European Review for Medical and Pharmacological Sciences 2020; 24: 7063-7076 In vitro redox activity of haemozoin and β-haemozoin interacting with the following antimalarials: artemether, lumefantrine and

J.L. IMBERT PALAFOX1, L. GONZÁLEZ LINARES1, V.E. REYES-CRUZ2, M.A. BECERRIL FLORES1, J.C. RUVALCABA LEDEZMA1, C.M. GONZALEZ ALVAREZ3, A. ARENAS FLORES2, J.E. BAUTISTA GARCIA1

1Área Académica Medicina, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Estado de Hidalgo, México 2Área Académica Ciencias de la Tierra, Materiales, Metalurgia, Instituto de Ciencias Básicas e Ingeniería, Universidad Autónoma del Estado de Hidalgo, Pachuca, Estado de Hidalgo, México 3Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Puebla, México

Abstract. – OBJECTIVE: parasites in- ronments suggest that the cell membranes can vade, grow and multiply inside erythrocytes and also be damaged by the unique presence of the obtain nourishment from haemoglobin. Then, antimalarial. the released haem group is oxidized to hae- matin and inert dimeric haemozoin bio-crys- Key Words: tals form, which provides the parasite a unique Haemozoin, β-Haemozoin, Antimalarials, Cyclic way to avoid the toxicity associated with the Voltammetry, Meccus longipennis. haem group. Therefore, antimalarial drugs are designed to inhibit dimer formation; however, recent electrochemical studies indicate that an inert dimer also promotes a toxic oxidizing en- Introduction vironment. Therefore, this work explores drug reactivity in the presence of monomers and di- mers to evaluate their contribution to redox ac- Resistance to new antimalarial drugs has be- tivity. come a research priority, and haemozoin inhibi- MATERIALS AND METHODS: Three medi- tion is a very attractive target because it appears cines mixed with haemozoin or β-haemozoin in to be an immutable pathway1. Due to an incom- carbon paste electrodes were tested using cy- plete understanding of the parasite processes that clic voltammetry. 2-4 RESULTS: The data indicated again that the lead to haemozoin formation a drug that specif- substances modify the natural redox state of ically targets the parasite factors responsible for haemozoin and β-haemozoin. This effect could their production has not yet been developed; thus, be attributed to the natural oxidation potential the topic has been a subject of intense debate. of the drugs. In addition, it was found that the Although a protein to detoxify the haem group oxidation potential decreased through quinine, (haem detoxifying protein) has been identified lumefantrine and artemether with the same ten- and characterized in falciparum, dency in the presence of haemozoin but with less current density. Additionally, it was ob- which is critical for the survival of the parasite, served that the oxidation response between the the systematic degradation of haemoglobin, the monomer haemozoin and antimalarial drugs is release of the haem group and the formation of carried out at more negative potentials. haemozoin [Hz] are not completely clear. Under CONCLUSIONS: Together, the total results in- this view, if HDP can be an important contributor dicate that antimalarials per se can contribute in achieving non-toxic Hz levels found in the par- to oxidation processes and that in combination asite, experimental support by classical molecu- with monomeric or dimeric haemozoin can in- 5 crease or decrease the oxidizing power of the lar, biochemical and cell biological approaches haemozoin forms. The various oxidizing envi- will be required.

Corresponding Author: Jose Luis Imbert Palafox, MD; e-mail: [email protected] 7063 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al.

The haematophagous hemipteran Rhodnius or experimental evidence of inert haemozoin prolixus or Meccus longipennis, such as Plas- crystals embedded within a nanosphere with modium, have evolved many genetic resources to those of their pro-oxidant effects and how the protect cells against haem toxicity. In this sense, haem group avoids oxidation15,16,20,21; therefore, haemoglobin degradation in Triatominae insects this research has the principal aim of determining is also a complex biochemical process, which whether there is a possible potential generation of takes place in an environment apparently differ- or an unbalanced redox state due ent from a digestive vacuole6-9. to the interaction of antimalarial drugs with Hz or The formation of a haemozoin-like crystallite β-Hz in a similar osmolar medium. is a vital process of insect vectors of Chagas dis- The subsidiary aims of the study are to under- ease10,11, Schistosomiasis12,13 and probably other stand and know more about the toxicity and in- diseases transmitted by the genus of haemato- stability aspects and explore whether antimalarial phagous mosquitoes, such as Aedes, Anopheles drugs may have potential secondary effects, such or Lutzomia. Notably, once the meal of the as ROS generation21. insect is digested in the stomach or anterior mid- gut, the haemoglobin is denatured, and then, the haem group is free to pass to the small intestine Materials and Methods or posterior midgut space where it is polymerized as nontoxic haemozoin6,10,11. Reagents and Drugs These apparently small and irregular frag- The reactive substances were analytical grade- ments that are found adhered to intestinal cells high purity and the deionised water had a specific provide strong evidence that Hz formation in resistance of 18.2 MWcm-1. Protoporphyrin IX®,

Rhodnius prolixus or haematophagous insects is (PPIX), (C34H34N4O4, MW 562.7) approx. ≥ 95%, a very efficient process and that haem crystalliza- catalog P8293; Quinine, (QN), (C20H24N2O2, MW tion favorably occurs10,11,14, in spite of the fact that 324.42) approx. 90%, catalog 145904; Amodi- the stomach and intestines are not a big digestive aquine, (AQ), (C20H22ClN3O*2HCl 2H2O, MW vacuole. 464.8) approx. ≥ 99%, catalog A2799; Chloro-

