Interaction of quinolines and artemisinin based antimalarials drugs with ferriprotoporphyrin IX

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4.4 Binding reaction of hemin with artemisinin drugs.

4.4.1 Binding reaction of hemin with artemisinin, dihydroartemisinin and artesunate in water-DMSO mixture.

Figure 4-4 Spectroscopic changes of Fe(III)PPIX at Soret band (402 nm) as a function of increasing concentartions of drug :

0 (a), 1.4 (b), 2.4 (c), 70 (d), 100 (e), 160 M of DHA (f) after 10 hours,

0 (a), 6 (b), 14 (c), 24 (d), 50 (e), 120 M of DHA (f) after 24 hours,

0 (a), 3 (b), 10 (c), 50 (d), 120 (e), 160 M of AST (f) after 24 hours,

0 (a), 14 (b), 18 (c), 70 (d), 100 (e), 160 M of ART (f) after 24 hours with 40 % DMSO aqueous as solvent and 0.1 M Tris-HCl buffer (pH 9).

Typical spectral changes observed upon hemin-sesquiterne endoperoxide lactone-based drugs complexation are shown in figure 4-4. They are similar to those observed on hemin-artesunate and hemin-artemisinin interactions in other mediums (Berman and Adams, 1997, Bilia et al., 2002).

The wavelength of 402 nm was selected to determine the constants of complexation. Complex formation was monitored by the decline in Absorbance (402 nm) whereas absorbance of hemin, incubated with drug, was essentially unchanged after 24 hrs.

The spectral changes resulting from addition of quinoline based drug to Fe(III)PPIX and those resulting from artemisinin drug-hemin interaction are different. The latter shows a substantial hypochromic effect of the hemin Soret band at 402 nm, accompanied with a red shift of 2-5 nm, whereas in bonding of hemin with quinoline, the peak maximums are still at about 396nm. It means that artemisinin and derivatives are auxochromes because their reaction to hemin leads to red shift, which is clearly apparent after 10 hours. The new peak around 407nm may belong to the complex.

About the decrease in absorbance of the Soret band of hemin, two possible processes can be envisaged which would lead to these changes. Either addition of micromolar concentrations of drug induces aggregation of hemin or the changes reflect drug association with hemin. While a large decrease in the absorbance of the Soret band is often an indication of aggregation, equally large decreases are caused by formation of p-p (donor-acceptor) complexes (Egan et al., 1997). Generally, spectral changes of iron porphyrins in the visible region vary depending on the conditions of solvents and pH and the nature of interacting species. The decrease of hemin absorbance is dependent on the drug concentration. A dilution experiment showed that Beer's law is strictly adhered to in the presence of 306 M drug and in the concentration range 0.99 to 48 M Fe(III)PPIX thus providing no evidence of hemin aggregation in this concentration range. Thus, the most reasonable explanation for these spectral changes is the presence of drug-hemin association. There is another feature on the titration curves of artemisinin and derivatives, i.e. a quite regular spectral changes over time with a well-defined isosbetic point around 325 nm. This behaviour is suggestive of an equilibrium between two species. These spectral modifications suggest a progressive disruption of delocalised -electron system of the hemin tetrapyrrole ring. In addition, another band at 290 nm with low intensity was also observed, the origin of that is unclear at this time. Without drug, the band of hemin was still unchanged after 24hours.

Typical kinetic analysis of the mixture of hemin plus artesiminin and derivatives are shown in Figure 9. For example, for 24 M of drug (at pH 9), the residual intensity of the soret band, compared to the control (hemin, 100%), is:

after 10h, 84% (ART), 83 % (AST), 59% (DHA)

after 24h, 66% (ART), 57 % (AST), 37 % (DHA)

Figure 4-5 Variation of absorbance at 402 nm observed when hemin is titrated with artemisinin derivatives (24 M) within 60 hours of observation in 0.1 M Tris buffer (pH 9, 37^oC).

At 37^oC, the curves of absorbance-time relationship for the complexation showed that the time taken to reach absorbance minimun is shorter for DHA. It was found that the reaction rate is faster with dihydroartemsinin, followed by artesunate than artemisinin did.

Some typical titration curves have been shown in Figures 4-6 and 4-7.

Figure 4-6 Variation in absorbance of hemin at 402 nm as function of total concentartion of artemisinin -based drugs after 10 and 24 hours of incubation at 37^oC (pH 9).

