Combination delivery of two oxime-loaded lipid nanoparticles: Time-dependent additive action for prolonged rat brain protection

Article abstract of DOI:10.1016/j.jconrel.2018.10.010

A novel approach for brain protection against poisoning by organophosphorus agents is developed based on the combination treatment of dual delivery of two oximes. Pralidoxime chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquin

Full text of DOI:10.1016/j.jconrel.2018.10.010

Accepted Manuscript
Combination delivery of two oxime-loaded lipid nanoparticles:
Time-dependent additive action for prolonged rat brain protection
Tatiana N. Pashirova, Anissa Braïki, Irina V. Zueva, Konstantin
A. Petrov, Vasily M. Babaev, Evgenia A. Burilova, Darya A.
Samarkina, Ildar Kh. Rizvanov, Eliana B. Souto, Ludovic Jean,
Pierre-Yves Renard, Patrick Masson, Lucia Ya. Zakharova, Oleg
G. Sinyashin
PII:
DOI:
Reference:
S0168-3659(18)30581-9
doi:10.1016/j.jconrel.2018.10.010
COREL 9493
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
15 May 2018
6 October 2018
8 October 2018
Please cite this article as: Tatiana N. Pashirova, Anissa Braïki, Irina V. Zueva, Konstantin
A. Petrov, Vasily M. Babaev, Evgenia A. Burilova, Darya A. Samarkina, Ildar Kh.
Rizvanov, Eliana B. Souto, Ludovic Jean, Pierre-Yves Renard, Patrick Masson, Lucia
Ya. Zakharova, Oleg G. Sinyashin , Combination delivery of two oxime-loaded lipid
nanoparticles: Time-dependent additive action for prolonged rat brain protection. Corel
(2018), doi:10.1016/j.jconrel.2018.10.010
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ACCEPTED MANUSCRIPT
Combination delivery of two oxime-loaded lipid nanoparticles: time-dependent additive action
for prolonged rat brain protection
Tatiana N. Pashirovaa*, Anissa Braïkib, Irina V. Zuevaa, Konstantin A. Petrova,c, Vasily M.
Babaeva, Evgenia A. Burilovaa,c, Darya A. Samarkinaa, Ildar Kh. Rizvanova, Eliana B. Soutod,e,
Ludovic Jeanb, Pierre-Yves Renardb, Patrick Massonc, Lucia Ya. Zakharovaa*, Oleg G.
Sinyashina
aArbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS,
Arbuzov St., 8, Kazan, 420088, Russia
bNormandie Univ, UNIROUEN, INSA Rouen, CNRS, COBRA (UMR 6014), Rouen, France
cKazan Federal University, Kremlyovskaya St., 18, Kazan, 420008, Russia
dDepartment of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra,
Coimbra, Portugal
eREQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University of
Coimbra, Coimbra, Portugal
Corresponding Authors
Tatiana N. Pashirova, Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan
Scientific Center of RAS, Arbuzov str. 8, Kazan, 420088, Russian Federation, tel: (007) 843 273
22 93, fax: (007) 843 273 22 53, E-mail: tatyana_pashirova@mail.ru
Lucia Ya. Zakharova, Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan
Scientific Center of RAS, Arbuzov St. 8, Kazan, 420088, Russian Federation, tel: (007) 843 273
22 93, fax: (007) 843 273 22 53, E-mail: lucia@iopc.ru
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ABSTRACT
A novel approach for brain protection against poisoning by organophosphorus agents is
developed based on the combination treatment of dual delivery of two oximes. Pralidoxime
chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA), have been loaded in solid-lipid
nanoparticles (SLNs) to offer distinct release profile and systemic half-life for both oximes. To
increase the therapeutic time window of both oximes, SLNs with two different compartments
were designed to load each respective drug. Oxime-loaded SLNs of hydrodynamic diameter
between 100 and 160 nm and negative zeta potential (-30 to -25 mV) were stable for a period of
10 months at 4 ºC. SLNs displayed longer circulation time in the bloodstream compared to free
3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous (iv) administration.
Paraoxon-poisoned rats (0.8×LD50) were treated with 3-HPA-loaded SLNs and 2-PAM+3-HPA-
loaded SLNs at the dose of 3-HPA and 2-PAM of 5 mg/kg. Brain AChE reactivation up to 30%
was slowly achieved in 5 hours after administration of 3-HPA-SLNs. For combination therapy
with two oximes, a time-dependent additivity and increased reactivation up to 35% were
observed.
Keywords: Solid-Lipid Nanoparticles, Blood-brain barrier, Acetylcholinesterase,
Organophosphorus agent, Oxime, Paraoxon.
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1. Introduction
Multidrug combination therapy is one of the promising approaches for treatment of chronic
diseases. Nanotechnological "two-in-one" approach using nanoparticles for packaging multiple
drugs in single carriers allows the improvement of drugs’ bioavailability, with the simultaneous
decrease of administered doses and adverse side-reactions [1-4]. Nanoparticles exhibit targeted
delivery of drugs and modified release profile and, when loaded with two drugs, may foster
synergistic therapeutic outcomes [2]. To date, prevention of irreversible brain damages after
acute organophosphorus (OP) poisoning is still a difficult problem, in particular in case of
delayed medical care [5]. OP threat has become increasingly important due to acts of terrorism
around the globe that have increased markedly in recent decades, and due to the proved use of
these agents against civilian populations in Iraq and Syria. The acute toxicity of OPs results from
covalent inhibition, phosphylation, of acetylcholinesterase (AChE). Low OP doses do not induce
clinical manifestations of cholinergic impairment. It is accepted that clinical signs of poisoning
appear when cholinesterase activity drops below 50% [6]. Severe signs, in particular
convulsions, appear when AChE activity of poisoned patients is below 10-20% [7]. In humans,
brain AChE activity less than 10% is associated with respiratory failure and death [8]. Thus, the
current emergency treatment of acute OP poisoning consists in administering a combination of
AChE reactivator (quaternary oximes), atropine as anticholinergic drug to protect muscarinic
receptors, and an anticonvulsant to prevent irreversible brain damage [5,9]. However, current
quaternary oximes are unable to reactivate phosphylated AChE in the central nervous system
(CNS) because they do not cross significantly the blood-brain barrier (BBB) [10]. Combination
of two oximes has been shown to improve the emergency treatment of OP poisoning by covering
a wide spectrum of organophosphorus agents [11-13]. Moreover, association of two oximes
revealed a beneficial effect by broadening the spectrum of the individual oximes [12].
Nanoparticles as oxime delivery vehicles are therefore promising for this purpose [14,15].
Recently, our group showed that 2-PAM-loaded solid lipid nanoparticles are able to protect
against paraoxon central toxicity [16]. Other researchers have also been working in that direction
[14]. However, due to rapid elimination of 2-PAM, the percentage of reactivation of central
AChE of the brain was low. To realize the potential of combination therapy at the present study,
design of formulations for delivery of two oximes with different physiochemical properties has
been investigated, in particular, the water-soluble charged oxime pralidoxime chloride (2-PAM)
and a poorly water soluble new uncharged hydrophobic oxime 6-(5-(6,7-Dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA) were combined.