Although the physical-chemical environments quine, (CQ), (C18H26ClN3 2H3PO4 , MW 515.86) are likely to be similar, the crystallization pro- approx. ≥ 99%, catalog C6628; Sodium Azide cesses of haematin in these insects are apparently (NaN3, MW 65.01) approx. 99.5%; Sodium hy- not the same; thus, the basic requirements of the droxide (NaOH, MW 40) approx. 98% and Sil- redox balance and pH equilibrium between the icon oil (SiO) (C7H8OSi, MW 136.22) approx. ≥ lipidic or aqueous medium may be similar from 99%, [the binding agent with 0.963 g/mL den- a biochemical point of view15,16. However, in the sity and 200 cSt viscosity at (25°C)] all of them faeces of these bugs, uniform crystallites do not were obtained from Sigma-Aldrich (St. Louis, occur, such as those observed in malaria samples; MO, USA). Arthemeter, (ART), (C12H26O5, MW interestingly, crystalline micro-arrays are pro- 298.4) approx. ≥ 99% and Lumefantrine, (LF), duced with remarkable uniformity in morphology (C30H32O1N14Cl3, MW 298.4) approx. ≥ 99% were and size in malaria samples17-19. obtained from Novartis Pharma AG (Campus The first description of Hz in a different - or Basel, Switzerland). Sodium chloride (NaCl, ganism from Plasmodium claims that “after a MW 58.44) 100.2%; Sodium nitrate (NaNO3, blood meal, R. prolixus midgut presents a huge MW 84.999) 100.7%; Hydrochloric acid (HCl, amount of Hz granules”; the midgut lumen con- MW 36.46) 35.5-38%; Absolute ethylic alcohol tains large electron-dense aggregates similar in (C2H5OH, MW 46.07) 99.5% and Ethylenedi- appearance to the haemozoin granules found in aminetetraacetic acid (EDTA, C10H16N2O8, MW malaria parasites10,11. The process of Hz forma- 292.25) approx. 99.4% were obtained from J. T. tion in S. mansoni and R. prolixus is similar14, Baker (Phillipsburg, NJ, USA). Graphite pow- but the differences do not seem to be derived der, 2-15 μm particle size, (C, MW 15.9999) from distinct atomic structures; instead, they are approx. 99.9995%, Alfa AesarTM (Thermo Fisher generated from the crystal growth conditions. Scientific, Waltham, MA; USA). , Thus, in part, this issue was elegantly resolved (Hb), (MW 64 kDa) approx ≥ 99% Becton & and showed that the haem crystals of the insect Dickinson (Franklin Lakes, NJ, USA). Haemo- 14 share the same unit cell and structure . It has zoin (Hz, Meccus longipennis), (C34H33FeN4O5, been challenging to reconcile the observations MW 633.50) approx. ≥50% and β-Haemozoin

7064 Redox potential of haem with antimalarials

(β-Hz, Meccus longipennis), (C68H66Fe2N8O10, electrons in 20 to 30 kV range and amplification MW 1264.00) approx. ≥50% were obtained from were of 5,500X for obtaining topographical imag- AAM-ICSA-UAEH, the present work. es. The retro dispersal electrons were from k level for microanalysis detecting the percentual weight Hz and β-Hz Preparation from of Elements (%) in a point area (100 nm2) at 30 M. Longipennis Kv, field depth 0.3 mm, ×10,000 times in 60 s. The techniques used were similar to referenc- The X-ray diffraction powder pattern was ob- es22,23. Briefly, 2.1 g of insect faeces and urine tained in equipment Inel, Equinox 2000. XRD were collected. The material was pulverized, and analysis was performed with CoKa1 X-rays, a then, dialyzed against water. After centrifugation step size of 0.02 and collection time of 1 s across at 2,500 rpm for 20 min, the dark supernatant was a 2θ range of 20-50º. The diffraction spectra were filtered (Whatman® grade I, catalogue No. 1001 processed using X’Pert High Score (Version 2.0) 125), and then, mixed with 1 M NaOH to obtain software with reference to the Powder Diffraction soluble Hz. To obtain b-Hz, the solution was File database (Version 4þ 2009) from the Inter- acidified with concentrated HCl, and the brown- national Centre for Diffraction Data (Newton ish precipitate was stored for 24 hours. Then, the Square, PA, USA)23. pellet was centrifuged at 2,500 rpm for 20 min and washed with deionized water (pH=6.0) 5 to Electrolytic Solutions, Electrochemical 8 times. Finally, the β-Hz nano-crystals and mi- Cell and Working Electrodes cro-crystals were separated from the water and The first solution was 0.1 M NaNO3, pH 6.50 allowed to dry at 32°C. Approximately 1.2 g of and second one was 0.15 M NaCl, pH 6.65; a typ- pure substance were obtained, representing 57% ical Pyrex brand cell was used, with three elec- of the original sample. trodes at room temperature and Nitrogen gas, the auxiliary or Opposite-Electrode was a graphite Preparation of Controls HZ and β-HZ bar. The Reference-Electrode was sulphates sat- ® from Hemoglobin of B&D urated (SSE) Hg/Hg2SO4(s)/K 2SO4 (sat), immerse The methods used were similar to referenc- in a “Luggin” capillary tube22. es22,23. All values of electrochemical assays were con- verted and reported using the Standard Hydrogen Characterization by Infrared and Electrode scale (SHE, 0.615 V total sum). The UV-Visible Spectrophotometry values of the electric current density were report- To perform the FTIR spectra and the spectral ed making a quotient between current flow and studies in UV-visible range, the methods used the Work-Electrode transversal surface work area were similar to references22,23. Results show the (0.0314 cm 2). average from three determinations for obtaining Buffers for pH regulation were not used be- statistical significance. cause the voltammetry assays were in micro-elec- trolysis conditions, and the changes were not Characterization by Zeta Potential appreciable. The Hz from M. longipennis 0.1 M, was dis- In order to obtain reliable results and to assure solved in deionized water and after that was realized the reproducibility of the voltammetry responses, the pH adjustment with NaOH 0.1 M or HCl 0.1 M it was used the Open Circuit Potential (OCP) or for obtaining pH values of 4, 5, 6, 7, 8 and 9 and Null Current (NC), and it was determined by fol- the Z potential in a 0 to -35 mV range was deter- lowing the Potential Variation Rate in function of mined. The equipment used was a Zetasizer, Brand time, until reaching a stable stationary state after Malvern, Model 3000 HSA and results show the being in contact with the solution. average of six determinations for each pH23. All the voltammetry assays were started with a specific (OCP) and scanning velocity (ν) of 20 Characterization through SEM, mVs -1 and the electro activity interval was es- EDS and XRD tablished between -1.5 and 2.0 V. The potential For Electron Micrograph and Energy Spectrum window for the NaCl medium was established of Hz and β-Hz molecules from M. longipennis, between two intervals of -1.5 to 2.0 V and -0.36 the equipment used was a JEOL 6300 Scanning to 1.6 V. Electronic Microscope, equipped with Energy The Carbon Paste Electrodes (CPE) were pre- Dispersive Spectrometer. Were used secondary pared with different compounds: Hz and β-Hz

