Malaria is one of the deadliest human infectious diseases, responsible for more than 600,000 deaths in 2021, most of them being young children in sub-Saharan Africa [1, 2]. Whereas a great majority of cases are uncomplicated and successfully treated with oral anti-malarials, severe malaria may occur in non-immune patients, including children under five years old living in high transmission endemic regions [1, 3,4,5]. A rapid and optimal anti-malarial treatment is critical to improve the outcome of severe cases. The first-line treatment recommended for severe malaria is intravenous artesunate, a semisynthetic derivative of artemisinin. In rural areas where intravenous treatments are not available, the World Health Organization (WHO) recommends the use of a pre-referral artesunate treatment before addressing the patient to the nearest hospital for appropriate care [1, 6]. Pre-referral artesunate treatment is a single dose of artesunate administered intrarectally to young children (< 6 years old) at risk of severe malaria when oral route is unavailable [1, 6]. This pre-referral artesunate treatment using suppositories was demonstrated to reduce the risk of death or permanent disability [6]. Pharmacokinetic studies showed large interindividual variability with a bioavailability of 11.7 to 54.4% (mean = 25.6%), but a 12-h parasite reduction ratio comparable to intravenous artesunate [7]. Among its advantages, the rectal route is non-invasive, usable in unconscious or vomiting patients, allows systemic drug absorption, and avoids at least partially the hepatic first-pass effect [8].
However, a negative cultural perception of the rectal route, as well as reports of melting artesunate suppositories under tropical and subtropical temperatures, and the possible expulsion of the suppositories could limit its use [8,9,10]. Although two artesunate suppositories have now been prequalified by the WHO, there is still little evidence for the implementation of this pre-referral treatment into endemic countries’ guidelines for severe malaria management [11, 12]. Consequently, the availability of rectal AS is reduced as observed in Ethiopia [13] and Kenya according to a recent national survey of primary public health facilities conducted between 2017 and 2021 [14]. In this context, there is a need for the development of an alternative artesunate pre-referral treatment.
The nasal route was described as an alternative to parenteral and oral routes [15]. It is better accepted than the rectal route and it avoids the first-pass hepatic metabolism. It allows to bypass the blood-brain barrier (BBB) and facilitates the diffusion of drugs directly inside the brain microvasculature constituted by endothelial cells held together by tight junctions leading to continuous and non-fenestrated vessels. This BBB restricts considerably the diffusion of molecules between blood and central nervous system (CNS) [16]. However, rapid alteration of the BBB is associated with a broad spectrum of diseases including cerebral malaria (CM). During CM, the neurovascular unit (NVU), defined as the interaction between of the microvasculature (endothelial and pericyte cells) and neural cells (neurons, astrocyte, microglia) is severely impacted by the local sequestration of infected red blood cells, leading to a neuroinflammatory process and BBB breakdown [17]. This process resulting in neurotoxicity and axonal injury associated with a compromised blood-nerve barrier [18, 19] and perivascular micro-haemorrhages may enable substance to cross the BBB by passive diffusion in both directions [20]. Taken together, these pathological events argued for the potential benefit of artesunate immediate access to the NVU during CM, providing a rapid reduction of the parasite burden and the resultant local inflammatory process. Indeed, the advantages of the nasal route include non-invasiveness, ease of drug administration, and fast drug absorption [15, 21]. The richly vascularized nasal mucosa is effective for drug absorption depending on the regional nasal deposition pattern [22]. The respiratory and the olfactory mucosa are of particular interest for drug absorption: the respiratory mucosa is the preferential site for systemic drug absorption [21, 23, 24], whereas the olfactory mucosa is the preferential site for nose-to-brain drug absorption [21, 24]. The olfactory zone provides a direct access to the brain through the olfactory and trigeminal nerve termination in the cavity. Nose-to-brain drug delivery use the endoneurial microvessels within the nerve fascicle and the perineurium [25, 26] to allow the passage of substances into the brain within minutes. Thus, nasal route allows to bypass the BBB that is of utmost importance in the case of cerebral malaria characterized by sequestration of infected red-blood cells in cerebral capillaries.
