Hindawi
Journal of Tropical Medicine
Volume 2020, Article ID 5278518, 9 pages
https://doi.org/10.1155/2020/5278518
Research Article
Quinine Sulphate Microparticles as Treatment for Leishmaniasis
Grace Lovia Allotey-Babington ,1 Seth Kwabena Amponsah ,2 Thomas Nettey,1
Clement Sasu,1 and Henry Nettey 1
1Department of Pharmaceutics andMicrobiology, School of Pharmacy, University of Ghana, P.O. Box LG 43 Legon, Accra, Ghana
2Department of Pharmacology and Toxicology, School of Pharmacy, University of Ghana, P.O. Box LG 43 Legon, Accra, Ghana
Correspondence should be addressed to Henry Nettey; hnettey@ug.edu.gh
Received 10 November 2019; Revised 16 March 2020; Accepted 30 March 2020; Published 30 April 2020
Academic Editor: Shyam Sundar
Copyright © 2020 Grace Lovia Allotey-Babington et al. +is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is
properly cited.
Background. Leishmaniasis is a neglected tropical disease caused by the Leishmania parasite and transmitted by the female
phlebotomine sandfly. +e disease can affect the skin (least fatal) or internal organs (most fatal). Current treatment options for
leishmaniasis have a number of adverse effects, and there appears to be resistance by the protozoan parasite (Leishmania spp.).
Reports suggest that quinine sulphate, not indicated for leishmaniasis, is effective in killing the Leishmania parasite. Indeed, the
efficacy of any drug is dependent on the concentration at the target site, which is also almost dependent on drug formulation. +e
current study assessed the pharmacokinetic profile of the microparticulate formulation of quinine sulphate and its in vitro and in
vivo efficacy against Leishmania donovani. Methods. Quinine sulphate was encapsulated in bovine serum albumin by the spray-
dryingmethod. Quinine sulphate microparticles were evaluated for size, zeta potential, drug content, encapsulation efficiency, and
in vitro release properties. Afterwards, the pharmacokinetic characteristics of quinine sulphate microparticles were estimated and
in vivo efficacy studies were also conducted. Results. +e size range of the quinine sulphate microparticles was between 2.0 and
5.0 µm. Microparticles had an average zeta potential of −35.2mV and an encapsulation efficiency of 94.5%. Also, Cmax, t1/2, and
AUC were all significantly desirable for quinine sulphate microparticles compared to the drug powder. Quinine sulphate
microparticles significantly reduced parasite load in rat organs than amphotericin B. Conclusion. Overall, quinine sulphate
microparticles had better pharmacokinetic profile and showed higher efficacy against Leishmania donovani parasites in vivo.+us,
quinine sulphate microparticles have the potential, especially, in treating visceral leishmaniasis.
1. Introduction parasite after a bite. Transmission of Leishmania species can
be from humans to animals and/or other humans
Leishmaniasis, a neglected tropical disease, is known to (anthroponotic) or from animals to humans (zoonotic) [5].
cause morbidity in more than 1 billion people worldwide [1]. Leishmaniasis presents in 3 major forms: visceral leish-
According to the World Health Organization (WHO), maniasis (highly fatal), also called kala-azar, which can
leishmaniasis is one of the major causes of death in a number destroy internal organs such as the liver, spleen, and lungs;
of resource-poor countries [2]. About 94% of new cases of mucocutaneous leishmaniasis (moderately fatal), which
leishmaniasis reported by the WHO in 2017 occurred in affects mainly mucous membranes; and cutaneous leish-
Africa, South America, and Asia, making it a disease of maniasis (least fatal), which mostly affects the skin and could
global concern [3]. lead to skin sores [6]. For many years, antimicrobial agents,
Leishmaniasis is caused by parasites from the genus notably the pentavalent antimonials, have been the mainstay
Leishmania, a subclass of the family Trypanosomatidae [4]. treatment options for leishmaniasis [7], however, with time
At least 20 Leishmania species (Leishmania donovani, the obligate intracellular parasites became resistant to these
Leishmania infantum, etc.) are known to infect humans, and agents. Currently, drugs like miltefosine, paromomycin, and
more than 90 sandfly species are known to transmit this amphotericin B are the gold standard in the treatment of
2 Journal of Tropical Medicine
leishmaniasis [8, 9]. Paromomycin and amphotericin B are 2.2. Equipment. +e Bilon-6000 Y Mini Spray Dryer was
administered parenterally. +is mode of administration is purchased from Shanghai Bilon Instrument Co., Ltd.