7065 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al. controls prepared from Becton & Dickinson®, Hz and β-Hz from M. longipennis alone or mixed with antimalarial drugs and complement- ed with Graphite powder and Silicon oil. The composition was: graphite, sample (Hz or β-Hz) or drug and silicon oil, and the weight propor- tions (wt.) were graphite: sample 50:50; graph- ite: drug 50:50; graphite: sample: drug 50:25:25. Two additional were: graphite: sample: drug 50:17:33 and 50:13:37 all the CPE were complet- ed with 0.3 ml oil22. The determinations were realized with a Po- tenciostat-Galvanostat, Mark PAR, Model 263A online with a Personal Computer and the runs, determinations, conditions and graphical steps were registered with the software Power Suite Version 3.122. Figure 2. Absorption spectra of Hz (Grey line) and β-Hz (Black line) from M. longipennis. Results

FTIR and UV-Visible Spectra which is similar to what was reported in the Figure 1 shows the infrared spectra for M. lon- literature and confirms two different chemical gipennis, where the classic bands corresponding structures. to carbonyl (C=O) and carboxylate (C-O) are ob- served at 1671 and 1127 cm−1 for Hz and at 1651 Zeta Potential and 1046 cm−1 for β-Hz; the signals match those In the curves of Figure 3, two slopes higher in the literature10,11,24-26. The absorption spectra than 900 can be observed between a pH inter- of Hz and β-Hz from M. longipennis in Figure val of 4.0 to 6.0; these two curves correspond 2 show the classical shift of the Söret band. The to β-Hz and the third slope at a pH interval band displacement, from 391 nm in an alkaline of 6.0 to 9.0 correspond to Hz. The slopes solution to 384 nm in an acidic medium, is due to the movement in the plane,

Figure 1. FTIR spectra of Hz (Black line) and β-Hz (Grey Figure 3. Zeta potential of Hz and β-Hz from M. line) from M. longipennis. longipennis.

7066 Redox potential of haem with antimalarials have a negative surface charge for the Hz- ions strongly bound to the surface, and at the βHz conjugated pair; the first one has a minor outer region, the ion distribution is determined or attenuating change from -23 mV towards by the balance between electrostatic forces and a negative potential close to -30 mV in a pH thermal movement. Therefore, the potential range of 9.0 to 6.0. The second one occurs when decreases when increasing the distance from the dimerization process begins. Although the the surface to a sufficiently large distance and molecules are not completely soluble and the thus achieves the solution zero potential. This pH changes to 6.0, a short predictable leap of potential reflects the effective charge on the -23 mV occurs, and the electric potential for particles and correlates with the electrostatic β-Hz formation has a value of -6.0 mV and pH repulsion between them. 5.0. The third change is when the pH changes Surprisingly, the negative surface charge of the from 5.0 to 4.0; the β-Hz molecules are less dimeric molecules reaches the values of -6.0 mV soluble, and the potential change is -6 to -3 mV. and -3.0 mV, which are close to electrical neutral- Most likely, the second and third slopes in the ity. This may mean that additional carboxyl group pH intervals of 6.0 to 5.0 and 5.0 to 4.0 show protonation occurs at pH values of 4.0 to 5.0. the union and microcrystallisation phases like as succeeding in the digestive vacuole of the Microscopical Description parasite where the pH is 4.5 to 4.97,18,19,27. These Figure 4 shows the control compounds ob- steps carry a notable calorific energy release tained from Haemoglobin® B&D using a USB in an assay titration and probably result from M-1 microscope (Cole-Parmer, amplified (×200)). electrostatic repulsion and attraction and van Characteristic brown and crystalline aspects can der Waals forces (personal observation). The be observed. Additionally, Hz has a large homo- Hz and β-Hz molecules in alkaline or acidic geneous crystalline form and β-Hz presents a solutions can acquire a surface electric charge, very small agglomerating particle. which can favor differential adsorption of ion Figure 5 shows the micrographs of Hz and solutions (antimalarials, for example) on the b-Hz crystals obtained from M. longipennis. In surface. The above adsorption affects the ion Figure 5A, there are many small plane crystals, distribution and increases the concentration of thin needle-like structures and the presence of counterions, thus forming a double electric lay- several particle sizes with average dimensions er at the interface. The internal region includes of 2 µm × 4-5 µm. The β-Hz image in Figure

Figure 4. Hz (a) and β-Hz crystals (b) ob- tained of Hemoglobine® Beckton and Dick- inson.

7067 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al.

Figure 5. SEM of A, Hz and B, b-Hz crystals from M. longipennis. Magnification 5, 500 ×, bar 1mm.