Among nasal route shortcomings, are the short retention time of drugs because of mucociliary clearance, metabolic degradation in the nasal cavity, and restricted dosing volume, mostly in children [21, 27, 28]. Over the last two decades, the nasal route was actively assessed for the delivery of various drugs, including vaccines, hormones (insulin, melatonin), opioids, and triptans [15, 29,30,31,32]. Nasal administration of sumatriptan powder is now recommended by the Food and Drug Administration (FDA) for the treatment of acute migraines in adults [33]. Compared to the oral route, the nasal route is associated with faster and greater relief of migraines symptoms [34]. Intranasal administration of artesunate for severe malaria treatment would allow both the systemic effects of artesunate, and the nose-to-brain delivery of the drug that would prevent cerebral malaria. This statement is based on preliminarydata obtained in a murine model of cerebral malaria that demonstrated the nasal route to prevent the development of complications including cerebral malaria [9]. It was demonstrated that dihydroartemisinin, the main metabolite of artesunate, was recovered into the brain of mice after artesunate intranasal treatment and that early artesunate treatment using intranasal route prevented the development of cerebral malaria.
In this context, there is evidence to further assess the nasal route for artesunate. The objectives of this study were, firstly, to assess artesunate permeation and cytotoxicity using a model of human nasal mucosa and, secondly, to study the nasal deposition pattern of the drug sprayed in a human nasal cast model. This would contribute to the establishment of the nasal route as an alternative for pre-referral treatment of malaria.
Methods
Chemicals, culture media, and drug solutions
Artesunate (Chemical Abstract Service CAS number: 88495-63-0; IPCA #19003AS6RII [1 kg]) was purchased from IPCA Laboratories Limited (Mumbai, India) through Hepartex (Saint-Cloud, France). Krebs-ringer buffer (KRB) was prepared by dissolving 6.8 g NaCl, 0.4 g KCl, 0.14 g NaH2PO4ּ H2O, 2.1 g NaHCO3, 3.575 g HEPES, 1.0 g D-glucose, 0.2 g MgSO4ּ 7H2O, and 0.26 g CaCl2ּ 2H2O in 1 L of distilled sterile water. HEPES, D-glucose, and NaHCO3, were purchased from Carl Roth GmbH (Karlsruhe, Germany). All other KRB reagents, 0.25% (w/v) trypsin-EDTA, Trypan blue stain (0.4%), phosphate buffer solution (PBS) tablets, fluorescein sodium salt (NaF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich Merck (Saint-Louis, Missouri, USA). Culture medium included minimum essential medium (MEM), 10% (v/v) heat-inactivated sterile foetal bovine serum (FBS), 1% (w/v) L-Glutamine, 1% (v/v) non-essential amino-acids (NEAA), and 20 µg/ml gentamicin. MEM, heat-inactivated FBS, L-glutamine, and NEAA, were purchased from Sigma-Aldrich Merck (Saint-Louis, Missouri, USA). Nasal epithelial cells RPMI 2650 (RMPI 2650 ECACC 88,031,602) were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton, Wiltshire, England). Falcon® Cell-culture flasks, Flacon® 96-well plates and Corning Costar® 12-well plates were purchased from Corning (Glendale, Arizona, USA). ThinCert® tissue-culture inserts for 12-Well plates (polyethylene terephthalate membrane, 1.13 cm², 0.4 mm pore size) were purchased from Greiner Bio-One (Kremsmünster, Austria). Artesunate and artesunate-d4 standards for mass spectrometry (MS) analysis were purchased from Alsachim (Illkirch-Graffenstaden, France). Ultrapure water was obtained from Biosolve (Dieuze, France) and Thermo Fischer Scientific (Massachusetts, USA). Mass spectrometry-grade (MS-grade) acetonitrile was obtained from Biosolve (Dieuze, France). MS-grade ammonium acetate, acetic acid and methanol were obtained from (Thermo Fischer Scientific (Massachusetts, USA). Formic acid and ammonium formiate were purchased from Sigma Aldrich (Saint-Louis, Missouri, USA).
MTT stock solution was prepared in PBS at a final concentration of 5 mg/ml. Artesunate stock solutions (30 mg/ml) for permeation and MTT tests were prepared into a 5% (w/v) NaHCO3 aqueous solution followed by dilutions into adequate media. Artesunate for MTT assays was diluted into culture medium to final concentrations of 16, 12, 6, 3, 1.5, and 0.75 µg/ml. The donor for artesunate permeation studies was either a 0.75 µg/ml artesunate solution or a 20 µg/mg powder mixture of artesunate and corn-starch. Corn-starch is an inert excipient that was used to dilute the artesunate content in the powder formulation, increase the weight of the formulation, and thus allow its reproducible weighing for the permeation study [35, 36]. The donor solution for NaF permeation tests was prepared by dissolving NaF into KRB (25 µg/ml). All molecules’ solutions were filtered before use (0.22 μm filter).
Artesunate formulations
Artesunate powder and solution formulations were prepared for the permeation studies.