invasive, painful, and requires the assistance from trained Shanghai, China. +e Malvern Zetasizer Nano ZS (for zeta
medical personel; thus, patient compliance is often poor. potential analysis) was obtained from Malvern Instruments
Miltefosine, an orally approved treatment for leishmaniasis Inc., Westborough, MA. USA. +e Phenom Desktop
[10], is relatively expensive and has adverse effects on the scanning electron microscope (SEM) was obtained from
reproductive and gastrointestinal systems [11]. +ere have +ermoFisher Scientific, Phoenix, AZ, USA. Dissolution
been recent reports that suggest treatment failures with the Apparatus I was obtained from Distek Inc. (North Bruns-
use of miltefosine and amphotericin B (both deoxycholate wick, New Jersey, USA). +e fluorescence plate reader was
and liposomal forms) [9]. Furthermore, resistance to some obtained from Tecan Infinite 200 PRO, Mannedorf,
previously used agents has been reported to be due to long Switzerland. ®
treatment duration and missed drug treatment regimens
among patients (probably due to the cost of some of the
drugs) [12, 13]. Additionally, a number of the antimicro- 2.3. Preparation and Characterization of Quinine
bials used currently in leishmaniasis treatment have narrow Sulphate Microparticles
therapeutic indices and thus have a high tendency to cause 2.3.1. Preparation of Microparticles. A homogenous mixture
toxicity [14–16]. +ere is, therefore, the need to find other of quinine sulphate and bovine serum albumin (BSA) was
agents that are effective against the Leishmania parasite and prepared in the ratio of 1 : 4. Briefly, the BSA solution was
also with high benefit to risk ratio. first prepared by completely dissolving 4 g of BSA powder in
In a publication by Nettey et al. in 2016, quinine sul- 40mL of distilled water. Quinine sulphate solution (0.2 g/
phate, among other antimicrobials, was found to inhibit the mL) was slowly added and stirred. Solubility of quinine
growth of Leishmania parasites in vitro. Quinine sulphate sulphate was enhanced by the addition of 0.1N hydrochloric
was found to be more effective than two standard anti- acid dropwise. +e final solution was made up to 50mL with
leishmania drugs, pentamidine and amphotericin B in vitro distilled water and allowed to equilibrate at room temper-
[17]. Quinine sulphate, however, has a short half-life and ature after constant stirring for two hours. +e resulting
often requires frequent dosing. To recommend quinine mixture was then cross-linked with 400 µL of glutaraldehyde
sulphate as a drug of choice for the treatment of leish- solution for an hour at room temperature. Excess glutar-
maniasis, an improved delivery system needs to be devel- aldehyde was neutralized by the addition of 1% sodium
oped to reduce the dosing frequency, amongst others, as this bisulphite. +e mixture was then spray-dried using a Bilon-
disease could be chronic. 6000Y Laboratory Spray Dryer to obtain quinine sulphate
Microparticulate drug technology is a science that adapts loaded microparticles. Microparticle controls (empty mi-
formulation of drugs intominute particles ranging from 0.1 to croparticles without quinine sulphate) were prepared by
100 μm in size. +ese drug particles exhibit unique physi- spray-drying BSA solution cross linked with glutaraldehyde.
cochemical properties such as ultrasmall size, large surface to
mass ratio, high reactivity, and unique interactions with bi-
ological systems. Additionally, these peculiar characteristics 2.3.2. Particle Size and Zeta Potential Determination.
are known to improve drug absorption, deliver an adequate Particle size distribution and zeta potential were determined
amount of drugs to cells of interest, and prolong systemic by weighing and suspending 1mg of the microparticles in
effects of drugs. +us, the quest to maximize efficiency and 10mL of distilled water. Part of the suspension (100 µL) was
reduce dosing frequency of drugs can be attained with further diluted to 1mL with distilled water, and the size and
microparticulate formulations [18]. charge (runs of three independent experiments) were de-
In the current study, we formulated a controlled delivery termined using a Malvern Zetasizer Nano-ZS (Malvern
system (microparticles) of quinine sulphate in an attempt to Instruments Inc., USA).+e equipment utilizes the principle
deliver quinine sulphate to target sites, maintain the desired of light scattering to measure the size of microparticles.
concentration over time, and improve the efficacy of the
drug. To evaluate this, the in vitro and in vivo antileishmania 2.3.3. Surface Morphology of Microparticles. To determine
activities of quinine sulphate microparticles were evaluated. surface morphology of the microparticles, a scanning
electron microscope (+ermoFisher Scientific, USA) was
2. Materials and Methods used. Briefly, microparticles were captured on carbon sheets
and observed at 5 kV.
2.1. Test Organism and Reagents. +e test organism Leish-
mania donovani (WHO strain DD8) was a gift from Dr.