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3-HPA displays a key 3-hydroxy-2-pyridine aldoxime structure as the reactivator moiety linked
to a dimethoxy dihydroisoquinolin as the AChE peripheral site binder in order to increase its
affinity for phosphylated enzyme. See Fig. 1 for structure of 3-HPA. 3-HPA showed an
exceptional potential for reactivation of VX-, paraoxon-, and tabun-phosphylated AChE [17].
Fig. 1. Structure of the oximes and synthetic route of hydrophobic oxime 6-(5-(6,7-Dimethoxy-
3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA).
Owing to the difficulty to reactivate cholinesterases inhibited by phosphoramidates such as tabun
as the prototype, 3-HPA constitutes a real progress in medical countermeasures of OP poisoning.
For this purpose, biocompatible and biodegradable solid-lipid nanoparticles (SLNs) [18,19],
prepared by the double emulsion method [20] with two different compartments to incorporate
both oximes with different release times, are appropriate. These encapsulated oximes would be
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suitable for intravenous (i.v.) administration, and in vivo efficient therapy of rats poisoned by
paraoxon. Here we have extended our previous work [16] and pursued several tasks that remain
to be solved: i) overcoming BBB for 2-PAM and its reactivating action of brain AChE; ii)
increasing the bioavailability of the novel oxime 3-HPA; iii) achieving sequential release of both
oximes and expanding the time window of oxime therapy.
2. Materials and methods
2.1. Chemicals
DYNASAN® 114 (Trimyristin) triglycerides was a gift from CREMER OLEO GmbH & Co.
KG (Hamburg, Germany), Lipoid® S75, 75% soybean phosphatidylcholine was a gift from
Lipoid GmbH (Ludwigshafen, Germany), Tween® 80 (Polysorbate 80) (BioXtra, Sigma-
Aldrich, product of France), Glycerol (≥ 99.5 %, Sigma-Aldrich ChemieGmbh, Steinheim,
Germany), Pyridine-2-aldoxime methochloride (2-PAM chloride, Pralidoxime chloride) (≥ 97 %,
Sigma-Aldrich), Dichloromethane for HPLC (PanReac AppliChem GmbH, Darmstadt,
Germany), Ultra-purified water (18.2 MΩcm resistivity at 25°C) was produced from Direct-Q 5
UV equipment (Millipore S.A.S. 67120 Molsheim-France). Solvents were purified by a dry
solvent station MB-SPS-800 (MBraun) immediately prior to use. Triethylamine was distilled
from CaH2 and stored over BaO or KOH. All reagents were obtained from commercial suppliers
(Sigma Aldrich, Acros, TCI) unless otherwise stated.
2.2. Synthesis of 3-HPA
Column chromatography purifications were performed on silica gel (40–63 μm) from Macherey-
Nagel. Thin-layer chromatography (TLC) was carried out on Merck DC Kieselgel 60 F-254
aluminium sheets. Compounds were visualized by UV irradiation and/or spraying with a stain
1
13
solution (KMnO4, ninhydrin or 2,4-DNP), followed by charring at 150 °C. H and C NMR
spectra were recorded with a Bruker DPX 300 spectrometer (Bruker, Wissembourg, France).
Chemical shifts are expressed in parts per million (ppm) from CDCl3 (δH = 7.26 ppm, δC = 77.16
ppm). J values are expressed in Hz. Mass spectra were obtained with a Finnigan LCQ Advantage
MAX (ion trap) apparatus equipped with an electrospray source. High-resolution mass spectra
were obtained with a Varian MAT 311 spectrometer using electrospray analysis. Analytical
HPLC was performed on a Thermo Electron Surveyor instrument equipped with a PDA detector
under the following conditions: Thermo Hypersil GOLD C18 column (5 µm, 4.6 x 100 mm)
with CH3CN and 0.1% aq. trifluoroacetic acid (TFA) as eluents [0.1% aq. TFA/ CH3CN (100/0)
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(5 min), followed by linear gradient from 0% to 100% of CH3CN (45 min), then 100% CH3CN
(10 min)], at a flow rate of 1.0 mL/min and UV detection Max Plot 220−360 nm.
Synthesis of Methyl 3-hydroxypicolinate (Compound 2). To a suspension of 3-
hydroxypicolinic acid 1 (10 g, 72 mmol, 1 equiv) in methanol (150 mL, 0.5 M) was added
dropwise at 0 °C a concentrated sulfuric acid (12 mL, 216 mmol, 3 equiv.). The obtained
solution was stirred at reflux 6 h. The cooled reaction mixture was concentrated under reduced
pressure. The pH was adjusted at 8.5 with an aqueous solution of saturated NaHCO3 and solid
NaHCO3. The aqueous layer was extracted with EtOAc and the combined organic layer was
washed with brine, dried over MgSO4 and concentrated under reduced pressure to give 2 as a
1
white solid (10.9 g, 99%). Rf =0.3 (Petroleum ether/EtOAc 1/1, v/v). m.p. = 74 °C. H NMR
(300 MHz, CDCl3): δ (ppm) = 4.06 (s, 3H), 7.34 (dd, J = 3.9, 8.4 Hz, 1H), 7.38 (dd, J = 1.5, 8.4
13
Hz, 1H), 8.38 (dd, J = 1.5, 3.9 Hz, 1H), 10.64 (s, 1H). C NMR (75 MHz, CDCl3): δ = 53.2,
126.3, 129.8, 130.2, 141.6, 158.9, 169.9. MS (ESI+): m/z (%): 154 (100) [M+H]+.
Synthesis of Methyl 3-hydroxy-6-bromopicolinate (Compound 3). To a solution of methyl 3-
hydroxypicolinate 2 (10 g, 65.36 mmol) in osmosed water (0.1 M) with crushed ice at 0 °C, was
added portionwise bromine (4 x 1.02 mL every 30 min, 78.4 mmol, 1.2 equiv) under vigorous
stirring. The mixture was vigorously stirred at 0 °C for 1-2 h. The solution was extracted by
dichloromethane and the combined organic layers were washed with brine, dried over MgSO4
and concentrated under reduced pressure to give 3 as a white-off solid (15.08 g, 99%). Rf = 0.3
1
(Petroleum ether/EtOAc 3/2, v/v). H NMR (300 MHz, CDCl3) δ (ppm) = 4.07 (s, 3H), 7.29 (d, J
13
= 8.7 Hz, 1H), 7.58 (dd, J = 0.3, 8.7 Hz, 1H), 10.72 (br s, 1H). C NMR (75 MHz, CDCl3) δ
(ppm) = 53.5, 129.5, 130.0, 130.7, 134.5, 158.5, 169.1. MS (ESI+): m/z (%): 234 (85) and 232
(100) [M+H]+ .