5B shows crystalline agglomerates with more acid remains were not observed and the sample homogeneous particle sizes that have cubic collection method was considered adequate. dimensions of 2 µm × 2 µm × 2 µm. Further- more, the crystals in Figure 5B are in large 3D Atomic and Molecular agglomerates, and the geometrical shape in- Spectroscopic Description cludes flat and well-formed faces with straight EDS tests for the Hz and β-Hz from the M. edges and sharp vertices. In both samples uric longipennis samples are shown in Figure 6. The

Figure 6. Elemental microanalysis of iron in Hz (a) and b-Hz (b) by EDS and XRS.

7068 Redox potential of haem with antimalarials

Table I. Elemental microanalysis with Scanning Energy Dispersive by X Ray (SED) from M. longipennis samples.

This work Reference (20)

Element Hz ß-Hz Hz ß-Hz

Iron (Fe) 0.54 4.57 0.36 2.38 Carbon (C) 37.44 52.32 34.21 38.13 Nitrogen (N) 24.59 20.26 29.35 21.03 Natrium (Na) 0.35 1 0.02 0.44 Chloride (Cl) 0 0.92 0.05 1.43 (O) 23.59 20.93 35.93 35.07 Total 86.51 100 99.92 98.48 samples are visualized through secondary elec- CDCC database produce different interplanar trons at 20KV 20KV, working distance of 17mm, distances and space groups. The crystal habit and at a 5,500×magnification. and cell parameters for β-Hz show two crystal- Elemental microanalyses of the same mole- line systems (monoclinic and tetragonal), anoth- cules by X-ray spectroscopy and energy disper- er two for the faeces sample (monoclinic and sive spectroscopy show the major signal for iron orthorhombic) and another (orthorhombic) for (Fe) in Hz (Figure 6A) and β-Hz (Figure 6B). Hz alone, as recorded in Table II. The associated bands of gold (Au) resulted from the coating process, and chloride and sodium Crystallographic Data were contaminants from solutions employed for It is interesting to see how the strong peak at their purification. With respect to the iron com- 27.60 (3,996) observed in the faeces sample ap- position, the β-Hz has a concentration 8.46 times pears in Hz at 28.03 and 28.69 peaks; whereas for greater than Hz from M. longipennis (Table I), and this difference in content in the dimer and monomer forms show the sample purity and efficiency in the sample collection procedure. It can be observed that the Hz and β-Hz amounts obtained from insect faeces are high, despite being in small amounts of sample, which is con- trary to the great amount of pure Haemoglobin® B&D used by the chemical method. This means that the biological digestion of blood from hema- tophagous insects is more efficient than chemical digestion.

Crystallographic Structure Figure 7 shows three diffraction patterns from M. longipennis samples: faeces, the Hz and β-Hz obtained for this study. An FCC (face centered cubic) crystalline structure with planes [1,1,1], [2,0,0], [2,2,0] and [3,1,1] can be observed Figure 7. XRD patterns comparison of Hz, b-Hz and from the β-Hz curve, and the analysis of the faeces. The 2q rank was 12° to 102o, with Cobalto radiation crystal alignment is in relation to these four re- (CoKa1=1.540560 A°). Reflection maximum values at 2Ɵ (º) and [inputs]. The curves have vertical displacement for flections. In their curve, the large signal of more better view. Faeces: 21.61, 24.01 (1,623), 27.60 (3,996), 38.20 than 11,000 inputs at peak 38.23 exactly matches (2,051), 44.28 (1,165), 77.77. Hz: 28.03, 28.69, 30.69 (1,253), with identical peaks of pure protein molecules, 31.44, 32.97 (1,222), 33.65, 44.63 (1,166), 45.26. b-Hz: such as trypsin and haemoglobin, which indi- 13.29 (1,769), 18.01 (1,861), 23.08 (2,377), 27.37 (2,344), cates that the arrangement of the β-Hz dimer 28.19, 29.08 (3,042), 38.23 (11,054), 44.38 (2,640), 77.71. Hemoglobin and trypsin samples were observed with face produces a preferred orientation with perfect cu- cubic center patterns, with only four peaks and (inputs): bic symmetry. In addition, the crystallographic 38.33 (11,472), 44.47 (2,919), 64.55 (752) and 77.68 (1,590) data according to the indexing software of the (data not showed).

7069 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al.

Table II. Crystallographic data of Hz and ß-Hz from Meccus longipennis.

Space Crystal d [Å]/Calculated value h k l Mult. group system Cell parameters

ß-Hemozoine M.l.

6.5864 1 1 0 4 C 1 c 1 (9) ++Monoclinic a=19.6441 Å b=7.0958 Å c=18.7029 Å β=115.692º 3.2040 0 2 1 4 P 1 21/m 1 (11) Monoclinic a=12.1011 Å b=8.5290 Å c=4.8841 Å β=96.240º 3.9265 1 1 0 4 I 4/m (87) Tetragonal a=5.5529 Å c=7.8993 Å 4.9705 2 0 0 2 P 1 21/c 1 (14) Monoclinic a=10.6776 Å b=6.9148 Å c=14.1193 Å β=111.407º

Faeces M.l.

3.2142 1 3 0 4 P 1 21/m 1 (11) Monoclinic a=5.4350 Å b=12.2410 Å c=6.9320 Å β=106.260º 2.3488 2 2 0 6 P b c a (61) Orthorhombic a=35.2450 Å b=7.7757 Å c=7.1720 Å

Hemozoine M.l.