Neelo Singh of the Leishmania Research Society, India. 2.3.4. Drug Loading of Microparticles. To determine the
Quinine sulphate standard was a gift from the Centers for amount of quinine sulphate in the microparticles, an esti-
Disease Control and Prevention, Atlanta, GA, USA. Bovine mated 10mg of the formulation (weighed in triplicate) was
serum albumin (BSA) powder, culture media (M199), crushed in a mortar. Phosphate-buffered saline (PBS, pH
Alamar blue, glutaraldehyde, sodium bisulfite, and all other 7.4) was added, and the contents were transferred into
reagents used for experiments were purchased from VWR Eppendorf tubes and centrifuged at 10,000 rpm for 10
International (Radnor, PA, USA). minutes. A portion of the supernatant was pipetted and
Journal of Tropical Medicine 3
further diluted in PBS to obtain an expected concentration of plates were washed three times with M199 to remove
20 µg/mL. Quinine sulphate in the microparticles was es- uninvaded parasites.
timated using a UV spectrophotometer at a wavelength of +e Leishmania-infected macrophages were then given
334 nm. various treatments. +e first group which served as the
negative control was treated with only culture media. +e
second group was treated with twice the IC50 concentrations
2.3.5. Encapsulation Efficiency of Microparticles. +e effec- of amphotericin B solution [17] to serve as positive control.
tiveness of the polymer in entrapping quinine sulphate was Quinine sulphate solution and quinine sulphate microparticle
determined by calculating the encapsulation efficiency. suspension were prepared in stock concentrations of 10mg/
Encapsulation efficiency is determined as a percent of the mL. +e stock preparations were further diluted with culture
theoretical drug content. Mathematically, it is estimated as media to obtain a concentration of 6.25 µg/mL, respectively.
actual drug loading +e third group received quinine sulphate solution (6.25 µg/
% encapsulation efficiency � × 100.
expected drug loading mL), while the fourth group received quinine sulphate mi-
1 croparticle suspension (6.25 µg/mL). +e number of wells per( ) group was 5 (n� 5). After 24 hours of incubation (at 37°C), the
wells of the plates were washed twice with PBS (pH 7.4). Care
was taken not to wash-off the cells at the bottom of the wells.
2.3.6. In Vitro Drug Release. In determining in vitro drug All the groups were then incubated with fresh culture media
release, 30mg of the prepared quinine sulphate micropar- for another 24 hours. Following that, cells were washed twice
ticles was loaded into hard gelatin shells. Equal amounts of with PBS andmacrophages fixed with paraformaldehyde for 5
quinine sulphate powder were similarly encapsulated and minutes. +e wells were then washed again with PBS and PBS
used as control. Six dissolution vessels were filled with with 0.1% Triton X (PBST) for 5 and 15 minutes, respectively,
500mL of phosphate buffer, pH 6.80. Triplicates of the 2 to eliminate the paraformaldehyde. +e plates were further
formulations were placed in baskets and allowed to rotate at incubated in 4′6′-diamidino-2-phenylindole for 10 minutes,
100 rpm at 37.1°C, at 0, 30, 60, 120, 240, 480, 720, and 1440 after which they were once again washed with PBS and PBST.
minutes. 5mL samples were drawn from the outer disso- +e parasite count in each well was determined with a
lution flasks and replaced with equal volume of phosphate fluorescent microscope at a magnification of 40x. Readings
buffer. +e contents of the triplicates of the 2 formulations were done by 2microscopists, and the average of readings was
were assayed using a UV spectrophotometer at a wavelength recorded.
of 334 nm.
2.5. Pharmacokinetic Profile of Quinine Sulphate
2.4. Effect of Quinine Sulphate Microparticles on Intra- Microparticles
macrophage Leishmania donovani (In Vitro). A sample of
Leishmania donovani promastigotes was cultured in an 2.5.1. Ethical Statement for Animal Use. +e protocol for the
M199 culture medium and incubated at 25°C. Growth of study was approved by the Centre for Plant Medicine Re-
parasite was monitored daily using the Neubauer counting search (CPMR), Mampong, Ghana (approval number CPM/
chamber to capture the stationary phase of growth. +e A.95/SF.6/111). +e European Community guidelines, as
concentration of the parasites was determined by the accepted principles for the use of experimental animals, were
formula: adhered to.