Synthesis of Methyl 3-(benzyloxy)-6-bromopicolinate (Compound 4). To a solution of methyl
3-hydroxy-6-bromopicolinate 3 (11.6 g, 49.8 mmol), in acetone (200 mL, 0.25 M) was added
successively K2CO3 (21 g, 149 mmol, 3 equiv) and benzyl bromide (12 mL, 100 mmol, 2.0
equiv). The heterogeneous reaction mixture was reflux overnight. Salts were removed by
filtration and the crude product was concentrated under reduced pressure. Purification by flash
chromatography (Petroleum ether/EtOAc 95/5 to 3/2, v/v) afforded the desired product 4 as a
1
white solid (15.2 g, 95%). Rf = 0.4 (Petroleum ether/EtOAc 8/2, v/v). H NMR (300 MHz,
CDCl3): δ (ppm) = 3.97 (s, 3H), 5.21 (s, 2H), 7.24 (d, J = 8.7 Hz, 1H), 7.47-7.34 (m, 5H), 7.51
13
(d, J = 8.7 Hz, 1H). C NMR (75 MHz, CDCl3): δ (ppm) = 52.9, 71.2, 125.0, 126.9, 128.4,
128.8, 131.2, 131.4, 135.2, 139.8, 154.0, 164.0. MS (ESI+): m/z (%): 324 (85) and 322 (100)
[M+H]+.
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Synthesis of Methyl 3-(benzyloxy)-6-(5-hydroxypent-1-ynyl)picolinate (Compound 5). To a
solution of methyl 3-(benzyloxy)-6-bromopicolinate 4 (3.19 g, 9.90 mmol) in dichloromethane
(66 mL) and triethylamine (33 mL) was added 4-pentyn-1-ol (650 µL, 9.93 mmol, 1 equiv). The
resulting mixture was degassed for 20 min with argon. CuI (200 mg, 1.02 mmol, 0.1 equiv.) and
Pd(PPh3)4 (527 mg, 0.50 mmol, 0.05 equiv.) were then poured and the solution was stirred under
argon at room temperature overnight. The reaction mixture was concentrated under reduced
pressure. Purification by flash chromatography (Petroleum ether/EtOAc 7/3 to 3/7, v/v) afforded
the desired product 5 as a yellow oil that crystallized upon standing (3.1 g, 96%). Rf = 0.33
1
(Petroleum ether/EtOAc 3/7, v/v). H NMR (300 MHz, CDCl3) δ (ppm) = 1.86 (qt, J = 6.4 Hz,
2H), 2.38 (br s, 1H), 2.54 (t, J = 7.0 Hz, 2H), 3.78 (t, J = 6.2 Hz, 2H), 3.96 (s, 3H), 5.20 (s, 2H),
13
7.30-7.45 (m, 7H). C NMR (75 MHz, CDCl3) δ (ppm) = 15.9, 31.0, 52.7, 61.4, 70.9, 79.6, 89.8,
121.9, 127.0, 128.3, 128.8, 130.1, 135.4, 135.6, 140.0, 153.0, 164.9. MS (ESI+): m/z (%): 673
(100) [2M + Na]+, 348 (95) [M+Na]+ and 326 (100) [M+H]+.
Synthesis of Methyl 3-(benzyloxy)-6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)pent-1-yn-1-yl)picolinate (Compound 6). Methanesulfonyl chloride (1.6 mL, 20.45 mmol,
1.5 equiv.) was added dropwise to a solution of methyl 3-(benzyloxy)-6-(5-hydroxypent-1-
ynyl)picolinate 5 (4.73 mg, 13.63 mmol) and triethylamine (5.6 mL, 40.89 mmol, 3 equiv) in dry
dichloromethane (0.1 M) at 0 °C. The solution was stirred room temperature for 2 h, washed
with brine, dried under MgSO4 and concentrated under reduced pressure. To a solution of the
crude product in dry CH3CN (0.1 M) were added 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (TIQ.HCl, 2.1 g, 1.5 equiv.) and K2CO3 (3.4 g, 3 equiv.). The mixture was
refluxed overnight. The crude mixture was filtered, concentrated under reduced pressure and
purified by flash chromatography on silica gel (CH2Cl2/MeOH, 100/0 to 95:5, v/v) to give the
1
desired product 6 (2.86 g, 74%) as an orangeous oil. Rf = 0.2 (CH2Cl2/MeOH 95/5). H NMR
(300 MHz, CDCl3) δ (ppm) = 1.93 (qt, J = 6.9 Hz, 2H), 2.51 (t, J = 6.9 Hz, 2H), 2.63 (t, J =
6.9 Hz, 2H), 2.72 (t, J = 5.4 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H), 3.57 (s, 2H), 3.83 (s, 3H), 3.84
(s, 3H), 3.96 (s, 3H), 5.19 (s, 2H), 6.53 (s, 1H), 6.60 (s, 1H), 7.25–7.45 (m, 7H).13C NMR (75
MHz, CDCl3) δ (ppm) = 17.4, 26.0, 28.7, 51.0, 52.7, 55.7, 55.8, 55.9, 57.2, 70.7, 79.5, 90.1,
109.4, 112.3, 121.7, 126.2, 126.6, 126.9, 128.2, 128.7, 130.0, 135.4, 135.5, 139.9, 147.1, 147.4,
152.9, 164.8. MS (ESI+): m/z (%): 501 (100) [M+H]+.
Synthesis
of
Methyl
6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-
hydroxypicolinate (Compound 7). To a solution of 6 (2.5 g, 5 mmol) in degassed methanol
(0.05 M) was added Pearlman's catalyst (500 mg, 20% Pd, moisture 50%). The solution was
bubbled with H2 and the reaction was stirred overnight at room temperature under H2 atmosphere
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(1 atm). The mixture was filtrated through celite and concentrated under reduced pressure to give
1
the desired product 7 as a orangeous oil that crystallized upon standing (2.07 g, 99%). H NMR
(300 MHz, CDCl3) δ (ppm) = 10.59 (s, 1H), 7.30 (br s, 1H), 7.26 (s, 1H), 6.58 (s, 1H), 6.51 (s,
1H), 4.04 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.53 (s, 2H), 2.87 – 2.74 (m, 4H), 2.68 (t, J = 5.7
Hz, 2H), 2.55 – 2.42 (m, 2H), 1.67 (m, 4H), 1.53 – 1.35 (m, 2H). 13C NMR (75 MHz, CDCl3) δ
(ppm) = 169.8, 156.9, 153.6, 147.7, 147.3, 129.0, 128.5, 126.4, 124.9, 111.1, 109.2, 56.9, 55.7,
55.6, 54.4, 52.9, 50.1, 37.2, 29.6, 27.0, 26.8, 25.9. MS (ESI+): m/z (%): 415 (100) [M+H]+.