3.2421 6 2 0 4 P b c a (61) Orthorhombic a=35.2450 Å b=7.7757 Å c=7.1720 Å

Haemoglobine® B&D and Trypsine® Sigma

2.3435/2.3480 1 1 1 8 F m-3 m (225) Cubic a=4.0590 Å 2.0295/2.0353 2 0 0 6 1.435 /1.4405 2 0 2 12 1.2238/1.2261 3 1 1 24 1.1717 2 2 2 8 1.0148 4 0 0 6

β-Hz, the strong peak is divided into 27.37, 28.19 still differences between synthetic and natural and 29.08. However, the large peak at 38.23 that is haemozoin (sHz & nHZ), for example, the size present in the β-Hz and faeces samples do not ap- and shape of the crystals may not be identical pear in purified Hz. This means that in the natural due to differences in the purification process faeces sample, there is also a face cubic centre in that is used. Natural haemozoin is composed of the cubic system (4 and 6) corresponding to iron small crystals measuring between 50 nm and 500 from β-haemozoin; therefore, both samples are nm, and synthetic β-haematin crystals can range identical. However, Hz presents a different orthor- from 50 nm to 20 μm, depending on the solvent hombic crystalline structure, as recorded in Table used28,29. Other results30,31 illustrate that the haem II. In both molecules, reflection peaks correspond- aggregates, which are precipitated by these sys- ing to aromatic compounds, such as C6H8N2 [00- tems, need to be carefully scrutinized, especially 004-0258], with signals at 33.75, 38.85 and 55.18, by powder diffraction, to ensure that the malaria and inorganic Fe3N [01-073-2101], with signals at pigment is present in the purest state. Finally, our 80.5, 92 and 103.56, were observed. Even though methodology is efficient, safe and effective, pro- there is a non-coincident peak at approximately ducing good material obtained from the natural 67°, these results apparently indicate a different faecal material of blood-sucking insects; there- crystalline structural characteristic, which also fore, the preparation of biological cultures of the confirms that it is a natural material with a high parasite (plasmodium) transmitter of the disease purity grade and demonstrates an adequate proce- is not necessary23 because we use them in this dure for obtaining the material. study as an alternative material for the evaluation It is important to note that some references28,29 of antimalarial drugs. mention that synthetic β-haematin crystals can be produced in vitro with the same nuclear magnetic Antimalarial Voltammetry resonance and X-ray diffraction patterns as the Determining the redox response of medicines haemozoin of the malaria parasite by adding a alone was necessary before studying the interac- whole trophozoite lysate that has been acidified tion between the antimalarial drugs and the Hz to a pH between 5 and 6. However, there are and b-Hz molecules.

7070 Redox potential of haem with antimalarials

Figure 8. CV on CPE-AQ (green curve, i), CPE-CQ Figure 9. CV of CPE-AQ-Hz (green curve, i), CPE- (blue curve, ii), CPE-QN (orange curve, iii), CPE-ART CQ-Hz (blue curve, ii), CPE-QN-Hz (orange curve, iii), (gray curve, iv) and CPE-LF (black curve, v) in the anodic CPE-ART-Hz (gray curve, iv) and CPE-LF-Hz (black direction in a 0.15 M NaCl medium at pH 6.5 (v= 20 mvs-1). curve, v).

Figure 8 shows the respective voltammograms in of each drug. It can be seen that at the same oxi- the anodic direction in a NaCl medium and shows dation potential of 1.3 V the current densities de- different oxidation current densities (curves i, ii, crease with values at 5 mAcm-2, 3.3 mAcm-2, and iii, iv and v). At the same oxidation potential of 1.3 0.6 mAcm-2 for AQ, CQ, and QN, respectively, V current densities of 23 mAcm-2, 4.8 mAcm-2, 1 and the current densities increase with values at mAcm-2, 0.009 mAcm-2 and 0.004 mAcm-2 for AQ, 0.15 mAcm-2 and 0.025 mAcm-2 for ART and LF, CQ, QN, LF and ART are produced, respectively. respectively. In this last case, the highest oxidiz- The decrease in the oxidation current density can ing power when Hz is present is for lumefantrine be attributed to 1) the chemical nature of the drugs and artemether. When compared with the drugs generating a less oxidizing environment or 2) the alone (Figure 8), the results indicate that anti- drugs diverting the oxidation processes of graphite malarials per se can contribute to the oxidation and the medium to more anodic potentials. With processes and, in combination, can also increase these considerations, lumefantrine and artemether or decrease the oxidizing power of Hz. have the weakest oxidizing environment. It is important to mention that the voltammet- Voltammetry of Antimalarials Mixed ric response of all drugs in the cathodic direction with β-Hz is equal or similar to the anodic direction, which In the case of b-Hz combined with five drugs may indicate that all of them are found in a rela- (Figure 10), it is possible to observe that the ox- tive redox equilibrium or are not inert substances. idation current densities majorly decrease with values at 2.8 mA cm-2 and 1.8 mA cm-2 for AQ Voltammetry of Antimalarials and CQ, respectively; QN stays at the same Mixed with Hz value of 0.6 mA cm-2 for and low values of 0.03 Due to the importance of the oxidation re- mA cm-2 and 0.03 mA cm-2 are observed for sponse in the generation of reactive oxygen spe- ART and LF, respectively. It is very interesting cies (ROS), which are the cause of damage to that the lowest oxidizing power is for lumefan- various biological molecules, we presented only trine and artemether. In this respect, it seems those results related to anodic behavior. that antimalarials can also be adjuvants to the Figure 9 shows five curves i, ii, iii, iv, and v of oxidation processes and attenuate the oxidizing the CPE-Hz from M. longipennis in the presence power.

7071 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al.

