Pre-referral intranasal artesunate powder for cerebral malaria: a proof-of-concept study

Abstract

Background: Malaria still kills young children in rural endemic areas because early treatment is not available. Thus, the World Health Organization recommends the administration of artesunate suppositories as pre-referral treatment before transportation to the hospital in case of severe symptoms with an unavailable parenteral and oral treatment. However, negative cultural perception of the rectal route, and limited access to artesunate suppositories, could limit the use of artesunate suppositories. There is, therefore, a need for an alternative route for malaria pre-referral treatment. The aim of this study was to assess the potential of intranasal route for malaria pre-referral treatment.

Methods: The permeability of artesunate through human nasal mucosa was tested in vitro. The Transepithelial Electrical Resistance (TEER) of the nasal mucosa was followed during the permeation tests. Beside, regional deposition of artesunate powder was assessed with an unidose drug delivery device in each nostril of a nasal cast. Artesunate quantification was performed using Liquid Chromatography coupled to tandem Mass Spectrometry.

Results: The experimental model of human nasal mucosa was successfully implemented. Using this model, artesunate powder showed a much better passage rate through human nasal mucosa than solution (26.8 ± 6.6% versus 2.1 ± 0.3%). More than half (62.3%) of the artesunate dose sprayed in the nostrils of the nasal cast was recovered in the olfactory areas (44.7 ± 8.6%) and turbinates (17.6 ± 3.3%) allowing nose-to-brain and systemic drug diffusion, respectively.

Conclusion: Artesunate powder showed a good permeation efficiency on human nasal mucosa. Moreover it can be efficiently sprayed in the nostrils using unidose device to reach the olfactory area leading to a fast nose-to-brain delivery as well as a systemic effect. Taken together, those results are part of the proof-of-concept for the use of intranasal artesunate as a malaria pre-referral treatment.

Keywords: Artesunate; Nasal cast; Nasal mucosa; Nose-to-brain delivery; Pre-referral treatment; Severe malaria.

Fig. 1

TEER measurement protocol.  ALI air-liquid interface culture, KRB krebs-ringer buffer, min minutes, TEER Transepithelial electrical resistance

Fig. 2

Nasal cast and spraying device (picture curtesy of Aptar pharma). a, b Pictures of the experimental set-up of artesunate powder deposition study. c Picture of the spraying device (UDSp). d Side picture of the nasal cast showing the nasal cavity divided in 4 blocks (in red): block 1 = nose, nasal valve, and frontal sinuses; block 2 = maxillary sinuses, frontal sinuses, nasal floor, turbinates, and ethmoids; block 3 = maxillary sinuses, sphenoids, nasal floor, ethmoids and turbinates; block 4: Sphenoids and nasopharynx

Fig. 3

3D representation of the nasal cast and spray device with the nasal delivery place (picture curtesy of Aptar pharma). a 3D representation of the experimental set-up of artesunate drug deposition study. b 3D representation of the experimental set-up of artesunate drug deposition study with the blocks of the nasal cast and corresponding nasal regions. block 1 = nose, nasal valve, and frontal sinuses; block 2 = maxillary sinuses, frontal sinuses, nasal floor, turbinates, and ethmoids; block 3 = maxillary sinuses, sphenoids, nasal floor, ethmoids and turbinates; block 4: Sphenoids and nasopharynx

Fig. 4

TEER during ALI culture of RPMI 2650 cells. TEER was measured on day 0, 4, 7, 11, and 14 during ALI culture of RMPI 2650 cells to monitor the formation of tight junctions. TEER values progressively increased and reached a mean value of 63 ± 4 Ω.cm² on day 14. Data are presented as the mean ± SD (n = 9). TEER Transepithelial Electrical Resistance, ALI Air-Liquide Interface

Fig. 5

RPMI 2650 cell viabilities according to artesunate concentration using a MTT assay. Artesunate cytotoxicity was assessed on RPMI 2650 cells prior to performing permeation tests with AS solutions. No cytotoxicity was recorded for the 0.75 µg/ml artesunate solution (cell viability > 80%). Data are presented as the mean ± SD (n = 9). DMSO dimethyl sulfoxide

Fig. 6

Artesunate permeation assay. a Permeation assay of artesunate in solution (0.75 µg/ml). b Permeation assay of artesunate in a powder (20 µg/mg). Artesunate permeation assays were performed on RPMI 2650 cell model using artesunate solution and powder formulations. Data are presented as the mean ± SD (n ≥ 3). AS artesunate, min minutes

Fig. 7

TEER measurements before and after the permeation assay of artesunate solution and powder formulations Cytotoxicity of artesunate was assessed according to TEER measurements before and after the permeation assay. Data are presented as the mean ± SD (n ≥ 3) TEER Transepithelial electrical resistance, TEER_i TEER before permeation assay, TEER_4 h TEER immediately after permeation assay, TEER_24 h TEER 24 h after permeation assay initiation

Fig. 8

Distribution of artesunate powder by regions of the nasal cast (nasal cast use courtesy of Aptar/DTF/Univ. of Tours). Deposition experimentations were performed three times. The mean amount of drug sprayed into each nostril of the nasal cast model was calculated to be 9.8 ± 0.4 mg. Data are presented as the mean ± SD (n = 2)