Synthesis
of
6-(5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-
hydroxypicolinaldehyde (Compound 8). To an ice-bath cooled solution of methyl 6-(5-(6,7-
dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxypicolinate 7 (2.07 g, 5.0 mmol, 1
equiv) in dry dichloromethane (0.05 M) were successively added dry triethylamine (1.6 mL, 11
mmol, 2.2 equiv.) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.3 mL, 5.5 mmol, 1.1
equiv.). The mixture was stirred at room temperature for 2 h under argon atmosphere. The
organic layer was washed with brine, dried over MgSO4 and concentrated under reduced
pressure. To a solution of the resulting residue in dry dichloromethane (0.05 M) at -78 °C, was
added dropwise DIBAL-H (12.5 mL, 1 M in dichloromethane, 12.5 mmol, 2.5 equiv.). Then, the
reaction mixture was stirred at this temperature for 15 min. The reaction was quenched with
methanol (12.5 mL) and the mixture was allowed to warm at room temperature. The organic
layer was washed with an aqueous solution of NaOH (1 M), dried over MgSO4 and concentrated
under reduced pressure. Then, tetrabutylammonium fluoride (5.5 mL, 1.1 equiv., 1 M in THF)
was added at 0 °C to the residue in dry THF (0.1 M) and the mixture was stirred for 30 min at 0-
5 °C. After concentration under reduced pressure, a purification by flash chromatography on
silica gel (CH2Cl2/MeOH 100:0 to 95:5, v/v) afforded 8 (1.29 g, 67%) as a yellow oil. Rf = 0.27
1
(CH2Cl2/MeOH, 95 :5). H NMR (300 MHz, CDCl3) δ = 10.03 (br s, 2H), 7.31 (d, J = 8.7 Hz,
1H), 7.27 (d, J = 8.7 Hz, 1H), 6.60 (s, 1H), 6.53 (s, 1H), 3.84 (d, J = 1.6 Hz, 6H), 3.59 (s, 2H),
13
2.95 – 2.67 (m, 6H), 2.63 – 2.41 (m, 2H), 1.90 – 1.57 (m, 4H), 1.46 (d, J = 7.0 Hz, 2H). C
NMR (75 MHz, CDCl3) δ = 198.5, 156.8, 154.7, 147.4, 147.1, 135.6, 129.6, 126.2, 126.1, 125.9,
111.2, 109.4, 77.1, 57.9, 55.8, 55.7, 55.5, 50.7, 37.1, 29.5, 28.3, 27.0, 26.8. MS (ESI+): m/z (%):
385 (100) [M+H]+.
Synthesis
of
6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy-2-
pyridine aldoxime (3-HPA). To a solution of aldehyde 8 (1.29 g, 3.35 mmol) in methanol
(stored on MS 3 Å, under argon, 0.1 M) were added successively NH2OH.HCl (245 mg, 3.52
mmol, 1.05 equiv.) and sodium acetate (305 mg, 3.68 mmol, 1.1 equiv.). The mixture was stirred
at room temperature under argon atmosphere until completion (3-4 h). After concentration under
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reduced pressure, the residue was purified by flash chromatography on silica gel (gradient
CH2Cl2/MeOH, 98 :2 to 90 :10) to give the desired oxime 3-HPA as a white solid (976 mg,
1
73%). Rf = 0.2 (CH2Cl2/MeOH 95:5). H NMR (300 MHz, CDCl3) δ (ppm) = 1.33–1.43 (m,
2H), 1.55–1.73 (m, 4H), 2.55–2.72 (m, 2H), 2.85–2.96 (m, 4H), 3.74 (s, 1H), 3.81 (s, 3H), 3.82
(s, 3H), 6.51 (s, 1H), 6.58 (s, 1H), 6.95 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 8.28 (s,
13
1H), 9.88 (br s, 1H). C NMR (75 MHz, CDCl3) δ (ppm) = 14.2, 22.7, 26.1, 26.9, 37.1, 50.3,
54.7, 55.8, 55.9, 57.3, 76.6, 109.3, 111.2, 123.8, 124.3, 125.1, 135.1, 147.5, 147.8, 152.4, 153.7,
160.6, 171.9. MS (ESI+): m/z (%): 400 (100) [M+H]+. HRMS (ESI+): m/z calcd for C22H30N3O4
400.2236; found: 400.2230. HPLC: tR = 19.15 min (purity = 97.83%).
2.3. Preparation of 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs
The multiple emulsion (w/o/w) method described by Pashirova et al [13] was chosen for
preparing SLNs. The hydrophobic oxime 3-HPA (0.04 g) was melted in the lipid phase
composed of lipid (Dynasan 114), 5 mL glycerol and Lipoid® S75. Temperature was 5-10°C
above the melting point of lipid. Then the aqueous phase (1 ml) was added to the lipid phase
composed of lipid (Dynasan 114), 5 mL glycerol, Lipoid® S75 and the hydrophobic oxime 3-
HPA (0.04 g) at the same temperature and homogenized with high shear homogenizer (Ultra-
Turrax®, IKA, T18, Germany) for 10 minutes, under stirring intensity of 10,000 rpm. The
mixture was kept at constant temperature during homogenization. Then, this w/o emulsion was
poured into a total weight (40g) of cooled solutions of surfactant (Tween® 80) under magnetic
stirring (750 rpm) (Heidolph, Germany) for 20 min to allow the formation of SLNs. Briefly, for
production of SLNs loading two oximes (hydrophilic oxime (2-PAM) and hydrophobic oxime 3-
HPA) the same method was used by replacing the inner aqueous phase (1 ml), which was
prepared by dissolving 2-PAM (0.04 g) in ultra-purified water. Dynasan 114 was selected as the
solid lipid and Tween 80 as surfactant added to the external aqueous phase of the multiple
emulsion.
2.4. Characterization of SLNs
The mean particle size, zeta potential and polydispersity index of SLNs were determined by
dynamic light scattering (DLS) measurements, using the Malvern Instrument Zetasizer Nano
(Worcestershire, UK). The measured autocorrelation functions were analyzed by Malvern DTS
software, applying the second-order cumulant expansion methods. The effective hydrodynamic
radius (RH) was calculated according to the Einstein-Stokes equation D=kBT/6πηRH, in which D
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is the diffusion coefficient, kB the Boltzmann’s constant, T the absolute temperature, and η the
viscosity. The diffusion coefficient was measured at least in triplicate for each sample. The
average error of measurements was approximately 4%. All samples were diluted with ultra-
purified water to suitable concentration and analyzed in triplicate.
2.5. Encapsulation efficiency and loading capacity
Encapsulation efficiency (EE, %) and loading capacity (LC, %) were assessed for samples
containing 2-PAM. These parameters were determined indirectly by filtration/centrifugation
technique, measuring free 2-PAM (non-encapsulated) by spectrophotometry. A volume of 0.5
mL of each 2-PAM+3-HPA-loaded SLNs was placed in centrifugal filter devices Ultracel 100K
(100,000 MWCO, Amicon Millipore Corporation, Bedford, Massachusetts) to separate lipid and
aqueous phases and centrifuged at 10,000 rpm, for 15 minutes (Eppendorf AG, Hamburg,
Germany). Free 2-PAM was quantified by measuring the absorbance using a Specord 250 Plus
(Analytik Jena AG, Germany) at 294 nm (the molar extinction coefficient of 2-PAM at 294 nm
-1
is 11962 M-1.cm at pH = 7.4). Encapsulation efficiency (EE, %) and loading capacity (LC, %)
were assessed for samples containing 3-HPA. These parameters were determined indirectly by
centrifugation technique, measuring free 3-HPA (non-encapsulated) by spectrophotometry. A
volume of 1 mL of each 3-HPA-loaded SLNs or 2-PAM+3-HPA-loaded SLNs were placed in 2
mL Eppendorf tube and was centrifuged (Eppendorf AG, Hamburg, Germany) for 10 min at
5000 rpm to remove precipitated free 3-HPA compound. A volume of 0.2 mL of each 3-HPA-
loaded SLNs was placed in new 2 mL Eppendorf tube and 1mL of dichloromethane was added
to each sample. Then extraction of 3-HPA was performed, using the Vortex shaker (Neutec
F202A0176 Model ZX3 Advanced Vortex Mixer) at 4000 rpm for 10 min. The mixture (∼1.2
mL) was centrifuged (Eppendorf AG, Hamburg, Germany) for 6 min at 5000 rpm to separate the
layers. The lower organic layer was transferred to a flask and evaporated to dryness under a
gentle stream of nitrogen. 3-HPA extraction procedure was repeated 2 times. The residue was
then reconstituted with 3 mL of dichloromethane. Loaded 3-HPA was quantified by UV
absorbance using a Specord 250 Plus (Analytik Jena AG, Germany) at 315 nm (the extinction
-1
coefficient of 3-HPA at 315 nm is 8686 M-1.cm (Fig. 1S, SM). The parameters were calculated
against appropriate calibration curve, using the following equation [21]:
(1)
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(2)
2.6. In vitro 2-PAM or 3-HPA release profile
The release of 2-PAM or 3-HPA from SLNs was performed using the dialysis bag diffusion
method. The dialysis bags retain SLNs and allow released 2-PAM or 3-HPA to diffuse into the
medium. The bags were soaked in Milli-Q water for 12 h before use. 5 milliliters of SLNs were
poured into the dialysis bag and the two ends of the bag were sealed with clamps. 50 mL
methanol and 50 mL of 0.025 M sodium phosphate buffer pH 7.4were used as the receiving
phase. The vessel was placed in a thermostatic shaker (New Brunswick, USA), at 37ºC, under a
stirring rate of 150 rpm. At predetermined time intervals, 1 mL samples were withdrawn, and
analyzed by determining the absorbance at 294 nm for 2-PAM and at 315 nm for 3-HPA (Fig.