For the powder formulation, pure artesunate powder and corn-starch were successively weighted in a plastic flask to a final concentration of 20 µg/mg artesunate in corn starch. The powder mixture was homogenized by shaking and the flask was stored at room temperature and protected from light until use. For the solution formulation, 30 mg of pure artesunate powder was weighted in a tinted glass vial and dissolved into 1 ml of 5% NaHCO3 (W/V) aqueous solution. The resulting solution was diluted in 2 ml of NaCl 0,9% (V/V) and then further diluted in KRB to a final artesunate concentration of 0.75 µg/ml. The solution formulation was prepared extemporaneously immediately before use.
Cell culture
Nasal epithelial cells RPMI 2650 were used between passage 11 and 25 for all experiments. They were routinely cultured into 25 cm² polystyrene cell-culture flasks under standard conditions (humid atmosphere at 37 °C and 5% CO2). For cell passaging, cells were washed with PBS at 37 °C and then detached with 0.25% (w/v) trypsin-EDTA at 37 °C seven days after previous seeding. Cell viability was assessed using a standard trypan blue staining procedure. After counting, cells were seeded in a new flask at a \(4 \times {10^4}{\text{cells}}/{\text{cm}}^{2}\) seeding density. Culture medium was changed every two to three days. Multilayer cell culture was performed into tissue-culture inserts according to the protocol previously described by Reich and Becker [37]. Briefly, \(2 \times {10^5}{\rm{cells}}/{\rm{cm}}^{2}\) were seeded on permeable ThinCert® insert membranes and cultured under liquid-covered conditions (LCC) for eight days. After this period, the inserts were lifted at the air-liquid interface (ALI) and cultured for 14 more days to allow the formation of multiple cell layers and tights junctions [37, 38]. Transepithelial electrical resistance (TEER) was measured every two to three days during ALI culture. Experiments were performed in triplicate during at least three independent assays.
Transepithelial electrical resistance (TEER)
TEER was measured using a Millicell® ERS-2 Voltohmmeter (Merck, Darmstadt, Germany) and the STX01 chopstick electrode according to the manufacturer’s instructions (Fig. 1). Briefly, culture medium or KRB was added to the apical and basolateral compartments of the cell culture to final volumes of 1 ml and 1.5 ml, respectively. Cultures were then left to equilibrate at 37 °C for 30 min before measurements. TEER readings were corrected by subtracting blank filters values and normalized to the surface area of the membrane (1.13 cm²). Cell cultures having TEER values of at least 60 Ω.cm² were used for permeation assays [39].
MTT cytotoxicity assay
A MTT cytotoxicity assay was performed to assess artesunate cytotoxicity on epithelial cells RPMI 2650 [40, 41]. Briefly, the cells were seeded at a density of \(1.5 \times {10^4}{\text{ cells}}/{\text{cm}}^{2}\) in 96-well plates. After 24 h, artesunate drug dilutions were added and cells were incubated for 24 h. Following drug exposure, cells were gently washed twice with 100 µl PBS at 37 °C, before adding 110 µl of MTT stock solution diluted in culture medium (final MTT concentration : ~ 0.5 mg/ml). After four hours of incubation, the MTT solution was discarded and 200 µl of DMSO was added to each well. After one hour of incubation, absorbances were read at 485 nm using a Tristar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Germany). Negative (cells without xenobiotics) and positive (cells with 25% v/v DMSO) controls were included. Results were expressed as cell viability (%) relative to negative control (100% viability). Acording to ISO 10993-5:2009, artesunate dilutions with cell viability percentages above 80% were considered as non-cytotoxic [42, 43]. Experiments were performed in triplicate during three independent assays.
In vitro permeation studies
The permeation of the sodium salt of fluorescein (NaF) was used to validate the human nasal mucosa model derived from the culture of nasal epithelial cells RPMI 2650. Before the permeation test, multilayer cultures were rinsed with preheated KRB (37 °C) and their TEER were measured. In another 12-Well plate, 0.5 ml of donor solution was added into the donor chamber and 1.5 ml of KRB (37 °C) in the receptor chamber. The plates were incubated for 1 h under orbital shaking (37 °C, 5% CO2, humid atmosphere, 100 rpm). Samples (100 µl) were collected from the receptor chamber at fixed time intervals and immediately replaced with fresh KRB (37 °C). Permeated NaF was quantified into each sample using a Tristar2 LB 942 Multimode Microplate Reader (Berthold Technologies, Germany) with excitation and emission wavelengths of 485 and 535, respectively. Experiments were performed with five replicates during two independent assays. The apparent permeability coefficient of NaF (Papp) was calculated with the following FDA approved equation and expressed in cm/s [39]:
$${P}_{app}=\left(\frac{V\times {C}_{f}}{{C}_{i}\times A\times t}\right)$$
V: volume of the acceptor chamber (cm3); Cf : drug concentration in the acceptor chamber at the end of the experiment (g/l or mol/l); Ci: initial drug concentration in the donor chamber (g/l or mol/l); A: surface area of the membrane (cm²); t: duration of the experiment (s).