N � (av) × 16 × 104 cells/mL, (2)
2.5.2. Animal Care. Male Sprague-Dawley (SD) rats with
where “N” is the concentration of parasites and “av” is the body weight ranging between 174 and 211 g and 6-7 weeks
average number of cells. old were used. +e animals were obtained from the Animal
Growth monitoring was continued until the stationary House, Centre for Plant Medicine Research (CPMR),
phase of the parasites was at a concentration of about 4 × Mampong Akuapem, Eastern Region, Ghana. +e rats were
107– 5 × 107 cells/mL. kept in an environmentally controlled breeding room
Rat peritoneal macrophages, which were to be the in (temperature 26± 2°C and a 12 h dark/light cycle) for 1 week.
vitro cells used, were cultured in an M199 culture medium +is was to make animals acclimatize with the environment
and incubated at 37°C and 5% CO2. Growth of macrophages before experimentation was started. Animals were fed with
was monitored until they were about 80% confluent. Sub- standard laboratory chow and given water ad libitum. All
sequently, 12-chamber well plates were seeded with 2 × 105 animal treatments and handling were done in accordance
cells/well of macrophages in 1mL of media and incubated with guidelines published by the National Institute of Health
overnight (to aid in equilibration). Macrophages were then for the Care of Laboratory Animals.
infected with Leishmania donovani by aspirating the growth
media from the wells and overlaying the macrophage cells
with 300 µL suspension containing 6 × 106 cells of L. 2.5.3. Drug Administration and Blood Sampling. To deter-
donovani. Plates were incubated at 37°C to allow for the mine the pharmacokinetic parameters of quinine sulphate
parasites to invade the macrophages. After 24 hours, the well microparticles compared to quinine sulphate powder
4 Journal of Tropical Medicine
formulation, SD rats were randomly put into two groups of solution. Animals in the fourth group were administered
five. Quinine sulphate powder was administered to rats in quinine sulphate microparticles corresponding to 10mg/kg
Group 1, while those in Group 2 were given quinine sulphate body weight of the entrapped drug. +ose in the fifth group
microparticle suspension. All injections were administered received amphotericin B solution at 3mg/kg body weight
via the intraperitoneal (IP) route using a 23-gauge needle. (the dose of amphotericin B for treating leishmaniasis), as
Each rat was dosed either with 30mg/kg of quinine sulphate positive control. For each group, treatment was done every
solution or microparticles. Serial tail vein blood samples other day for a period of two weeks. On day 14, tail vein
were collected predose and then at 0.5, 1.5, 2, 4, 8, 12, 24, 48, blood samples were obtained, and parasite load (described in
and 72 hours following IP administration of a single dose of detail subsequently) was determined. After this, the animals
drug solution or microparticles. Each sample was collected were sacrificed, and their livers and spleens were removed
into EDTA tubes and stored at −20°C until analyzed. and weighed. Portions of the organs were washed with
normal saline, weighed, and homogenized in 10mL of
normal saline using a sterile glass Potter-Elvejhem tissue
2.5.4. Estimation of Quinine Sulphate Level in Blood. grinder. +e homogenates were centrifuged at 1000 rpm,
Frozen blood samples were thawed, and 100 µL was pipetted and the supernatant from these homogenates was analyzed
into Eppendorf tubes. Distilled water (200 µL) was added to for parasite load (described in detail subsequently).
each tube and vortex-mixed. A volume of 400 µL of diethyl
ether [19] was then added, mixed, and kept at room tem-
perature for 10 minutes. +is was later centrifuged at 2.6.3. Parasite Load in Blood and Organs. To determine the
10,000 rpm for 5 minutes. +e upper (organic) layer was concentration of parasites in blood and organs, a standard
transferred into separate Eppendorf tubes and evaporated at curve of L. donovani concentration was obtained. +is was
65°C in a water bath. +e dried sample was reconstituted done by first serially diluting a known stock suspension
with 300 µL of 0.1M H2SO4, and 200 µL was transferred into (1× 107 cells/mL) of L. donovani in a 96-well plate with
96-well plates. Samples were then analyzed under fluores- M199. A volume of 10 µL of Alamar blue dye was added to
cence at 355 nm excitation and 450 nm emission. Drug each well and left at 25°C for 4 hours. +e ability of viable
standards were prepared by spiking whole blood with cells to reduce resazurin (the active component in Alamar
various concentrations of quinine sulphate, and extraction blue) to resorufin, a red highly fluorescent compound, was
was done using the same method as described earlier. Both determined by measuring fluorescence at an excitation
standards and samples were analyzed on the same plate. wavelength of 544 nm and emission wavelength of 590 nm.