2S, SM) using a Specord 250 Plus (Analytik Jena AG, Germany) and HPLC-MS. At
predetermined time intervals, 10 μL of samples were diluted in methanol and water (70:30, v/v).
Details of HPLC-MS analysis are described below (in section 2.8). Calibration curve of 3-HPA
in sodium phosphate buffer (0.025 M) is given in Fig 3S. All samples were analyzed in triplicate.
2.7. Pharmacokinetic and tissue distribution study
All experiments involving animals were performed in accordance with the guidelines set forth by
the European Communities Council Directive of November 24, 1986 (86/609/EEC) and the
protocol of experiments approved by the Animal Care and Use Committee of Kazan Federal
University.
Wistar rats of both sexes were purchased from the Laboratory Animal Breeding Facility (Branch
of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Puschino, Moscow Region,
Russia) and were allowed to acclimate to their environment in the vivarium for at least 1 week
before experiments. Animals were kept in sawdust-lined plastic cages in a well-ventilated room
at 20–22 °C in a 12-h light/dark cycle, 60−70% relative humidity and given ad libitum access to
food and water.
The maximum recommended volume for i.v. administration into rats is 1-2 ml [22]. Proceeding
from this maximum recommended volume, the dose of 5 mg/kg 2-PAM and/or 5 mg/kg 3-HPA
was selected. Sample was slowly administered into the tail vein of rats weighing 200-250g.
Two groups of animals were set: 3-HPA solution (DMSO-water (50 w/w) or 3-HPA-SLNs or 2-
PAM+3-HPA-SLNs, respectively at a dose of 5 mg/kg (~ 1 ml 0.1% w/w of 3-HPA) were
11

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injected in the tail vein of each group. At predetermined time intervals (5, 15, 30, 45, 60, 120,
180, 240 and 300 min after injection), five rats at each time point from each group were deeply
anesthetized by isoflurane inhalation until the tail-pinch reflex disappeared, and blood samples
(1ml) were collected, placed into heparinized test tubes and centrifuged (at 12,000 rpm, 10 min,
4ºC) to get plasma. Thereafter, animals were quickly decapitated and tissue samples (whole
brain) were immediately collected, washed with physiological saline (NaCl 0.9%) and dried with
filter paper. Brain samples were weighed and homogenized with a nine-fold aliquot of saline
(NaCl 0.9%) under ice bath. Tissue homogenates were processed similarly as plasma samples.
Plasma and tissue samples were frozen and stored at -80°C until analysis.
2.8. Assay of 3-HPA in plasma and brain tissue
HPLC-MS was used to analyze 3-HPA in samples. Briefly, 100 μL plasma samples (or 500 μL
brain homogenate) of were transferred into 2 mL Eppendorf tubes. A volume of 200 μL (or 1 ml
in case brain homogenate) of dichloromethane was added to each sample and mixed, using a
vortex shaker (Neutec F202A0176 Model ZX3 Advanced Vortex Mixer, VELP Scientifica,
Italy) at 4,000 rpm for 10 min. Then, the mixture was centrifuged (Eppendorf AG, Hamburg,
Germany) for 6 min at 5,000 rpm to separate the layers. The lower organic layer was transferred
to a flask and evaporated to dryness under a gentle stream of nitrogen. 3-HPA extraction
procedure was repeated 2 times. The residue was then reconstituted with 1mL of
dichloromethane, and centrifuged at 12,000 rpm for 5 min.
An aliquot (100 μL) of the clear supernatant was diluted in methanol and water (70:30, v/v). A
system from the Agilent HPLC 1200 series (Agilent Technologies, USA) equipped with binary
pump, vacuum degasser and autosampler was used to inject the samples. Aliquots of 10 µL were
run using isocratic mobile phase consisting of methanol and water (70:30, v/v) pumped at a
constant flow rate of 0.3 mL/min.
Quantitation was achieved by MS-MS detection in positive mode using AmazonX (Bruker
Daltonik GmbH, Germany) mass spectrometer equipped with an electrospray ionization (ESI)
source. The optimized operational parameters for MS analysis were −4500 V as the voltage on
the capillary and 10 L/min the flux of nitrogen as a drying gas (250°С). Detection of ions was
performed in the multiple reaction monitoring (MRM) mode, the transition pair of 3-HPA at the
m/z 400 precursor ion to the m/z 382 product ion (Fig. 2).
The analytical data were processed using the Bruker Daltonics software. Calibration curves were
acquired by plotting the peak area of the transition pair of analyte against the nominal
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concentration of 3-HPA calibration standards in plasma (or brain homogenate). The
concentrations used were 5, 10, 20, 50, 80 ng/mL. The calibration curve was linear over the
concentration range of 5-100 ng/mL of 3-HPA in both rat plasma and in brain homogenate (Fig.
2d).
Fig. 2. Full scan mass (a) and product ion (b) mass spectra of 3-HPA in positive ionization
mode. Typical MRM chromatogram of 3-HPA (c). Calibration curve of 3-HPA in brain
homogenate (d).
2.9. Measurement of brain AChE inhibition and reactivation level
For brain AChE inhibition assays, rats were poisoned by 0.8×LD50 of POX (600 μg/kg, i.p.).
Whole brains were collected 1 h, 6 hrs or 8 hrs after POX administration and frozen in liquid
nitrogen. Whole brains of control group of rats were collected 1h after intraperitoneal (i.p.)
injection of physiological saline. Thereafter, AChE activity of brain homogenates was analyzed.
The mean brain AChE activity of poisoned animals (8 rats per group) was compared with mean
brain AChE activity of control group (8 rats).