Artesunate permeation was evaluated in a similar way as described above for NaF. Aliquots of solution and powder formulations, equivalent to 0.357 and 200 µg of artesunate, respectively, were added into the donor chamber. Each permeation test lasted for 4 h and 200 µl samples were regularly collected from the acceptor chamber. TEER measurements were performed immediately before (TEERi), after (TEER4 h), and 24 h after (TEER24 h) test initiation. Artesunate permeation tests were performed in triplicate during three independent tests.
Artesunate in vitro deposition study
In vitro deposition of artesunate nasal powder was characterized in an adult male nasal cast (courtesy of Aptar/DTF/Univ. of Tours) with chemical quantification (Fig. 2). The nasal cast model used was designed from Computed Tomography images (CT scan) of a plastinated head model [44] and was previously validated as a predictive model for nasal aerosol deposition [45, 46].
Six unidose (UDSp) devices supplied by Aptar Pharma (Le Vaudreuil, France) were filled with 10 ± 0.5 mg of artesunate powder. One device per nostril was manually actuated into the nasal cast with a fixed insertion depth of 1.5 cm, delivery angle (horizontal plane) of 45° and angle from the centre wall of 4°, after being humidified for 10 min at 3 lpm with a AMGH nebuliser (DTF, Saint-Etienne, France) (Fig. 3). The delivered dose was assessed by weighing each device before and after testing. The different regions of interest (Nose, Olfactory Zone, Turbinates, Nasal Floor, Nasopharynx and Lungs) were rinsed with a PBS solution at pH 8 and frozen at -80 °C to ensure artesunate remained stable.
Analytical methods
For the permeation study, artesunate quantification was performed with an ultra-high performance liquid chromatography system (AcquityTM, Waters, Massachusetts, USA) coupled to a tandem mass spectrometer (XEVO-TQ-MS, Waters). Chromatographic separation was performed using an Ethylene Bridged Hybrid (BEH) C18 column (1.7 μm, 2.1 × 100 mm, Waters). The mobile phase consisted of 10 mM ammonium formiate in water (pH = 3) and 0.1% (v/v) formic acid in acetonitrile. Calibrators were prepared in KRB at concentrations ranging from 0.5 to 25 000 ng/ml. Before quantification, 20 µL of acetonitrile containing artesunate-d4 internal standard (at a concentration of 2.5 µg/mL) were added to 180 µL of each sample.
For the nasal deposition study, the samples were, firstly, thawed in a water bath at room temperature (20 °C). One millilitre (5 × 200 µl) of each sample was deposited on five filter membranes mounted on hydrophobic supports (OmniporeTM 10.0 μm, 47 mm). The membranes were thereafter left to dry overnight at room temperature. Artesunate was eluted from the membranes with 5 ml of methanol for each sample and the resulting solutions were analysed for drug determination. Artesunate quantification was performed using an Ultra-High Performance Liquid Chromatography system (Ultimate 3000, Thermo Fischer Scientific, Massachusetts, USA) coupled to a quadrupole-time-of-flight mass spectrometer (Impact II, Bruker, Massachusetts, USA). Chromatographic separation was performed using a Luna Omega Polar C18 column (1.6 μm, 2.1 × 50 mm, Phenomenex, California, USA). The mobile phase consisted of solvent (A), 1 mM ammonium acetate in ultra-pure water and 0.05% (v/v) acetic acid and solvent (B) 1 mM ammonium acetate in methanol and 0.05% (v/v) acetic acid. The lower limit of quantification (LOQ) of artesunate was set at 0.05 µg/g of solution. The drug deposition in each region of the nasal cast was expressed as the fraction (%) of the actual dose (100%) delivered with the device.
Statistical analysis
All data management was performed with Microsoft Office Excel and GraphPad Prism 9.2.0 and 9.4.1 software. Statistical analyses, including Kruskal-Wallis test with Dunn’s multiples comparison test and Mann-Whitney test were performed with the significance threshold of α = 0.05. Data are presented as the mean ± standard deviation (SD) of at least two replicates.