To determine the parasite load, the blood cells and tissue
cells (samples) had to be lysed to release the intracellular
2.6. Effect of Quinine Sulphate Microparticles on Leishmania parasites. To do this, equal volumes of each sample (blood or
donovani-Infected Rats organ suspension (homogenate)) were added separately to
2.6.1. Animal Care and Drug Preparation. Six-week-old the M199 growth medium containing Triton X (0.1%) in
Sprague-Dawley (SD) rats (weighing 190± 20 g) were ob- different tubes and centrifuged at 3000 rpm to lyse cells of
tained from the Centre for Plant Medicine Research (CPMR), samples. Volumes of 100 μL of the supernatants were
Mampong, Ghana. +e animals were kept in a pathogen-free transferred into a 96-well plate. A volume of 10 µL of Alamar°
facility and handled according to institutional guidelines, blue dye was added to each well and left at 25 C for 4 hours.
under a 12/12 hour light/dark cycle, at a temperature of Samples were analyzed under fluorescence using a fluo-
26± 2°C. Rats were fed with a standard diet and given water rescence plate reader set at an excitation wavelength of
ad libitum. Quinine sulphate solution and quinine sulphate 544 nm and emission wavelength of 590 nm, as done for
microparticle suspension were prepared with normal saline to standard samples. +e fluorescence readings were obtained
obtain final concentrations of 1mg/mL. for test samples (after 14 days of treatment). +e readings
were normalized against readings obtained using samples
from non-Leishmania-infected rats. Parasite load of test
2.6.2. Infection of Rat with L. donovani and Treatment with samples were determined from the standard curve
Quinine Sulphate Formulations. Sprague-Dawley rats, 25 in constructed.
number (each weighing 190± 20 g), were administered in-
traperitoneally 0.2mL suspension of 2×107 cells of Leish- 3. Statistical Analysis
mania donovani. Blood samples were taken weekly to check
for serum load of L. donovani in the rats. Four weeks after Data were expressed as mean values± standard error of
infection, the parasite load (described in detail subsequently) mean. Dunnett’s multiple comparison test was used to
in the blood of rats was determined and recorded (to serve as compare mean values within various groups at 95% confi-
baseline). After this, the rats were divided randomly into five dence interval (CI). +e t-test was also used to assess the
groups (5 in each group). Each rat in the first group was difference between quinine sulphate microparticles and
administered intraperitoneal injections of normal saline. solution, at certain instances.
Rats in the second group were administered blank micro- +e pharmacokinetic parameters of quinine sulphate mi-
particles by the same route.+ose in the third group received croparticles and solution in the rat model were derived by
intraperitoneally 10mg/kg body weight of quinine sulphate assuming that the drug was administered into a single
Journal of Tropical Medicine 5
release of quinine sulphate from the microparticles was
biphasic, with burst release of 40% in the first hour, followed
by a continuous steady release of the drug over the next 24
hours. +us, the matrix used in the formulation of the
microparticulate was able to control the release of the drug
agent. A cumulative release of 91% of the drug was calculated
at the end of the 24 hours (Figure 2).+e study was extended
to 48 hours, and this yielded an additional release of 8%.
Figure 1: Scanning electron microscope (SEM) images of quinine 4.2. In Vitro Activity of Quinine Sulphate Formulations
sulphate microparticles. Scanning electron micrograph of quinine (Microparticles and Powder) against Intramacrophage L.
sulphate-BSA microparticles observed at 5 kV. +e image shows donovani. +e susceptibility of L. donovani amastigotes to
particles that are irregular and porous. quinine sulphate was determined by measuring the per-
centage reduction of parasites compared to untreated in-
fected control cultures. +e results show that all 3 agents
1: Characteristics of quinine sulphate microparticles. used, quinine sulphate powder, quinine sulphate micro-Table particles, and amphotericin B, were able to inhibit the ac-
Average particle size 2–5 µm
Zeta potential 35.2mV tivity of L. donovani in vitro (Figure 3). +e parasite load−
Drug loading 18.9% reduction in the groups treated with quinine sulphate mi-
Entrapment efficiency 94.5% croparticles and amphotericin B was much higher than that
Average size of microparticles was determined to be between 2 and 5 of the quinine sulphate powder group. +e difference was
microns with a polydispersity index (PDI) of 0.31. found to be statistically significant (p< 0.05). Quinine sul-
phate microparticle formulation was, however, comparable
to amphotericin B, the positive control.
compartment. +e maximum quinine sulphate plasma con-
centration (Cmax) and its corresponding time (Tmax) were de-
termined by inspection of the concentration-time curves. +e 4.3. Pharmacokinetic Profile of Quinine Sulphate Micropar-
elimination rate constant (Kel) was assessed by linear regression ticles and Powder. A calibration curve for quinine sulphate
analysis of the terminal part of the log plasma concentration- standard had a linearity (r2) of 0.9932.+e concentrations of
time curve. Area under the drug concentration-time curves the quinine sulphate in the individual blood samples were
(AUC) was calculated by the linear trapezoidal rule. AUC was obtained by interpolating from the standard curve.