For in vivo brain AChE reactivation assay, 1 h after poisoning by 0.8×LD50 of POX, rats were
i.v. injected (8 rats per each group) 3-HPA-loaded SLNs, 3-HPA solution (DMSO-water (50
w/w), 2-PAM water solution and 2-PAM+3-HPA-loaded SLNs at the same dose of 3-HPA (5
mg/kg) 2-PAM (5 mg/kg). Whole brains were then collected at different time intervals after
administration of free 3-HPA or 2-PAM+HPA-SLNs or 3-HPA-SLNs and frozen in liquid
nitrogen. The mean activity of brain AChE after injection of free 3-HPA or 3-HPA-loaded-SLNs
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or free 2-PAM or 2-PAM+3-HPA-SLNs were compared to the mean brain AChE activity of
poisoned group.
Whole brains homogenates were prepared in a Potter homogenizer with 0.05 M Tris-HCl, 1 %
Triton X-100, 1 M NaCl, 2 mM EDTA; pH 7.0 (1 volume of brain for 2 volumes of buffer) at
4°C. The homogenates were centrifuged (10,000 rev/min, at 4ºC) for 10 minutes using
Eppendorf 5430R centrifuge with FA-45-30-11 rotor (Eppendorf AG, Hamburg, Germany).
For AChE activity assay, 50µl supernatant was incubated with 5µl of tetra-isopropyl
pyrophosphoramide (iso-OMPA) – as a butyrylcholinesterase specific irreversible inhibitor - in
final concentration 0.1 mM, for 30 minutes. Then, AChE-catalyzed hydrolysis of substrate was
started by adding of 10 µl of acetylthiocholine iodide (final concentration 1 mМ). After 10, 20 or
30 min of hydrolysis at 25°C, reactions were stopped by adding the carbamylating agent
neostigmine (0.1 mM). Samples were diluted in 50 mM phosphate buffer (pH 8.0) and DTNB
(0.1 mM) was added. AChE activity was measured according to the Ellman method [23] by
determining the production of yellow 5-thio-2-nitro-benzoate anion, resulting from reduction of
DTNB by thiocholine (the product of substrate hydrolysis) at 412 nm by spectrophotometry
(PerkinElmer λ25). The amount of thiocholine produced during 20 min (10th–30th min) was
calculated. Sample without substrate was used as a blank. Three samples of each brain
homogenate were measured independently. AChE activity was expressed in relation to the
amount of total protein, which was determined by the Bradford method [24]. Data were analyzed
using Origin 8 and expressed as the mean ± SEM. Two group comparisons were performed
using Mann-Whitney test. P< 0.05 were considered statistically significant.
3. Results and discussion
3.1 Synthesis of 3-HPA
The synthesis of 3-HPA has been improved in comparison with those reported in [17]. The
synthetic routes of 3-HPA are shown in Fig. 1. All the compounds were carefully purified and
1
characterized by H NMR and 13C NMR. The synthesis started with the esterification of
commercially available 3-hydroxypicolinique acid 1 followed by regioselective bromination of
pyridine and finally benzylation of the 3-hydroxy function. Previous synthesis procedure of
compound 4 was obtained with a non-optimized yield from 35 to 40 % (3 steps). Modification of
the procedure allowed robust and easily upscalable production of 4 with 95% to quantitative
yield, mainly by running the bromination step (c) at 0°C instead of room temperature, allowing a
complete regioselectivity for the bromination reaction. Sonogashira cross-coupling reaction
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leaded to the alcohol 5 with excellent yield (from 96 to 99%). Formation of the corresponding
mesylate
followed
by
a
nucleophilic substitution with 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride (TIQ.HCl) furnished the compound 6 with an acceptable
yield of 74 % (63 % previously reported). Then concomitant reduction of the alkyne and
deprotection of the phenol function was carried out using Pearlman’s catalyst under a hydrogen
atmosphere and MeOH as sole solvent to obtain compounds 7 quantitatively. Catalytic
hydrogenation was then followed by a sequence of three reactions comprising (i) the protection
of the phenol group with TBDMSOTf, (ii) the reduction of the methyl ester to aldehyde upon
DIBAL-H treatment, and finally (iii) the deprotection of the TBDMS ether under the influence of
TBAF. These transformations led to the aldehyde 8 in 67 % over three steps versus 11% over
four steps previously reported, by changing the reaction conditions of protection step, adaptating
the reduction step reaction time and equivalents of DIBALH, and performing the deprotection
step at 0°C. The last step consisted of the formation of the oxime function with the condensation
of hydroxylamine onto the aldehyde (76%). Altogether, the desired oxime has been obtained in
eleven steps with 34-36% overall yield from 1 versus 5 % from 5 as previously reported.
3.2. Characterization of oxime-loaded lipid nanoparticles and study of in vitro oxime release
To prepare 3-HPA- loaded SLN and SLN with dual delivery of oximes (2-PAM and 3-HPA) we
used the technique described earlier [13]. To characterize stability of oxime-loaded SLNs,
particle sizes and zeta potential by dynamic light scattering during time were evaluated. Results
are presented in Table 1 and Supplementary material (SM) (Fig. 3S, 4S).
The size of 3-HPA-SLNs is around 100 nm and it increased to 160 nm for dual delivery of 2-
PAM+3-HPA-SLNs. Polydispersity index of 2-PAM-3-HPA-loaded SLNs is 0.61, it is higher
than for empty SLNs (70 nm). The zeta potential of nanoparticles is reduced with increasing of
the concentration of 3-HPA (Fig. 4S, SM) and in the case of dual delivery SLN. After 10-month
storage at 4 ºC 3-HPA-SLNs and 2-PAM+3-HPA-SLNs showed a good stability. The size and
polydispersity index did not change, the zeta potential slightly decreased. The encapsulation
efficiency for 2-PAM is around 95%; and it is comparable with previous 2-PAM-loaded SLNs
formulation. The encapsulation efficiency for 3-HPA of 3-HPA-SLNs and 2-PAM-3+HPA-
SLNs formulations equals 52±2 % and 63.1±3%, respectively. The loading capacity is close to
4% for 3-HPA and 6% for 2-PAM.
Table 1.
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Size (Hydrodynamic diameter), Polydispersity index (PdI), Zeta potential (ZP), encapsulation
efficiency (EE, %), and loading capacity (LC, %) of nanoparticles (data are expressed as mean ±
SD)
Formulation
1-day storage
10-month storage
Size, nm PdI
EE, %
LC, %
Size,
nm
PdI
ZP, mV
ZP, mV
2-PAM
3-HPA
2-PAM
3-HPA
Empty SLN
68±13
0.26±0.01
-55±1.5
-30.0±0.7
-25±5
-
-
-
-
-
3-HPA-SLN
106±19
164±23
0.38±0.02
0.61±0.02
106±19
106±16
0.39±0.1
0.49±0.2
-19.2±0.5
-10.3±0.3
-
52±2
63.1±3
3.5±0.1
4.2±0.2
2-PAM+3-HPA-SLN
94.7±2
6.3±0.1
In vitro release of oximes was analyzed by HPLC-MS and spectrophotometry methods. As seen
in Fig. 3A, the faster release of 2-PAM was observed for 2-PAM+3-HPA-loaded SLNs (Fig. 3A
curve 2) compared to 2-PAM-loaded SLNs (Fig. 3A curve 1): 50% of 2-PAM was released in 10
minutes and 15 minutes (Fig. 3A curve 1), respectively.