β-haemozoin with lumefantrine and artemether in three different proportions can be viewed in Figures 11A, 11B, 11C and 11D. The varying weight ratios are listed in the following: graphite 2, 2.94, 3.84/Hz or β-Hz 1, 1, 1/drug 1, 1.94, 2.84, respectively. Figures 11A and 11B show that there is a minor modification in the oxidation current densities of Hz with variable amounts of lumefantrine (elec- trophilic molecule with 3 chlorides directly influ- encing the pi bond of a benzene ring) and with variable amounts of artemether. In the β-Hz case Figures 11 (c) and (d), there are probably major modifications of the oxidation current densities with lumefantrine, but there are also those with artemether. In general, a drug ratio of 50:25:25 is very stable because the increase in drug no longer contributes to the oxidizing power of the arte- Figure 10. CV of CPE-AQ-b-Hz (green curve, i), CPE- mether without aromatic groups (no pi bonds), CQ-b-Hz (blue curve, ii), CPE-QN-b-Hz (orange curve, iii), which can interact with the p-p bond of Hz. The CPE-ART-b-Hz (gray curve, iv) and CPE-LF-b-Hz (black drug that decreases the oxidizing power of Hz the curve, v). most is lumefantrine since for the same oxidation potential of 1 V, a low oxidation current density is obtained with respect to the artemether drug It is important to note that the oxidation cur- (0.02 mAcm-2 and 0.09 mA cm-2, respectively). rent density, for a potential of 1.3 V with Hz-AQ and Hz-CQ are higher than those with b-Hz-AQ and b-Hz-CQ. These results are similar to assays Discussion of Hz and b-Hz without drugs and indicate that the most reactive molecule is Hz7,21,22. The b-Hz The mechanism of action of artemisinin (ART) in the presence of drugs also has oxidizing power, drugs is still under debate, but a widely held and these are contradictory results. It is assumed view is that ART is a pro-drug that is activated that b-Hz is an inert molecule, which is formed by reductive cleavage of an endoperoxide ring; by the parasite so that it does not suffer damage the resulting free radical is thought to react with by the haem group, once the haem group is di- susceptible groups within a range of essential merized and “polymerized”. parasite proteins, thus leading to cellular damage It has been demonstrated that both molecules and death36-38. Direct insights into the nature of are indeed “true” p-p dimers (pi bonds), and the ART activator in vivo is unknown, but some in this case, variable electronic transfers occur studies suggest that it is important to maintain re- depending on which drugs can be employed, dox homeostasis. The major disadvantage of ART according to the recent theories of chemical and drugs is that they have very short half-lives in electronic bonding32,33. vivo (0.5-2 hours). Indeed, it has been suggested that one reason for the short half-lives of endop- Voltamperometric Study of Hz and eroxide antimalarials is premature opening of the β-Hz With Lumefantrine and endoperoxide ring when the drug is located away Artemether at Variable Concentrations from the active site in the parasite37. A mixed preparation of antimalarials, to check The crystalline structure of haemozoin shows the stability of the redox potential, was conduct- the presence of dimers stabilized by p-p interac- ed for a recommended treatment with the drug tions that are also present in solution and have AL® [Coartem; Novartis, tablet contains 20 mg been proposed as an alternative nucleation unit. artemether and 120 mg lumefantrine (proportion So, the beginning of haemozoin nucleation in 1:6)]. In this six-day treatment, the total dose the detoxification process requires the internal administered was 0.480 g plus 1.920 g34,35. In lipid membrane of the digestive vacuole17-19 and this manner, the reactivity of haemozoin and an efficient proton pump that acidifies it39. These

7072 Redox potential of haem with antimalarials

Fi g u r e 11. The four trials had the same proportions 50:25:25 (blue curve, i), 50:17:33 (brown curve, ii), and 50:13:37 (gray curve, iii) and were with anodic direction on NaCl medium 0.15 M, pH 6.6 (v= 20 mvs-1). nano-nuclear structures support the mechanism led Lonsdale40 in 1968 to suggest that epitaxial of action of antimalarial drugs that inhibit the relationships between these crystalline phases growth of haemozoin. Recent studies have shown could be an important factor contributing to their that the direct observation of intact cells, in a formation. Epitaxy is defined as the growth of vitrified state, (it means without fixation process) one crystal on the substrate of another, so there locates and guide the haemozoin crystallization is at least one preferred orientation and a near inside the inner membrane of digestive vacuole, geometrical fit between the contacting surface and preferably occurs in aqueous phase rather lattices41. When lattice-matched “seeds” are pres- than in lipid phase and facing the membrane in ent in solution, the barrier to nucleation can be the common orientation face ×100ý. In addition, significantly reduced, so crystallization occurs in the haemozoin crystals are clearly not engulfed environments that have otherwise not met critical in any lipid sphere; rather, it is proposed that the supersaturating conditions. A similar scene may haemozoin crystals form on an acylglycerol lipid be present for haemozoin and antimalarial drugs. film adsorbed to the inner leaflet of the DV phos- Quinolines also form various complexes with pholipid membrane17-19. soluble haematin, but complexation is insufficient The common coexistence of these crystalline to suppress haem detoxification and is a poor in- species in physiological deposits and the observa- dicator of drug specificity. tion that the nucleus of a stone is often chemically The divergent hypotheses on the inhibition different than the material in subsequent layers of haematin crystallization posit that drugs act