determined till the last measurement point and was also ex- +e concentration-time curve for quinine sulphate mi-
trapolated to infinity AUC0⟶∞. +e elimination half-life (t1/2) croparticles showed a sharp spike within the first hour after
was calculated by equating t −11/2e to 0.693Kel .+e statistical test administration compared to the powder (Figure 4). +e
of significance was performed on the various pharmacokinetic pharmacokinetic parameters calculated for the two groups
parameters using an unpaired t-test at 95% CI. are shown in Table 2. All the parameters investigated except
t1/2 were found to differ significantly among the two groups.
4. Results Peak quinine sulphate blood concentration (Cmax) was about
2-fold higher with quinine sulphate microparticles than
4.1. Characterization of Quinine Sulphate Microparticles powder form. Total drug exposure (AUC) was about 3-fold
4.1.1. Particles Size Distribution, Zeta Potential, and En- greater with quinine sulphate microparticles than powder
capsulation Efficiency. +e average size of quinine sulphate- form.
BSAmicroparticles obtained ranged between 2.0 and 5.0 µm,
with a polydispersity index (PDI) of 0.31. +e value of the
PDI indicates that the size distribution may be broad. +e 4.4. Activity of Quinine Sulphate Formulations in Leishmania
scanning electron micrograph (Figure 1) showed that the donovani-Infected Rats. After two weeks of treatment with
particles were irregularly shaped and porous. +e SEM the various drug formulations, blood samples and tissue
image, additionally, showed a couple of large particles, samples showed that quinine sulphate microparticle for-
explaining the value of PDI obtained. +e encapsulation mulation was best at reducing parasite load. Quinine sul-
efficiency was calculated to be about 95%. Other charac- phate microparticles were found to significantly (p< 0.05)
teristics of quinine sulphate microparticles are summarized reduce L. donovani in blood and tissues (the liver and spleen)
in Table 1. of infected rats compared to amphotericin B.+e rats treatedwith quinine sulphate powder eliminated more parasites in
the blood than in the tissues. +is probably was due to rapid
4.1.2. In Vitro Release of Quinine Sulphate Formulations absorption of the unformulated drug (quinine sulphate
(Microparticles and Powder). A high amount (98%) of powder) into circulation. A summary of parasite clearance in
quinine sulphate was released into the dissolution medium blood and tissues (the spleen and liver) with different
from the powder formulation within the first 8 hours. +e treatments is shown in Figures 5(a)–5(c).
6 Journal of Tropical Medicine
Release profile of quinine sulphate Concentration-time profile of
formulations quinine sulphate formulations in serum
100 10.0
80 8.0
60 6.0
40 4.0
20
2.0
0
0 5 10 15 20 25 30 0.0
Time (hours) 0 10 20 30 40 50
Time (hours)
QS MP
QS powder QS powder
QS MP
Figure 2: In vitro cumulative percent release of quinine sulphate
formulations. +e microparticle formulation (QS MP) showed Figure 4: +e mean plasma drug concentration-time profile of the
extended release profile (91% over 24 hours) compared with the quinine sulphate formulations. Sharp spike in serum concentration
drug powder (QS powder) (98% over 8 hours), n� 3. Release of was observed with the microparticulate formulation (QSMP). Area
quinine sulphate from the albumin matrix was biphasal. A burst under the curve (AUC) was significantly higher in the micro-
release of about 40% was observed in the first hour. particulate formulation group than in the powder group (p≪ 0.01
in Table 2).
In vitro evaluation of quinine sulphate formulations
on intracellular L. donovani
80 encapsulation efficiency of approximately 95%, an indication∗
of the high efficiency of the spray-drying technique.
∗
60 +e release of quinine sulphate from the microparticle∗∗
polymer matrix occurred in two phases: first, an initial burst
40 release of the unentrapped drug (quinine sulphate thatadhered to the outer surface of the matrix during the
manufacturing process), followed by a slower continuous
20 release from the polymer matrix due to its hydration and
diffusion of drug through pores. +e porous nature of the
0
Control QS powder QS MP AMB microparticles as confirmed in the scanning electron mi-
Treatment groups crograph image must have contributed significantly to the
Figure 3: Comparative studies on parasite load reduction in rat effective release of the drug (91%) in 24 hours. Kesse et al.
peritoneal macrophages using various drug formulations. AMB, conducted a similar study, where they formulated amo-
amphotericin B; QS powder, quinine sulphate powder; QS MP, diaquine and quinine sulphate microparticles using BSA as
quinine sulphate microparticles; Control: untreated group (n� 5). their matrix. +e average particle size, zeta potential, and in
Quinine sulphate significantly reduced parasite load in the mac- vitro release profile they reported were similar to those
rophage cells used. Formulated QS MP were much more effective obtained in this current study [20]. Although the formu-
than the pure quinine sulphate powder. Effect of formulated lation and evaluation of the microparticles prepared are
quinine sulphate microparticles was comparable to the standard similar to the work done by Kesse et al., this study further
drug amphotericin B, the positive control (∗p< 0.05 and investigated the in vivo release of the formulated micro-
∗∗p< 0.01). particles and conducted efficacy (in vitro and in vivo) studies
against the leishmania parasites.