Fig. 3. In vitro release of 2-PAM (A) (1-3) and 3-HPA (B) (4, 5, 6) using the dialysis bag
method (n =3) from 2-PAM-loaded SLNs (1); 2-PAM+3-HPA-loaded SLNs (2, 3, 6); 3-HPA-
loaded SLNs (4, 5). The concentration of 2-PAM and 3-HPA is 0.1% (w/w). Phosphate buffer,
0.025 M (1, 2, 4), methanol-phosphate buffer (50% v/v) (3, 5, 6), pH = 7.4, 37 °C.
In vitro release time of 3-HPA was not dependent on the type of delivery formulation (Fig. 3B
curves 5,6), i.e. 3-HPA-loaded SLNs versus 2-PAM+3-HPA-loaded SLN, but it was two orders
slower than for 2-PAM: 50% of 3-HPA was released in 15 hours. The full release time of 3-HPA
was more than 80 hours (Fig. 3B curve 4).
3.3. Pharmacokinetic study
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The concentration of 3-HPA in plasma after one single intravenous injection of 3-HPA in
DMSO-water solution (5 mg/kg) and loaded in SLNs with the same dose is shown in Fig. 4A.
Fig. 4. Concentration of 3-HPA in rat plasma (A) and in rat brain homogenate (B) after
intravenous injection of 3-HPA-loaded SLNs (1) free 3-HPA (2) in DMSO-water solutions (50%
w/w). The dose of 3-HPA is 5 mg/kg for 3-HPAin DMSO-water solutions and 3-HPA-loaded-
SLNs. Each point represents the mean ± SD in five rats.
The pharmacokinetics of 3-HPA in rat bloodstream can be described by a two-compartment
model (3),

C
 Aet  Bet
(3)

t
where [C]t is 3-HPA concentration in plasma at time t, α and β are distribution and elimination
rate constants, respectively. The distribution phase α is very fast. Thus, we only monitored the
elimination phase (β). At 5 min, the 3-HPA maximum concentration in plasma was 640 ng/ml; it
progressively decreased to 6 ng/ml over 60 min.
Calculated pharmacokinetic parameters for elimination are summarized in Table 2. The
elimination half-time (t1/2β) for 3-HPA is 18 min, which is similar to elimination half-time of 2-
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PAM (t1/2β = 24 min). The 3-HPA-loaded-SLNs exhibited an elimination half-time (t1/2β) of 154
min, i.e. 8.5-fold longer than for free 3-HPA.
Table 2.
Pharmacokinetic parameters observed in rats after intravenous injection of 3-HPA in DMSO-
water solutions (50% w/w), 3-HPA-loaded SLNs. The dose of 3-HPA is 5 mg/kg for 3-HPA in
DMSO-water solutions (50% w/w) and in 3-HPA-SLNs preparation. Results represent the mean
± SE for five rats.
*
Samples
β, min-1
t1/2β , min
3-HPA solution
0.03833±0.004 18±1
3-HPA-SLNs
0.00451±0.002 154 ±10
*t1/2β= ln2/β, according to Eq.1 in which β is the elimination rate (min-1) from blood.
To determine the ability of 3-HPA to cross the blood-brain barrier, 3-HPA in DMSO-water
solution and 3-HPA-loaded SLNs were analyzed in brain tissues (Fig. 4B). It is clearly observed
that the concentration of 3-HPA in the brain tissue in the case of 3-HPA-loaded SLNs is much
higher than for injected free 3-HPA in DMSO-water solution.
3.4. Reactivation of Brain AChE in vivo
To test the in vivo reactivation of brain AChE, we used a rat model for OP toxicity using POX.
One hour after poisoning by 0.8×LD50 of POX (600 μg/kg, i.p.), brain samples were collected
and AChE activity was analyzed. One hour after administration of this sub-lethal dose of POX,
the residual activity of brain AChE was 31±4% (Fig. 5, Fig. 6 dotted line). One hour after
injection, the irreversible inhibitory action of POX on brain AChE reached a plateau, as
evidenced by the level of AChE inhibition after one, six and eight hours (Fig. 5). Thus, recovery
of AChE brain activity in the absence of AChE reactivators, due to possible de novo enzyme
synthesis, was not significant (p > 0.05, Mann-Whitney test).
One hour after POX (600 μg/kg, i.p.), free 3-HPA (5 mg/kg) in DMSO-water solution (50%
w/w), 3-HPA-loaded SLNs at the same dose (5 mg/kg), 2-PAM-loaded SLNs (5 mg/kg) and 2-
PAM-3-HPA-loaded SLNs (5 mg/kg for 2-PAM and 5 mg/kg for 3-HPA) were injected. At
different times after injection (0.5, 1, 3, 5 and 7 hours), brain samples were collected and activity
of AChE was measured. We observed that after injection of free 3-HPA, phosphylated brain
AChE was poorly reactivated: residual activity of brain AChE was about 34±3% and reactivation
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was less than 5%. (Fig. 6, line 4). Likely, low brain AChE reactivation mediated by free 3-HPA
is associated with its fast elimination rate (Fig. 4).
Fig. 5. Brain AChE activity 1h, 6 hrs, 8 hrs after poisoning of rats by POX (600 mg/kg, i.p.) and
7 hrs after i.v. injection of 2-PAM (5 mg/kg) in water, 3-HPA (5 mg/kg) in DMSO-water (50%
w/w), DMSO-water (50% w/w) alone or empty SLNs to rats poisoned by POX (1 h, 600 mg/kg,
i.p.). Mean AChE activity of brain homogenates measured in control group of rats was taken as
100%.
On the other hand, with 3-HPA-SLNs, 30% brain AChE reactivation is slowly achieved in 5
hours (Fig. 6, curve 1). After 7 hours the same level of reactivation was detected. It should be
noted that this is the first example of high reactivation of phosphylated brain AChE. But there is
a threshold (until 3 hours after 3-HPA-loaded SLNs injection) when brain AChE reactivation
was only a few percent (Fig. 6). This could be attributed to possible in vivo very slow release of
3-HPA from SLNs (in vitro release takes more than 80 hours (Fig. 3B). The effect of 2-PAM-
loaded SLNs (dose of 2-PAM 5 mg/kg) on brain AChE reactivation reached a plateau (15±3%) 1
hour after injection (Fig. 6 curve 2), i.e. the plateau appears earlier than for 3-HPA. Though the
release of oximes from SLNs can be different in vitro and in vivo, in vivo reactivation kinetics of
phosphylated brain AChE correlates with in vitro faster release time of 2-PAM from SLNs
compared to release of 3-HPA (Fig. 3A). Therefore, it is assumed that the design of SLNs
formulation for dual delivery of two oximes (2-PAM+3-HPA-loaded SLNs) with different
release properties for both oximes makes it possible to increase the therapeutic time window for
oximes. As we can see for combinational therapy with two SLN-encapsulated oximes, brain
AChE reactivation takes place from 30 minutes to 7 hours and reaches 40±3% (Fig. 6 curve 3).