7073 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al. either by the sequestration of soluble haematin by Authors’ Contribution forming non crystallizable complexes or the inhi- J.L. Imbert Palafox designed and directed the project and bition is due to the drug interaction with crystal wrote the final manuscript. L. González Linares made the surfaces. However, drug-crystal interactions are experimental materials and did the Voltammetry assays. not the dominant mechanism of haematin growth V.E. Reyes-Cruz conducted and demonstrated the manage- 42 ment and use of the Voltammeter. A. Arenas Flores con- inhibition . ducted and analyzed the XRD equipment and its respective The redox potential variability from insoluble software. J.E. Bautista Garcia prepared and cultivated the and nonconductive organic molecules, such as Hz insect’s colony and C.M. Gonzalez Alvarez made the spec- and β-Hz in the presence of antimalarial drugs trophotometric assays. M.A. Becerril Flores and J.C. Ruv- has been quantified through an electrochemical alcaba Ledezma read and re-wrote the initial manuscript. All authors read and agree on the submitted version of the array with a carbon paste electrode (CPE). manuscript. Finally, the authors have shown the viabil- ity of conducting quantitative characterization studies of insoluble drugs, such as antimalarial artemether and lumefantrine using cyclic voltam- References metry with carbon paste electrodes in a fast and inexpensive manner; furthermore, this technique 1) Thomas V, Góis A, Ritts B, Burke P, Hänscheid T, Mc- is able to consider the more complex planar aro- Donnell G. A novel way to grow hemozoin-like matic structures and the presence of fluorine and crystals in vitro and Its use to screen for hemozo- in Inhibiting Antimalarial Compounds. PLoS One chlorine, which are very electronegative atoms. 2012; 7: e41006. 2) Slomianny C. Three-dimensional reconstruction of the feeding process of the malaria parasite. Blood Conclusions Cells 1990; 16: 369-378. 3) Rosenthal PJ, Meshnick SR. Hemoglobin catabolism Clinically, the toxicity and side effects of arte- and iron utilization by malaria parasites. Mol Bio- mether and lumefantrine are presents. Some pa- chem Parasitol 1996; 83: 131-139. tients complained of dizziness, headache, nausea, 4) Abu Bakar N, Klonis N, Hanssen E, Chan C, Til- vomiting, abdominal, pain, diarrhoea, and palpi- ley L. Digestive-vacuole genesis and endocyt- tation. A few patients had rashes and others had ic processes in the early intraerythrocytic stages of . J Cell Sci 2010; 123: reticulopenia, leukopenia, and increased SGPT, 441-450. and urea nitrogen, sinus bradycardia, arrhythmia 5) Sullivan DJ Jr, Guzman IY, Goldberg DE. Plas- or premature ventricular beat. To maintain the modium hemozoin formation mediated by his- intracellular redox balance is very important, so tidine-rich proteins. Science 1996; 271: 219- indicate treatments that could potentially alter it, 22. (artemether) or (benflumetol,) should consider 6) Stiebler R, Timma BL, Oliveira PL, Hearne GR, Egan that maybe there will be a imbalance. A better TJ, Oliveira MF. On the physico-chemical and physiological requirements of hemozoin forma- guide for rational application and standardize tion promoted by perimicrovillar membranes in malaria treatment should be include to evaluate Rhodnius prolixus midgut. Insect Biochem Mol the drugs redox potential43. Biol 2010; 40: 284-292. 7) Goldberg DE, Slater AFG, Cerami A, Henderson GB. Hemoglobin degradation in the malaria par- asite plasmodium falciparum: an ordered pro- Conflict of Interest cess in a unique organelle. PNAS 1990; 87: The Authors declare that they have no conflict of interests. 2931-2935. 8) Kollien AH, Schaub GA. The development of Try- panosoma cruzi in Triatominae. Parasitology To- Acknowledgements day 2000; 16: 381-387. To the Ministry of Public Education (SEP-PROMEP: Secre- 9) Caiaffa CD, Stiebler R, Oliveira MF, Lara FA, Pai- taría de la Educación Pública-Programa de Mejora del Pro- va-Silva GO, Oliveira PL. Sn-protoporphyrin inhib- fesorado) for the financial support to Biomedical Research its both degradation and hemozoin forma- Academic Group, in the 2013 call for Consolidate Research tion in Rhodnius prolixus midgut. Insect Biochem Groups. The authors would like to acknowledge and tru- Mol Biol 2010; 40: 855-860. ly thank the collaboration of Yesenia Elizabeth Ruvalcaba 10) Oliveira MF, Silva JR, Dansa-Petretski M, de Souza W, Cobián who holds a B.A in Teaching English as a Foreign Lins U, Braga CMS, Masuda H, Oliveira PL. Haem Language, for her contributions on the revision and trans- detoxification by an insect. Nature 1999; 400: 517- lation of the article. 518.