5. Discussion +e pharmacokinetic characteristics of the quinine
sulphate formulations were evaluated in SD rats. Formu-
Quinine sulphate had earlier been shown to be effective, in lating the drug into microparticles enhanced the extent of
vitro, against L. donovani [17]; however, due to its short half- absorption from the site of administration into systemic
life, frequent dosing will be necessary in order to maintain circulation. +is was exhibited by both high Cmax and AUC
steady concentrations of the drug in the blood. To reduce values for the microparticle formulation compared to those
this pharmacokinetic challenge, a microparticulate delivery for quinine sulphate powder. +e high Cmax of the micro-
system of quinine sulphate was formulated. Quinine sul- particle formulation, which was achieved in a short time,
phate was entrapped in a BSA matrix that controlled the coupled with the high total drug exposure (AUC) is in-
release of the drug. +e average size of particles prepared dicative that the microparticles were absorbedmuch and to a
ranged between 2 and 5 μm, and had a negative zeta potential greater extent from the peritoneal cavity. +e microen-
(−35.2mV). SEM monograph revealed irregular and porous capsulation matrix used (BSA) affected the way the drug was
particles. Particles with such properties usually exhibit good released into circulation. +e degradation process can be
flow in the tablet manufacturing process. +e amount of categorized as either bulk-eroding or surface-eroding
quinine sulphate loaded into the microparticles yielded an [21–23]. Bulk-eroding polymers exhibit a peculiar
Cumulative percent
Parasite load reduction (%) release (%)
Concentration of quinine
sulphate (μg/mL)
Journal of Tropical Medicine 7
Table 2: Pharmacokinetic parameters of quinine sulphate formulations.
Pharmacokinetic QnSO4 powder, mean± SE of QnSO4 microspheres, mean± SE of Significantly different
parameter mean mean p value (p< 0.05)
Cmax (µg/mL) 3.73± 0.403 6.81± 0.977 0.0194 Yes
Tmax (hr) 0.700± 0.200 2.40± 0.696 0.0470 Yes
t1/2 (hr) 9.7± 2.4 45.4± 16.1 0.0931 No
AUC0⟶72 (µg·hr/mL) 50.2± 5.40 154± 14.6 0.0002 Yes
AUC0⟶∞ (µg·hr/mL) 90.9± 39.8 266± 61.8 0.0443 Yes
Pharmacokinetic properties were higher in the microparticulate formulation of quinine sulphate than in the powder (n� 5).
Parasite load reduction in blood samples Parasite load reduction in the liver
80.0 80.0 ∗∗
60.0 60.0
40.0 40.0
20.0 20.0
0.0 0.0
Control Bank MP QS powder QS MP AMB Control Bank MP QS powder QS MP AMB
Treatment groups Treatment groups
(a) (b)
Parasite load reduction in the spleen
80.0 ∗
60.0
40.0
20.0
0.0
Control Bank MP QS powder QS MP AMB
Treatment groups
(c)
Figure 5: Comparative studies on percent parasite reduction in blood and tissues. In all the tissues studied, treatment with quinine sulphate
microparticles resulted in more than 60% reduction in parasite load (n� 5). +e reduction observed was about 15–18 percentage points
better than the standard drug “amphotericin B.” Difference observed was significant (∗p< 0.05). AMB, amphotericin B; QS MP, quinine
sulphate microparticles; QS powder, quinine sulphate powder; Blank MP, blank microparticles; Control, untreated group. (a) Blood. (b)
Liver. (c) Spleen.
phenomenon known as the “burst release” kinetics. Bovine Results from this study indicated that quinine sulphate
serum albumin (BSA) is a bulk-eroding polymer, explaining microparticles reduced tissue L. donovani levels signifi-
the initial rise in the plasma concentration of the drug within cantly compared to the positive control (amphotericin B).
