developed varied
from a minimum of 7 days (Mendes et al., 2013) up to a maximum of
60 days, but at pH 4.2–4.4 (Allen and Erickson, 2011) or 4.0 (Binson
et al., 2019).
Visual inspection of the different solutions did not reveal any sig-
nificant modifications of their organoleptic properties such as odour,
clarity and colour, or formation of precipitates during the considered
period. Moreover, pH determinations showed that there was less than
0.5 pH unit change throughout the entire study period for all the pre-
parations.
Storage stability studies of syrups (Fig. 3) were carried out only at
4 °C, since these formulations displayed problems of microbiological
stability within the first two weeks if stored at room temperature.
Syrups without preservative showed a stability substantially similar to
6
International Journal of Pharmaceutics 587 (2020) 119692
Fig. 3. Stability of the different hydrochlorothiazide (HCT) pH 5.5 buffered
syrups stored at 4 °C in light-protected glass vials, in the absence (A) or in the
presence (B) of Na Benzoate as preservative.
that of the corresponding solutions stored at 4 °C, maintaining residual
HCT concentration values clearly higher than 90%, up to 4 weeks
(Fig. 3A). Nonetheless, the presence of Na Benzoate was less effective in
prolonging the stability of the preparations, allowing an extension of
the stability period of only two further weeks. (Fig. 3B). Moreover,
differently from the case of the simple HCT solutions, no stability dif-
ferences were observed among the different syrup formulations by
varying the CD type (HPβCD or SBEβCD) or its concentration.
No modifications in organoleptic properties such as odour, clarity
and colour, or precipitation phenomena or mold formation, or pH
changes were observed in both the series of formulations (without or
with preservative) during these storage periods (4o 6 weeks, respec-
tively). Mold formation was instead detected in both cases after these
periods.
3.4. In vivo studies
On the basis of the stability study results, the formulations con-
taining 25 mM SBEβCD, both in form of solution and syrup, were se-
lected for in vivo studies on rats, in order to evaluate the HCT diuretic
effect, in comparison to the effect evoked by a simple HCT suspension.
All formulations were orally administered at the same dose of
10 mg kg−1. The induced diuretic effect was measured as total volume
of excreted urine, diuretic activity (volume of excreted urine per 100 g
body weight), and diuretic index, with respect to a control group
treated with an equal volume of saline solution. The gastrointestinal
motility was also evaluated for safety assessment of the developed
formulations with regard to their potential side effects on gastro-
intestinal motility, since one of the most common collateral reactions
M. Cirri, et al. International Journal of Pharmaceutics 587 (2020) 119692

caused by oral CD intake is an increase in gastrointestinal propulsions,

causing diarrhea (Lovatti Alves et al., 2019). The increased gastro-
intestinal motility could be due to the complex formation of bile salts
with CDs, leading to enhanced intestinal lipids concentrations (Stevens,
1999). Moreover, the concomitant presence of maltitol in the developed
syrups could exert a laxative function and emphasize such an effect. The
gastrointestinal motility was evaluated both assessing possible episodes
of diarrhea and measuring the number of faecal pellets produced by the
animals during the entire experiment, at the same time points con-
sidered for the diuresis.
Concerning the diuretic activity, the SBEβCD-based HTC solution
showed in the first 6 h a more pronounced effect compared to HCT
suspension, probably due to the increased drug solubility, and then
higher drug bioavailability, obtained by CD complexation. An analo-
gous result, in terms of faster effect and increased diuretic activity was
obtained after administration to rats of HCT as βCD complex rather
than as plain drug (Mendes et al., 2016). Surprisingly, the SBEβCD-
based HTC syrup showed, in the first 2 h after administration, values
analogous to those obtained with HTC suspension. This result could be
attributed to a slower drug absorption rate from the syrup, with respect
to the simple solution, due to its higher viscosity. Moreover, both so-
lution and syrup gave rise to more reproducible results than the sus-
pension, due to the better dose uniformity assured by the complete drug
dissolution obtained in such formulations (Fig. 4 A, B). Furthermore,
unlike the suspension, the diuretic activity values obtained by both
formulations containing the drug as SBEβCD complex were still sig-
nificantly different (p < 0.05) from the control up to 24 h.
As for possible gastrointestinal side effects of the formulations ex-
erted by the presence of CDs and/or maltitol, in vivo experiments re-
vealed that none of the tested treatments affected the gastrointestinal
motility with respect to the control group (Table 3). No episodes of
diarrhea occurred, and no increase in the faecal pellets number was
observed, thus allowing to rule out any possible gastrointestinal side
effect of the developed formulations due to the presence of SBEβCD
and/or the concomitant presence of maltitol.