The solid lines are best fits of the data to a 1:1 association model obtained by nonlinear least-squares analysis. Association constants for all of the compounds are summarized in Table 4-2. It should be pointed out that the conditions under which the association constants have been measured are of course quite different from those in food vacuole of the parasite (pH~ 5.4, acidic aqueous medium). These are only apparent or conditional association constants. It can be seen that the extinction of the hemin solution decreases with increasing in total drug concentration. Generally, at drug concentrations smaller than 50 M (molar ratio hemin / drug =1:17), there is a significantly and progressive decay in Soret absorbance, more perceptible in the case of DHA. At highest concentrations of drug (molar ratio hemin / drug 1: 25), this decrease is less significant, indicating the saturation of drug binding to heme. Correlation coefficients of the non linear fittings are better than 0.9, which implies that the titration curves can be well describes by the 1:1 association between drug and Fe(III)PPIX. Similar variation in absorbance of hemin at 402 nm as function of total drug concentration has been obtained at pH 7.4 and the results are also consistent with the formation of 1:1 complex. Values of binding constants at related pH obtained from these titration curves are summarized in Table 4-2.

Figure 4-7 Variation in absorbance of hemin at 402 nm as function of total concentration of artemisinin-based drugs after 24 hours of incubation at 37^oC (pH 9).

Table 4-2 Binding constant of hemin-drug complexes in 40 % DMSO, pH 9,

0.1M Tris-HCl buffer

4.4.2 Binding reaction of hemin with artesunate in water-propylene glycol mixture.

10 hrs

Because of insolubility of artesiminin and dihydroartemisinin in water-propylene glycol mixture, we only reported the bonding reaction of hemin with artesunate in this medium. Figure 4-8 shows typical spectral changes observed when hemin is titrated with artesunate. The wavelength of 398nm was selected to determine the constants of complexation. The main spectroscopic changes consist of a red shift of about 3-4 nm and a decrease in intensity of the Soret band. In addition, the spectra of artesunate showed another absorption peak at 290 nm, already observed in DMSO aqueous medium. The titration curves showed, in the range 250-650 nm, a quite regular spectral change over time with a well-defined isosbetic point around 330 nm.

Figure 4-8 Spetroscopic changes of Fe(III)PPIX Soret band (398nm) as a function of increasing concentartions of drug :

AST (10 h): 0 (a), 1.4 (b), 2.4 (c), 70 (d), 120 (e), 160 M of AST (f) after 10 hours,

AST (24 h): 0 (a), 6 (b), 14 (c), 50 (d), 70 (e), 160 M of AST (f) after 24 hours with water-propylene glycol mixture as solvent and 0.1 M Tris-HCl buffer (pH 9).

24 hrs

Similar variation in absorbance of hemin at 402 nm as function of total drug concentration has been obtained at pH 7.4 and the results are also consistent with the formation of 1:1 complex as shown in Figure 4-9.

Figure 4-9 Variation in absorbance of hemin at 398 nm as function of total concentration of artesunate (pH 9) after 10 or 24 hours of incubation at 37^oC.

Values of binding constants at related pH obtained from these titration curves are summarized in Table 4-3.

Table 4-3 Binding constant of hemin-drug complexes in water-

propylene glycol at pH 9, in 0.1M Tris-HCl buffer.

In the light of above results, we observed that fixing the concentration of hemin at 3 M and varying drug from 0 to 200 M, the Soret band of hemin decreased. Along with the increase in incubation time and drug concentration, it arises a new peak centred at 290 nm, indicating that the peak belongs to artemisinin compounds. The origin of which is unclear at this time but we suggested that it is a decomposed product of artemisinin as a result of reaction between hemin and drugs. The spectral propriety of this product with strong absorbance is similar to that observed by Green and co-workers (Green et al., 2000) caused by alkali decomposition of ART. They called it ARTS 290 and have described this compound as an enolate/carboxylate. Enolates readily couple to electrophilic dyes such as diazonium salts (Zollinger, 1991), therefore it is possible that

Figure 4-10. Spectral changes of the alkali decomposition product of 0.001 M artesunate to 1 M NaOH after 20 min.

the alkali decomposition product of ART may be similar in structure. In order to verify this assertion, we mixed 0.001 M of AST with 1M of NaOH at room temperature for 20 min. We observed exactly a peak at 290 nm, which has the same characteristic of ART 290 as shown in figure 4-10.