the first 30 minutes of administration. Furthermore, the +is outcome confirms findings of previous work by Nettey
surface drug, which caused 40% burst release in the in vitro et al., who demonstrated that amodiaquine (drug) in
study, wouldmake quinine sulphate readily available. It may, particulate form had a high potential for the treatment of
however, be desirable when high levels of the drug are re- visceral leishmaniasis [25]. Furthermore, it was observed
quired to kill parasites, followed by sustained therapeutic that blank microparticles, which did not contain any drug,
levels. Also, the longer elimination half-life (t1/2) of the showed some levels of antileishmania activity in the organs
microencapsulated quinine sulphate would translate into compared to the negative control (normal saline). Mi-
less frequent dosing. croparticles by their unique morphology resemble some
+e percent parasite load in the blood and organs after types to microorganisms, and thus, their presence in the
quinine sulphate administration was determined by finding body can stimulate an innate immune response. Phagocytic
the percentage of parasite load of the treated cells to that of cells, such as macrophages are attracted to the site to
the untreated cells (negative control). +e percent parasite eliminate the foreign substance (microparticles). +is
reduction was then determined relative to untreated [24]. immunological response generated could explain the
Parasite load reduction
(%)
Parasite load reduction
(%)
Parasite load reduction
(%)
8 Journal of Tropical Medicine
clearance of the parasites by the blank (empty) CDC: Centers for Disease Control and Prevention
microparticles. SEM: Scanning electron microscope
Quinine sulphate solution was observed to reduce the RPM: Rotations per minute
parasite load in the blood by 42.9%; however, in the tissues, PBS: Phosphate-buffered saline
the reduction in parasite load was about 33%. +is could be PBST: Phosphate-buffered saline with Triton X
due to the fact that quinine sulphate solution is readily UV: Ultra violet
absorbed into circulation. Concentration of the drug solu- CPMR: Centre for Plant Medicine Research
tion in the tissues may not have been as high as in the blood SD: Sprague-Dawley
to reduce the parasite load. In the groups treated with the IP: Intraperitoneal
microparticulate formulation, a higher reduction of para- EDTA: Ethylenediaminetetraacetic acid
sites in the blood and tissues were observed. +e most CI: Confident interval
plausible explanation for better parasite clearance observed Cmax: Maximum serum concentrationfor quinine sulphate microparticles both in vitro and in vivo T : Time taken to reach the maximum serum
may be due to the fact that more quinine sulphate particles max concentration
may have been engulfed by peritoneal macrophages ANOVA: Analysis of variance
(phagocytic cells) just after the IP administration. Phago- PDI: Polydispersity index
cytic cells usually migrate to the secondary lymphoid organs t : Half-life
such as the spleen after taking up foreign substances from 1/2K : Elimination rate constant
the body for further processing before finally ending up in elGLAB: Grace Lovia Allotey-Babington
the liver. Due to the fact that these microparticles are loaded SKA: Seth Kwabena Amponsah
with quinine sulphate, the concentration of the drug in these TN: +omas Nettey
organs tends to be high. +is could explain the high re- CS: Clement Sasu
duction in parasite load in the above mentioned organs as HN: Henry Nettey.
compared to the blood. Coincidentally, macrophages are
among the cells which serve as hosts to Leishmania parasites
in the body. A statistically significant difference (p< 0.05) in Data Availability
parasite load was observed between the formulated micro- +e data that support the findings of this study are available
particles and the positive control, amphotericin B, after 2 from the corresponding author upon reasonable request.
weeks of treatment in both blood and tissues investigated.
+is can be speculated to be main because of poor ab-
sorption of amphotericin B into cells or effective efflux by the Conflicts of Interest
cells. Clearing of parasite load in the blood as well as tissue is All the authors have no conflicts of interest to declare.
very critical in eliminating Leishmania parasites. +is is
because parasites left in tissues after treatment could lead to Authors’ Contributions
future reinfection.
GLAB and HN were involved in conceptualization of the
6. Conclusion research. GLAB, SKA, TN, CS, and HN conducted the re-
search, data analysis, and interpretation, while GLAB, SKA,
Microencapsulation of quinine sulphate using the spray- and HN drafted the manuscript. All authors reviewed, ed-
drying process produced microparticles with an efficient ited, and approved the final manuscript.
release profile. +ese properties of the microparticles were
exhibited both in vitro and in vivo (pharmacokinetic and Acknowledgments
efficacy studies). Additionally, the microparticulate form of
quinine sulphate was more effective in clearing intratissue L. +e authors acknowledge Dr. Neelo Singh of the Leishmania
donovani than the conventional drug (powder form). From Research Society, India, who gifted us with the Leishmania
the current study, quinine sulphate microparticles showed donovani parasite.
potential as a drug of choice for leishmaniasis (especially
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