This high reactivation level is clearly the result of the use of oxime nanoformulation, since
injection of either free 2-PAM in water, 3-HPA in DMSO-water, only DMSO or empty SLNs
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did not lead to significant increase in brain AChE reactivation of POX poisoned rats, 7 hours
after injection of tested compounds (Fig. 5).
Fig. 6. Brain AChE reactivation level as a function of time after poisoning of rats by POX (600
mg/kg, i.p.) and subsequent i.v. injection 3-HPA-loaded SLNs (1), 2-PAM-loaded SLNs (2) or 2-
PAM-3-HPA-loaded SLNs (3) and free 3-HPA in DMSO-water solution (4). Mean AChE
activity of brain homogenates measured in control group of rats was taken as 100%. Data are
expressed as mean ± SEM. The dotted line shows the level of residual brain AChE activity one
hour after POX poisoning, without administration of reactivators.
It is important to note that 30 minutes after injection of 3-HPA-loaded SLNs or 2-PAM-loaded
SLNs (dose 5 mg/kg of 2-PAM and 3-HPA, respectively), no reactivation of AChE was
observed. Whereas injection of 2-PAM+3-HPA-loaded SLNs (dose of 2-PAM 5 mg/kg, 3-HPA
5 mg/kg) resulted in reactivation of 10% AChE activity (Fig. 6 curve 3 and Fig. 6S). Thus, in the
initial part of reactivation curve an additive action of the two encapsulated oximes on
phosphylated brain AChE is observed. It is important to note that for the other time points the
additive effect of the two oximes is not observed. It also should be noted that the characteristics
of oxime-loaded lipid nanoparticles (mean diameter around 100 nm and negative charge around
−30 mV) are only slightly different between the two types of SLNs (single encapsulated oxime
or two encapsulated oximes). Therefore, the additive action of oximes on AChE reactivation at
30 min-time point could be the result of in vivo alteration in the release time of 2-PAM and 3-
HPA between single and double nano-formulations (cf. Fig. 3A for in vitro release). A possible
interplay of both oximes on phosphylated AChE the 30 min-time point can also be considered.
Given that the bimolecular rate constant of 3-HPA for POX-phosphylated AChE is higher than
that of 2-PAM [17], an allosteric potentiation of 3-HPA-mediated reactivation of AChE by 2-
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PAM may occur through binding of positively charged 2-PAM on the peripheral site of AChE.
Further study should clarify this hypothesis.
The interactions between nanoparticles and biological environment in brain are complex and
poorly known. There is little evidence that nanoparticles remain stable under conditions of the
brain microenvironment [25]. The efficiency of delivery to the brain depends on numerous
factors such as the ability of nanoparticles to move within the parenchyma [26], and the effective
diffusion within the extracellular matrix [27]. In addition, it is well known that functionalization
of SLNs can enhance brain drug delivery (uptake mechanism and transport pathways) [28]. The
highest potential for brain delivery was found for SLNs stabilized by polysorbate 80 [29,30] and
other poloxamers [31]. High delivery was also shown to be associated with difference in
adsorption of apolipoprotein E (apoE) and BBB specific proteins on the surface of SLNs [29-32].
ApoE adsorbed on nanoparticles mediates their interaction with LDL (low density lipoproteins)
or scavenger receptors located on the BBB. Then, brain uptake processes take place [33]. Protein
crown bound on nanoparticles affects different processes of biological interest [34,35], including
improvement of nanoparticle accumulation in the brain [36]. At the moment, we do not know
either the mechanism of oxime-loaded SLNs transfer across the BBB or the release process of
oximes. Further studies are needed to shed light on these mechanisms and to improve efficiency
of brain AChE reactivation by optimizing nano-formulations. Special attention will be paid to
characterization of the behavior and diffusive capability of nanoparticles as a function of their
size, surface charge, surface coating under brain environment conditions.
4. Conclusions
In conclusion, several tasks were solved: i) encapsulation of 3-HPA in solid lipid nanoparticles
increased the bioavailability (solubility) of 3-hydroxy-2-pyridine aldoxime (3-HPA) and
prolonged circulation time in the bloodstream by 8.5 times compared to free 3-HPA; ii) 3-HPA-
SLNs showed high reactivation of brain AChE (30%) inhibited by paraoxon; iii) overcoming
BBB for 2-PAM and achieve sequential release of both oximes (2-PAM and 3-HPA) and
prolongation of the time window of oxime therapy leading up to 35% brain AChE reactivation
were reached. Further studies are needed to determine the mechanisms of crossing the BBB and
in vivo release of oximes from SLNs. Also, it will be of paramount interest to determine whether
SLNs association of 2-PAM and 3-HPA can reactivate in vivo tabun-inhibited AChE and AChE
inhibited by other potent phosphoramidates [37,38].
Acknowledgements
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Biochemical experiments was supported by the Russian Science Foundation, grant No. 14-50-
00014 to Irina V. Zueva and Konstantin A. Petrov. This work was in part supported by the
Direction Générale de l’Armement (through PhD Fellowship to Anissa Braïki), the Agence
Nationale de la Recherche (ANR) (project ReCNS-AChE, grant number ANR-13-ASTR-0002-
02) to Pierre-Yves Renard and Ludovic Jean. This work was also partially supported by INSA
Rouen, Rouen-Normandie University, Centre National de la Recherche Scientifique (CNRS),
Région Haute-Normandie (CRUNCh network), and the LabexSynOrg (ANR-11-LABX-0029) to
Anissa Braïki, Pierre-Yves Renard and Ludovic Jean.
E. B. Souto acknowledges the financial support received from Portuguese Science and
Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) under
the project references M-ERA-NET/0004/2015-PAIRED and UID/QUI/50006/2013, co-financed
by FEDER, under the Partnership Agreement PT2020.
P. Masson was supported by the Russian Government Program of Competitive Growth of Kazan
Federal University.
The authors gratefully acknowledge the CSF-SAC FRC KSC RAS for providing mass
spectrometer access.
Author Contributions
Tatiana N. Pashirova developed preparation and characterization of SLN and performed in vitro
drug release experiment; Irina V. Zueva and Konstantin A. Petrov performed biochemical
experiments; Vasily M. Babaev and Ildar Kh. Rizvanov developed the technique of assay of 3-
hydroxy-2-pyridine aldoxime in plasma and brain tissue by the HPLC-MS and analyze data
obtained performed the experimental work on mass spectrometer equipped; Evgenia A. Burilova
and Darya A. Samarkina performed extraction experiment for plasma and brain tissue; Anissa
Braïki synthesized oxime 3-hydroxy-2-pyridine aldoxime and characterized properties of this
oxime; Pierre-Yves Renard and and Ludovic Jean proposed the idea of synthesis of oxime 3-
hydroxy-2-pyridine aldoxime and controlled it; Eliana B. Souto controlled the synthesis of
SLNs; Lucia Ya. Zakharova proposed the idea of physico-chemical part of the work and
controlled it; Patrick Masson designed experiments; Oleg G. Sinyashin proposed the idea of the
whole work.
Conflict of interests
None
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Abbreviations
AChE, acetylcholinesterase; BBB, blood brain barrier; OP, organophosphorus agent; BChE,
butyrylcholinesterase; 2-PAM, Pralidoxime chloride; SLN, solid lipid nanoparticle; SNC, central
nervous system.
Supplementary material. Supplementary data are available in the online version, at
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