7074 Redox potential of haem with antimalarials

11) Oliveira MF, Silva JR, Dansa-Petretski M, de Souza and Characterization of Haemozoin and ß-Hae- W, Braga CMS, Masuda H, Oliveira PL. Haemozo- mozoin from Meccus longipennis. Iran J Parasitol in formation in the midgut of the blood-sucking 2019; 14: 59-67. insect Rhodnius prolixus. FEBS Lett 2000; 477: 24) Slater AFG, Cerami A. Inhibition by 95-98. of a novel haem polymerase activity 12) Oliveira MF, d’Avila JCP, Torres CR, Oliveira PL, Tem- in malaria trophozoites. Nature 1992; 355: 167- pone AJ, Rumjanek FD, Braga CMS, Silva JR, Petretski 169. MD, Oliveira MA, de Souza W, Ferreira ST . Haemo- 25) Slater AFG, Swiggard WJ, Orton BR, Flitter WD, zoin in mansoni. Mol Biochem Par- Goldberg DE, Cerami A, Henderson GB. An iron-car- asitol 2000; 111: 217-221. boxylate bond links the heme units of malaria pig- 13) Chen MM, Shi L, Sullivan DJ, Jr. Haemoproteus ment. PNAS 1991; 88: 325-329. and Schistosoma synthesize heme sim- 26) Bendrat K, Berger BJ, Cerami A. Haem polymeriza- ilar to Plasmodium hemozoin and beta-hematin. tion in malaria. Nature 1995; 378: 138-139. Mol Biochem Parasitol 2001; 113: 1-8. 27) Olliaro PL, Goldberg DE. The plasmodium diges- 14) Oliveira MF, Kycia SW, Gomez A, Kosar AJ, Bohle tive vacuole: metabolic headquarters and choice DS, Hempelmann E, Menezes D, Vannier-Santos MA, drug target. Parasitology Today 1995; 11: 294- Oliveira PL, Ferreira ST. Structural and morpho- 297. logical characterization of hemozoin produced 28) Coronado LM, Nadovich CT, Spadafora C. Malari- by and Rhodnius prolixus. al hemozoin: from target to tool. Biochim Biophys FEBS Lett 2005; 579: 6010-6016. Acta 2014; 1840: 2032-2041. Oliveira MF, Timm BL, Machado EA, Miranda K, At- 15) Egan TJ, Chen JY, de Villiers KA, Mabotha TE, tias M, Silva JR, Petretski, de Oliveira MA, de Souza 29) Naidoo KJ, Ncokazi KK, Langford SJ, McNaugh- W, Pinhal NM, Souza JJF, Vugman NV, Oliveira PL. ton D, Pandiancherri S, Wood BR. Haemozoin (be- On the pro-oxidant effects of haemozoin. FEBS ta-haematin) biomineralization occurs by self-as- Lett 2002; 512: 139-144. sembly near the lipid/water interface. FEBS Lett 16) Graça-Souza, AV, Maya-Monteiro C, Paiva-Silva GO, 2006; 580: 5105-5110. Braz GR, Paes MC, Sorgine MH, Oliveira MF, Ol- 30) Vekilov PG, Rimer JD, Olafson K, Ketchum M. Lipid iveira PL . Adaptations against heme toxicity in or aqueous medium for hematin crystallization? blood-feeding arthropods. Insect Biochem Mol Cryst Eng Comm 2015; 17: 7790-7800. Biol 2006; 36: 322-335. 31) Bohlea DS, Kosara AD, Stephens PW. Phase ho- Kapishnikov S, Berthing T, Hviid L, Dierolf M, Men- 17) mogeneity and crystal morphology of the malar- zel A, Pfeiffer F, Als-Nielsen J, Leiserowitz L. Aligned ia pigment ß-hematin. Acta Crystallogr 2002; 58: hemozoin crystals in curved clusters in malari- 1752-1756. al red blood cells revealed by nanoprobe X-ray Kapetanaki S, Varotsis C Fe fluorescence and diffraction. PNAS 2012; 109: 32) . Ferryl-oxo heme interme- 11184-11187. diate in the antimalarial mode of action of arte- misinin. FEBS Lett 2000; 474: 238-241. 18) Kapishnikov S, Weiner A, Shimoni E, Guttmann P, Webster GT, Tilley L, Deed S, McNaughton D, Wood Schneider G, Dahan-Pasternak N, Dzikowski R, Leise- 33) BR rowitz L, Elbaum M. Oriented nucleation of hemo- . Resonance Raman spectroscopy can detect zoin at the digestive vacuole membrane in Plas- structural changes in haemozoin (malaria pig- modium falciparum. PNAS 2012; 109: 11188- ment) following incubation with chloroquine in in- 11193. fected erythrocytes. FEBS Lett 2008; 582: 1087- 1092. 19) K apishnikov S, Weiner A, Shimoni E, Schneider Khandave, SS, Joshi, SS, Sawant, SV and Onk ar, G, Elbaum M, Leiserowitz L 34) . Digestive vacuole SV membrane in plasmodium falciparum-infect- . Evaluation of bioequivalence and car- ed erythrocytes: relevance to templated nucle- dio-hepatic safety of a single dose of fixed ation of hemozoin. Langmuir 2013; 29: 14595- dose combination of artemether and lume- 14602. fantrine. J Bioequivalence Bioavailab 2010; 2: 081-085. 20) Vincent SH. Oxidative effects of heme and porphy- World Health Organization rins on proteins and . Semin Hematol 1989; 35) . Guidelines for the 26: 105-113. treatment of malaria. WHO/Global Malaria Pro- gram. Information Note on Delayed Haemolyt- Rifkind JM, Nagababu E, Ramasamy S, Ravi LB. 21) He- ic Anaemia following Treatment with Artesuna- moglobin redox reactions and oxidative stress. te, 2 October 2013. https://www.who.int/malar- Redox Rep 2003; 8: 234-237. ia/publications/atoz/who_note_delayed_hae- 22) Reyes CVE, Urbano GR, Veloz RMA, Imbert PJL. molytic_anaemia_oct13.pdf?ua=1 [cited 2019, Analysis of the electrochemical reactivity of nat- March 9]. ural hemozoin and β-hemozoin in the presence 36) Loup C, Lelievre J, Benoit-Vical F, Meunier B. Triox- of antimalarial drugs. Electrochim Acta 2011; 56: aquines and heme-artemisinin adducts inhibit the 9762-9768. in vitro formation of hemozoin better than chlo- 23) González LL, Reyes CVE, Velóz RMA, Urbano RG, roquine. Antimicrobial Agents Chem 2007; 51: Imbert PJL, Cobos MJA. New Method of Production 3768-3770.

7075 J.L. Imbert Palafox, L. González Linares, V.E. Reyes-Cruz, M.A. Becerril Flores, et al.

37) Chen Y, Zheng J, Zhu S, Chen H. Evidence for he- 40) Lonsdale K. Epitaxy as a growth factor in uri- min inducing the cleavage of peroxide bond or ar- nary calculi and gallstones. Nature 1968; 217: temisinin (Qinghaosu): cyclic voltammetry and in 56-58. situ FT IR spectroelectrochemical studies on the 41) Frincu, MC, Fogarty CE, Swift, JA. Epitaxial rela- reduction mechanism of artemisinin in the pres- tionships between uric acid crystals and miner- ence of . Electrochim Acta 1999; 44: 2345- al surfaces: a factor in urinary stone formation. 2350. Langmuir 2004; 20: 6524-6529. 38) Klonis N, Darren JC, Tilley L. Iron and heme 42) Olafson KN, Nguyen TQ, Rimer JD, Vekilov PG. Anti- metabolism in Plasmodium falciparum and the malarials inhibit hematin crystallization by unique mechanism of action of artemisinins. Curr Opin drug-surface site interactions. PNAS 2017; 114: Microbiol 2013; 16: 722-727. 7531-7536. 39) Ginsburg H. Abundant proton pumping in Plasmo- 43) Chang Ch. Development of antimalarial drugs and dium falciparum, but why? Trends Parasitol 2002; their application in China: a historical review. In- 18: 483-486. fect Dis Poverty 2014; 3: 9.

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