Exploring the repositioning of the amodiaquine as potential drug against visceral leishmaniasis: The in vitro effect against Leishmania infantum is associated with multiple mechanisms, involving mitochondria dysfunction, oxidative stress and loss of cell cycle control.

Visceral leishmaniasis (VL) is a progressive, debilitating, and potentially fatal disease if left untreated. As a neglected tropical disease (NTD), the available treatment is restricted to a few drugs, which typically must be administered over a long period but are associated with serious adverse effects and have variability in efficacy. In this sense, drug repositioning has been considered an excellent strategy in the search for alternative treatments, especially in reducing the time and cost of the research. In this work, the repositioning potential of amodiaquine (AQ), a well-known antimalarial drug, was investigated for the treatment of VL. AQ showed significant and selective activity against promastigotes (IC50 = 11.6 µg/mL) and intracellular amastigotes (IC50 = 2.4 µg/mL) of L. infantum, being 10 times more destructive to the intracellular parasites than the host cell. In addition, pre-treatment of macrophages with AQ caused a significant reduction in the infection index, indicating a prophylactic effect of this drug. SEM images showed that AQ induces strong shape alterations of the promastigotes with an increase in cell volume with rounding and ribbing (vertical ridges), as well as a shortened flagellum. In addition, AQ induced depolarization of the ΔΨm, an increase in ROS and neutral lipids levels, and changes in the cell cycle in promastigotes, without alterations to the permeability of the parasite plasma membrane. L. infantum-infected macrophages treated with AQ induced the activation of oxidative mechanisms by infected host cells, with an increase in ROS and NO levels. Finally, in vitro interactions between AQ and miltefosine were found to have an additive effect in both biological stages of the parasite, with the ∑FIC50 values ranging from 0.74 to 1.16 µg/mL and 0.54-1.11 µg/mL for promastigotes and intracellular amastigotes, respectively. Overall, these data highlight the utility of drug repurposing and indicate future preclinical testing for AQ itself or in combination as a potential VL treatment.

Assessment of NR4A Ligands That Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1.

Nurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a drug target for neurological disorders including Alzheimer's and Parkinson's diseases. Previous studies identified small-molecule NR4A nuclear receptor modulators, but it remains unclear if these ligands affect transcription via direct binding to Nurr1. We assessed 12 ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and the ability to bind the Nurr1 ligand-binding domain (LBD). Protein NMR structural footprinting data show that amodiaquine, chloroquine, and cytosporone B bind the Nurr1 LBD; ligands that do not bind include C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), and three high-throughput screening hit derivatives. Importantly, ligands that modulate Nurr1 transcription also show Nurr1-independent effects on transcription in a cell type-specific manner, indicating that care should be taken when interpreting the functional response of these ligands in transcriptional assays. These findings should help focus medicinal chemistry efforts that desire to optimize Nurr1-binding ligands.

Beneficial Effect of Chloroquine and Amodiaquine on Type 1 Diabetic Tubulopathy by Attenuating Mitochondrial Nox4 and Endoplasmic Reticulum Stress.

BACKGROUND: Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury. METHODS: Human renal proximal tubular epithelial cells (hRPTCs) were cultured in high-glucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks. RESULTS: CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment. CONCLUSION: We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD.

Synthesis of stable isotope-labeled chloroquine and amodiaquine and their metabolites.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism.

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer's disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aß), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aß release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aß peptide release (in the same range compared to AQ), and a 3-10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100 µM.

Deconstructing Quinoline-Class Antimalarials to Identify Fundamental Physicochemical Properties of Beta-Hematin Crystal Growth Inhibitors.

A versatile approach to control crystallization involves the use of modifiers, which are additives that interact with crystal surfaces and alter their growth rates. Elucidating a modifier's binding specificity to anisotropic crystal surfaces is a ubiquitous challenge that is critical to their design. In this study, we select hematin, a byproduct of malaria parasites, as a model system to examine the complementarity of modifiers (i.e., antimalarial drugs) to ß-hematin crystal surfaces. We divide two antimalarials, chloroquine and amodiaquine, into segments consisting of a quinoline base, common to both drugs, and side chains that differentiate their modes of action. Using a combination of scanning probe microscopy, bulk crystallization, and analytical techniques, we show that the base and side chain work synergistically to reduce the rate of hematin crystallization. In contrast to general observations that modifiers retain their function upon segmentation, we show that the constituents do not act as modifiers. A systematic study of quinoline isomers and analogues shows how subtle rearrangement and removal of functional moieties can create effective constituents from previously ineffective modifiers, along with tuning their inhibitory modes of action. These findings highlight the importance of specific functional moieties in drug compounds, leading to an improved understanding of modifier-crystal interactions that could prove to be applicable to the design of new antimalarials.