It has been postulated that the interaction of artemisininn with the target heme or hemin in vivo proceeds through the complexes, in which the peroxide bridge of trioxanes coordinates with the iron of hemin, leading to the formation of drug-hemin adduct.

This long-lived intermediate has the spectral characteristics of a heme peroxide (Berman and dams, 199)] compound analogous, in which the hemin is covalently bound to artemisinin via a iron-oxygen-carbon bond, formed by attack of the iron center on the endoperoxide group of drug. This metastable species gradually decays over the ensuing (24 hours for drug highest concentration or 48 hours for drug lowest concentration), to final product devoid of Soret absorbance, indicative of disruption of the heme tetrapyrrole ring system.

Based on data in Tables 4-2 and 4-3, AST should have greater affinity with hemin, then DHA and finally ART. In regard to their constants bindings values, AST and DHA should have the highest efficiency than ART. Practically, it has been shown that artemisinin derivates are at least five times higher than artemisinin. In regard to their molecular structure (Scheme 4-2) it is probably that succininic group of artesunate interacts with hemin propionic group via intra-molecular hydrogen bonding and make this bonding stronger than that of artemisinin does. In addition, in vivo metabolism, artemisinin and derivatives (artesunate, arteether, artemether) are hydrolyzed mainly to the active derivative DHA. DHA, a first generation analogous and metabolite of ART, is known with artesunate to be therapeutically more active than artemisinin (China Cooperative Research Group, 1982, Kamchonwongpaisan and Meshnick, 1996). It was demonstrated too by analyzing the reaction products of DHA plus hemoglobin and of DHA plus globin, DHA reacted rapidly with hemoglobin (as shown in spectral changes in Figure 4-4) but not with globin. This may be a major reason for fast action of these drugs compared to quinoline antimalarials. This is supported equally by the binding constants values (calculated in water propylene glycol mixture) of artesunate (0.43) which are higher than chloroquine (0.17) and quinine (0.05) do. In pratical applications, a similar trend is observed because artemisinin compounds are now at the top of list of new antimalarial drugs in terms of efficacy, rapidity of action and first order pharmacokinetic against chloroquine -resistant strains of plasmodium.

Our results showed that the physical property of the medium of reaction affects the rate of interaction between hemin and drug. We supposed that because of the weak dielectric constants of DMSO and PREG compared to that of water, the bonding constant values of the related artemisinin drug would be more greater in aqueous medium.

Water solubility of artesunate may be due to the presence of succinc group, which increases its polarity make it distinct from other derivatives. DMSO which is highly polar aprotic solvent, with negligible hydrogen bond donation compared to water, may be solvate artesunate anion only through ion-dipole interactions. Less an anionic ligand is solvated, the more readily the complex is formed. From literature, it is known aprotic dipolar solvent permit good complexation while allowing also reasonable solubility of the reacting species (Marcus, 1999). Then, artesunate could coordinate strongly with hemin. The structure of artemisinin-hemin complexes are not well known, may be they are transfert-charge complexes.

In water-DMSO mixture, the binding of hemin to artesunate is better than that in water-propylene glycol. In fact, the propylene glycol, with high viscosity, may be decrease significantly the reactivity of artesunate in this medium than water-dimethyl sulfoxide does.

The interaction of hemin and artemisinin compounds is supported too by molecular mechanic calculations. The molecular electrostatic potentiel (MEP) of artemisinin showed that it has two main region with negative potential which may interact with the positively charged iron ion of the heme, one of these regions covers endoperoxide oxygen O1 and O2 whereas the other encompasses the carboxyl oxygen atom 011 and O14 (Scheme 2-2). This suggests that electrostatic interaction between artemisinin and heme can involve large part of the artemisinin structure. However, the carboxyl and peroxide oxygen atoms, which been the most electronegative part of molecule, will probably be more competitive for complexation (Shukla et al., 1995). In addition, Tonmunphen and co-workers reported automated molecular docking of artemisinin to heme. The docking between artemisinin and heme indicated that artemisinin approaches heme by pointing O1 at the peroxide linkage toward the iron center, a mechanism that is controlled by steric hindrance whith a lowest binding energy of -33.13 kcal.mol^-1 (Tonmunphen et al., 2001, Taranto et al., 2001). This is probably due to pyramidal geometry of hemin (where iron is out of the main plan formed by the four pyrrole N atoms) which facilitates the approach of Fe to the endoperoxide moiety.