4. Conclusions

The objective of developing an oral pediatric liquid HCT formula-

tion at the target concentration of 2 mg/mL has been successfully
reached. Complexation with CD, in the presence of PVP as ternary
component, not only allowed to increase HCT solubility up to the de-
sired target value, but also to improve its stability in solution against
hydrolysis, enabling to obtain a solution stable up to 3-months at pH
5.5, in the presence of a safe preservative such ad 0.05 %w/v Na
Benzoate.
The developed oral pediatric HCT solution offers numerous benefits
with respect to the previously developed HCT suspensions (Tagliari
et al., 2009; Allen and Erickson, 2011, Santoveña et al., 2012; Mendes
et al., 2013; Binson et al., 2019), in terms of easier administration (also
by enteral feeding tubes), better patient acceptability, lower dosing
errors (due to possible not-well re-dispersion of the sediment) and then
more reproducible therapeutic effect, more accurate dose dilution,
better tolerability in terms of pH (5.5 vs 3.0–3.5), and, additionally,
higher stability on storage (3 months vs maximum 2 months), despite
the higher pH.
Moreover, the developed HCT solution provides several advantages
also compared to the 0.5 mg/mL solution present in the European
Paediatric Formulary, such as higher drug concentration in solution
(and then lower dose volume to be administered), higher versatility,
allowing accurate dose adjustments by simple dilution, better patient
Fig. 4. Diuretic effect in rats as excreted urine total volume (A), diuretic ac-
tivity (excreted urine volume /100 g body weight) (B) and diuretic index (mean
urine volume treated group/mean urine volume control group) (C) after a
single oral administration (10 mg/Kg−1) of hydrochlorothiazide (HCT) as pH
5.5 buffered solutions or syrups, both in the presence of 25 mM SBEβCD, or as
aqueous suspension, compared to a control group treated with a same volume
of physiological saline (0.9% NaCl). *P < 0.05 and **P < 0.01 vs control
group; ^P < 0.05 vs HCT suspension (values shown are the mean ± SEM of 5
rats for group).
Table 3
Effects of a single oral administration (10 mg kg−1) in rats of HCT as pH 5.5
buffered solution or syrup, both in the presence of 25 mm SBEβCD, or as
aqueous suspension on the gastrointestinal motility (measured as number of
pellets) vs a control group treated with a same volume of physiological saline
(0.9% NaCl) (Values shown are the mean ± SEM of 5 rats for group).
Treatments time (h)
1 2 4 6

tolerability, due to the less acidic pH (5.5 vs 3.2), high safety, due to the Control 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.6 1.0 ± 0.9
absence of potential harmful co-solvents such as propylene glycol HTC + SBEβCD solution 0.0 ± 0.0 1.3 ± 1.6 1.7 ± 1.4 3.0 ± 2.2
(EMA, 2006), as well as of parabens (considering the potential toxic HTC + SBEβCD syrup 0.3 ± 0.3 0.3 ± 0.3 0.3 ± 0.3 2.0 ± 1.2
effects of such preservatives (Nowak et al., 2018). Furthermore, it is HTC suspension 0.3 ± 0.3 0.3 ± 0.3 0.3 ± 0.3 2.0 ± 1.2
suitable also for children with diabetic diseases and does not present

7
M. Cirri, et al.
cariogenicity problems, due to the absence of sucrose (EMA, 2013).
Moreover, the lack of side effects on the gastrointestinal motility was
also proved.
Furthermore, the simple developed preparation method could be
employed to draw up a Standard Operating Procedure (SOP) easy to use
by both community and hospital pharmacies to prepare a safe HCT oral
pediatric solution, able to assure the correct dose administering, im-
prove the efficiency of the treatment and guarantee the formulation
stability up to 3 months.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial or not-for-profit sectors.
CRediT authorship contribution statement
Marzia Cirri: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Project administration, Supervision,
Writing - original draft, Writing - review & editing. Francesca
Maestrelli: Data curation, Formal analysis, Writing - review & editing.
Natascia Mennini: Data curation, Formal analysis, Investigation.
Lorenzo Di Cesare Mannelli: Data curation, Formal analysis,
Investigation, Methodology. Laura Micheli: Formal analysis,
Investigation. Carla Ghelardini: Project administration, Resources,
Supervision. Paola Mura: Conceptualization, Data curation,
Supervision, Project administration, Resources, Writing - original draft,
Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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