Human glutathione S-transferases- and NAD(P)H:quinone oxidoreductase 1-catalyzed inactivation of reactive quinoneimines of amodiaquine and N-desethylamodiaquine: Possible implications for susceptibility to amodiaquine-induced liver toxicity.

Amodiaquine (AQ), an antimalarial drug, widely prescribed in endemic areas of Africa and Asia, is used in combination with artesunate as recommended by the WHO. However, due to its idiosyncratic hepatotoxicity and agranulocytosis, the therapeutic use has been discontinued in most countries. Oxidative bioactivation to protein-reactive quinonimines (QIs) by hepatic cytochrome P450s and myeloperoxidase (MPO) have been suggested to be important mechanisms underlying AQ idiosyncratic toxicity. However, the inactivation of the reactive QIs by detoxifying enzymes such as human glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreducatase 1 (NQO1) has not been characterized yet. In the present study, the activities of 15 recombinant human GSTs and NQO1 in the inactivation of reactive QIs of AQ and its pharmacological active metabolite, N-desethylamodiaquine (DEAQ) were investigated. The results showed that GSTP1-1, GSTA4-4, GSTM4-4, GSTM2-2 and GSTA2-2 (activity in decreasing order) were active isoforms in catalyzing GSH conjugation of reactive QIs of AQ and DEAQ. Additionally, NQO1 was shown to inactivate these QIs by reduction. Simulation of the variability of cytosolic GST-activity based on the hepatic GST contents from 22 liver donors, showed a large variation in cytosolic inactivation of QIs by GSH, especially at a reduced GSH-concentration. In conclusion, the present study demonstrates that a low hepatic expression of the active GSTs and NQO1 may increase the susceptibility of patients to AQ idiosyncratic hepatotoxicity.

The effect of milk thistle (Silybum marianum) and its main flavonolignans on CYP2C8 enzyme activity in human liver microsomes.

Milk thistle is a widely-consumed botanical used for an array of purported health benefits. The primary extract of milk thistle is termed silymarin, a complex mixture that contains a number of structurally-related flavonolignans, the flavonoid, taxifolin, and a number of other constituents. The major flavonolignans present in most extracts are silybin A, silybin B, isosilybin A and isosilybin B, silydianin, silychristin and isosilychristin. Silymarin itself has been reported to inhibit CYP2C8 activity in vitro, but the effect of the individual flavonolignans on this enzyme has not been studied. To investigate the effects of milk thistle extract and its main flavonolignans (silybin A, silybin B, isosilybin A and isosilybin B) on CYP2C8 activity at relevant concentrations, the effect of milk thistle extract and the flavonolignans on CYP2C8 enzyme activity was studied in vitro using human liver microsomes (HLM) incorporating an enzyme-selective substrate for CYP2C8, amodiaquine. Metabolite formation was analyzed using liquid chromatography-tandem mass spectrometry (LC/MS-MS). The concentration causing 50% inhibition of enzyme activity (IC50) was used to express the degree of inhibition. Isosilibinin, a mixture of the diastereoisomers isosilybin A and isosilybin B, was found to be the most potent inhibitor, followed by isosilybin B with IC50 values (mean ± SE) of 1.64 ± 0.66 µg/mL and 2.67 ± 1.18 µg/mL, respectively. The rank order of observed inhibitory potency after isosilibinin was silibinin > isosilybin A > silybin A > milk thistle extract > and silybin B. These in vitro results suggest a potentially significant inhibitory effect of isosilibinin and isosilybin B on CYP2C8 activity. However, the observed IC50 values are unlikely to be achieved in humans supplemented with orally administered milk thistle extracts due to the poor bioavailability of flavonolignans documented with most commercially available formulations.

Influence of LAR and VAR on Para-Aminopyridine Antimalarials Targetting Haematin in Chloroquine-Resistance.

Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aromatic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol substituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logResistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

Interactions of the antimalarial amodiaquine with lipid model membranes.

A detailed molecular description of the mechanism of action of the antimalarial drug amodiaquine (AQ) is still an open issue. To gain further insights on that, we studied the interactions of AQ with lipid model membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) by spin labeling electron spin resonance (ESR) and differential scanning calorimetry (DSC). Both techniques indicate a coexistence of an ordered DPPS-rich domain with a disordered DPPC-rich domain in the binary DPPC/DPPS system. We found that AQ slightly lowered the melting transition temperatures associated to both domains and significantly increased the enthalpy change of the whole DPPC/DPPS phase transition. DSC and ESR data also suggest that AQ increases the number of DPPC molecules in the DPPC-rich domains. AQ also causes opposing ordering effects on different regions of the bilayer: while the drug increases the ordering of the lipid acyl chains from carbon 7 to 16, it decreases the order parameter of the lipid head group and of carbon 5. The gel phase was mostly affected by the presence of AQ, suggesting that AQ is able to influence more organized lipid domains. Moreover, the effects of AQ and cholesterol on lipid acyl chain ordering and mobility were compared at physiological temperature and, in a general way, they are similar. Our results suggest that the quinoline ring of AQ is located completely inside the lipid bilayers with its phenol ring and the tertiary amine directed towards the head group region. The nonspecific interaction between AQ and DPPC/DPPS bilayers is a combination of electrostatic and hydrophobic interactions.

Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes.

1. Herbal supplements widely used in the US were screened for the potential to inhibit CYP2C8 activity in human liver microsomes. The herbal extracts screened were garlic, echinacea, saw palmetto, valerian, black cohosh and cranberry. N-desethylamodiaquine (DEAQ) and hydroxypioglitazone metabolite formation were used as indices of CYP2C8 activity. 2. All herbal extracts showed inhibition of CYP2C8 activity for at least one of three concentrations tested. A volume per dose index (VDI) was calculated to determine the volume in which a dose should be diluted to obtain IC50 equivalent concentration. Cranberry and saw palmetto had a VDI value > 5.0 l per dose unit, suggesting a potential for interaction. 3. Inhibition curves were constructed and the IC50 (mean ± SE) values were 24.7 ± 2.7 µg/ml for cranberry and 15.4 ± 1.7 µg/ml for saw palmetto. 4. The results suggest a potential for cranberry or saw palmetto extracts to inhibit CYP2C8 activity. Clinical studies are needed to evaluate the significance of this interaction.

Involvement of myeloperoxidase and NADPH oxidase in the covalent binding of amodiaquine and clozapine to neutrophils: implications for drug-induced agranulocytosis.

Amodiaquine (AQ) and clozapine (CLZ) are associated with a relatively high incidence of idiosyncratic agranulocytosis, a reaction that is suspected to involve covalent binding of reactive metabolites to neutrophils. Previous studies have shown that both AQ and CLZ are oxidized to reactive intermediates in vitro by activated neutrophils or by the combination of hydrogen peroxide and myeloperoxidase (MPO). Neutrophil activation leads to an oxidative burst with activation of NADPH oxidase and the production of hydrogen peroxide. However, the importance of this pathway in covalent binding in vivo has not been examined. In this study, we found that the binding of both AQ and CLZ to neutrophils from MPO knockout mice ex vivo decreased approximately 2-fold compared to neutrophils from wild-type mice, whereas binding to activated neutrophils from gp91 knockout (NADPH oxidase null) mice decreased 6-7-fold. When the AQ studies were performed in vivo, again the binding was decreased in MPO knockout mice to about 50% of the binding in wild-type mice; however, covalent binding was significant in the absence of MPO. Surprisingly, there was no significant decrease in covalent binding of AQ to neutrophils in vivo in gp91 knockout mice. In addition, there was extensive binding of AQ to many types of bone marrow cells and to peripheral lymphocytes. These results indicate that MPO is not the only neutrophil enzyme involved in the oxidation of AQ and that NADPH oxidase is not the major source of peroxide. There was also no decrease in AQ binding to neutrophils in COX-1 or COX-2 knockout mice. We were not able to readily reproduce the AQ in vivo studies with CLZ because of its acute toxicity in mice. These are the first studies to examine the enzymes involved in the bioactivation of AQ by neutrophils in vivo.

Heat stabilization of blood spot samples for determination of metabolically unstable drug compounds.

BACKGROUND: Sample stability is critical for accurate analysis of drug compounds in biosamples. The use of additives to eradicate the enzymatic activity causing loss of these analytes has its limitations. RESULTS: A novel technique for sample stabilization by rapid, high-temperature heating was used. The stability of six commercial drugs in blood and blood spots was investigated under various conditions with or without heat stabilization at 95°C. Oseltamivir, cefotaxime and ribavirin were successfully stabilized by heating whereas significant losses were seen in unheated samples. Amodiaquine was stable with and without heating. Artemether and dihydroartemisinin were found to be very heat sensitive and began to decompose even at 60°C. CONCLUSION: Heat stabilization is a viable technique to maintain analytes in blood spot samples, without the use of chemical additives, by stopping the enzymatic activity that causes sample degradation.

In-vitro inhibitory effect of Tualang honey on cytochrome P450 2C8 activity.

OBJECTIVES: To investigate the effect of Tualang honey on cytochrome P450 2C8 (CYP2C8) activity in vitro using an amodiaquine N-desethylase assay. METHODS: CYP2C8 and NADPH cytochrome P450 reductase was cotransformed, expressed and harvested. The incubation assay contained expressed proteins, MgCl(2), NADP, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, potassium phosphate buffer, and amodiaquine. The rate of conversion of amodiaquine to desethylamodiaquine, the metabolite, was determined using a high performance liquid chromatography (HPLC) method. The inhibition parameters, IC50 (concentration of inhibitor causing 50% inhibition of original enzyme activity) and apparent inhibition constant (K(i) ) values were determined. KEY FINDINGS: The recombinant proteins were successfully expressed and used to investigate the effect of Tualang honey on CYP2C8 activity. The activity was measured by the rate of metabolism of amodiaquine to desethylamodiaquine determined using a successfully developed HPLC method. Kinetic parameters as determined by nonlinear least-squares regression and evaluated with Aikeike's goodness of fit criteria revealed that Tualang honey competitively inhibited CYP2C8 activity in a dose-dependent manner. Maximum inhibition of 80% occurred at 0.01% honey. The IC50 and K(i) values were (10.0 ± 3.0) × 10(-3) % and (5.1 ± 0.5) × 10(-3) % w/v, respectively. CONCLUSIONS: This study has provided evidence for the in vitro inhibition of CYP2C8-mediated amodiaquine N-desethylase activity by Tualang honey. It revealed that honey, through this inhibition, may have the potential to cause in-vivo drug-food interaction with drugs metabolized by CYP2C8.

Confirmation of Frm2 as a novel nitroreductase in Saccharomyces cerevisiae.

Nitroreductases comprise a group of FMN- or FAD-dependent enzymes that reduce nitrosubstituted compounds by using NAD(P)H, and are found in bacterial species and yeast. Although there is little information on the biological functions of nitroreductases, some studies suggest their possible involvement in oxidative stress responses. In the yeast Saccharomyces cerevisiae, a putative nitroreductase protein, Frm2, has been identified based on its sequence similarity with known bacterial nitroreductases. Frm2 has been reported to function in the lipid signaling pathway. To study the functions of Frm2, we measured the nitroreductase activity of purified Frm2 on 4-nitroquinoline-N-oxide (4-NQO) using NADH. LC-MS analysis of the reaction products revealed that Frm2 reduced NQO into 4-aminoquinoline-N-oxide (4-AQO) via 4-hydroxyaminoquinoline (4-HAQO). An Frm2 deletion mutant exhibited growth inhibition in the presence of 4-NQO. Thus, in this study, we demonstrate a novel nitroreductase activity of Frm2 and its involvement in the oxidative stress defense system.

Investigation of the interaction between amodiaquine and human serum albumin by fluorescence spectroscopy and molecular modeling.

The interaction of amodiaquine (AQ) with human serum albumin (HSA) has been studied by fluorescence spectroscopy. Based on the sign and magnitude of the enthalpy and entropy changes (ΔH(0) = -43.27 kJ mol(-1) and ΔS(0) = -50.03 J mol(-1) K(-1)), hydrogen bond and van der Waals forces were suggested as the main interacting forces. Moreover, the efficiency of energy transfer and distance between HSA and acceptor AQ was calculated. Finally, the binding of AQ to HSA was modeled by molecular docking and molecular dynamic simulation methods. Excellent agreement was found between the experimental and theoretical results. Both experimental results and modeling methods suggested that AQ binds mainly to the sub-domain IIA of HSA.

Distribution of human CYP2C8*2 allele in three different African populations.

BACKGROUND: The aim of this study was to investigate cytochrome P450 2C8*2 (CYP2C8*2) distribution and allele frequency in three populations from West and East Africa exposed to Plasmodium falciparum malaria. CYP2C8 enzyme is involved in the metabolism of the anti-malarials amodiaquine and chloroquine. The presence of the CYP2C8*2 defective allele has been recently associated to higher rate of chloroquine-resistant malaria parasites. METHODS: A total of 503 young subjects were genotyped for the single nucleotide polymorphism rs11572103 (A/T). Eighty-eight were from southern Senegal, 262 from eastern Uganda and 153 from southern Madagascar. The PCR-RFLP technique was used to discriminate the wild-type (A) from the defective allele (T). RESULTS: A CYP2C8*2 (T) allele frequency of 0.222 ± 0.044 was detected in Senegal, 0.105 ± 0.019 in Uganda and 0.150 ± 0.029 in Madagascar. CONCLUSIONS: This study demonstrated that CYP2C8*2 allele is widespread in Africa. This allele occurs at different frequency in West and East Africa, being higher in Senegal than in Uganda and Madagascar. These data indicate that an important fraction of the populations analysed has a decreased enzymatic activity, thus being at higher risk for drug accumulation with two possible consequences: i) an exacerbation of drug-associated adverse side effects; ii) an increase of drug-resistance selection pressure on P. falciparum parasites.

A ferrocene-based reagent for the conjugation and quantification of reactive metabolites.

In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma-mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.