Discovery and Mechanistic Analysis of Structurally Diverse Inhibitors of Acetyltransferase Eis among FDA-Approved Drugs.

Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis-proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.

OCT1 Deficiency Affects Hepatocellular Concentrations and Pharmacokinetics of Cycloguanil, the Active Metabolite of the Antimalarial Drug Proguanil.

Cycloguanil, the active metabolite of proguanil, acts on malaria schizonts in erythrocytes and hepatocytes. We analyzed the impact of the organic cation transporter OCT1 on hepatocellular uptake and pharmacokinetics of proguanil and cycloguanil. OCT1 transported both proguanil and cycloguanil. Common variants OCT1*3 and OCT1*4 caused a substantial decrease and OCT1*5 and OCT1*6 complete abolishment of proguanil uptake. In 39 healthy subjects, low-activity variants OCT1*3 and OCT1*4 had only minor effects on proguanil pharmacokinetics. However, both, cycloguanil area under the time-concentration curve and the cycloguanil-to-proguanil ratio were significantly dependent on number of these low-functional alleles (P = 0.02 for both). Together, CYP2C19, CYP3A5, OCT1 polymorphisms, and sex accounted for 61% of the variation in the cycloguanil-to-proguanil ratio. Most importantly, in vitro OCT1 inhibition caused a fivefold decrease of intracellular cycloguanil concentrations in primary human hepatocytes. In conclusion, OCT1-mediated uptake is a limiting step in bioactivation of proguanil, and OCT1 polymorphisms may affect proguanil efficacy against hepatic malaria schizonts.

Application of molecular docking and PSO-SVR intelligent approaches in antimalarial activity prediction of enantiomeric cycloguanil analogues.

A series of antifolate compounds, i.e. 1-(4-chlorophenyl)-6,6-dimethyl-1,3,5-triazine-2,4-diamine, or cycloguanil analogues, have shown effective inhibiting properties against Plasmodium falciparum dihydrofolate reductase (PfDHFR). In this work, the stereoselectivity of PfDHFR to the R and S enantiomer of cycloguanil analogues was obtained from molecular docking calculations and integrated into QSAR study to obtain a more accurate prediction model. Results indicate that PfDHFR can bind to cycloguanil analogues in the R and S enantiomers. Cycloguanil analogues with alkyl chain substituent prefer the R enantiomer over S because they do not experience steric hindrance with the Phe58 side chain, while cycloguanil analogues with phenol chain substituent prefer the S enantiomer over R because they do not experience steric hindrance with Leu46 and Met55 side chains. Particle swarm optimization and support vector regression were used to select relevant descriptors and generate the effective prediction model, with a high statistical significance level (r2training = 0.941; r2test = 0.884).

Proguanil and cycloguanil are organic cation transporter and multidrug and toxin extrusion substrates.

BACKGROUND: Malaria, HIV/AIDS, and tuberculosis endemic areas show considerable geographical overlap, leading to incidence of co-infections. This requires treatment with multiple drugs, potentially causing adverse drug-drug interactions (DDIs). As anti-malarials are generally positively charged at physiological pH, they are likely to interact with human organic cation transporters 1 and 2 (OCT1 and OCT2). These transporters are involved in the uptake of drugs into hepatocytes and proximal tubule cells for subsequent metabolic conversion or elimination. This efflux of cationic drugs from hepatocytes and proximal tubule cells into bile and urine can be mediated by multidrug and toxin extrusion 1 and 2-K (MATE1 and MATE2-K) transporters, respectively. METHODS: Here, the interaction of anti-malarials with these transporters was studied in order to predict potential DDIs. Using baculovirus-transduced HEK293 cells transiently expressing human OCT1, OCT2, MATE1 and MATE2K uptake and inhibition was studied by a range of anti-malarials. RESULTS: Amodiaquine, proguanil, pyrimethamine and quinine were the most potent inhibitors of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) transport, a known substrate of OCT1/2, resulting in half maximal inhibitory concentrations (IC50) of 11, 13, 1.6, and 3.4 µM, respectively. Only quinine had a drug-drug index higher than the cut-off value of 0.1 for OCT2, therefore, in vivo pharmacokinetic studies focusing on DDIs involving this compound and other OCT2-interacting drugs are warranted. Furthermore, proguanil appeared to be a substrate of OCT1 and OCT2 with affinities of 8.1 and 9.0 µM, respectively. Additionally, MATE1 and MATE2-K were identified as putative transport proteins for proguanil. Finally, its metabolite cycloguanil was also identified as an OCT1, OCT2, MATE1 and MATE2-K substrate. CONCLUSION: Anti-malarials can reduce OCT1 and OCT2 transport activity in vitro. Furthermore, proguanil and cycloguanil were found to be substrates of OCT1, OCT2, MATE1 and MATE2-K, highlighting the importance of these transporters in distribution and excretion. As these compounds shares substrate overlap with metformin DDIs can be anticipated during concurrent treatment.

Microbial Metabolism of Atovaquone and Cytotoxicity of the Produced Phase I Metabolite.

BACKGROUND AND OBJECTIVES: Atovaquone is a hydroxynaphthoquinone with selective action in the mitochondrial respiratory chain of malaria parasite. It is employed for both the treatment and prevention of malaria, in a combination with proguanil. The aim of this study was to elucidate the in vitro metabolites from atovaquone and to evaluate their cytotoxic activities. METHODS: The biotransformation of atovaquone was performed using Mucor rouxii NRRL 1894, Cunninghamella echinulata var. elegans ATCC 8688a and C. elegans ATCC 10028b, which have been reported as microbial models of mammalian drug metabolism. Experiments were also carried out with two probiotic strains from the human intestinal tract: Bifidobacterium sp. and Lactobacillus acidophilus. The phase I metabolite was isolated, its chemical structure was elucidated and its toxicity was evaluated using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. Cell cytotoxicity assays were also carried out with atovaquone. RESULT: Only the fungi were able to convert atovaquone to metabolite trans-3-[4'-(4″-chlorophenyl)cyclohexyl)-1,2-dioxo-dihydro-1H-indene-3-carboxylic acid. The metabolite displayed 50 % inhibitory concentration (IC50) values of 110.20 ± 2.2 and 108.80 ± 1.5 µmol/L against breast cancer cell line SKBR-3 and fibroblasts cell line GM07492-A, respectively. The IC50 values of atovaquone were 282.30 ± 1.8 and 340.50 ± 1.4 µmol/L against breast cancer and normal fibroblasts cell lines, respectively. CONCLUSIONS: The produced metabolite was more toxic than atovaquone and was not selective to normal or cancer cell lines. The present study is the first to report the production of atovaquone metabolite.

Formation of the diuretic chlorazanil from the antimalarial drug proguanil--implications for sports drug testing.

Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance.

The antimalarial drug proguanil is an antagonist at 5-HT3 receptors.

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 µM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 µM), and radioligand competition (Ki = 2.61 µM) with the 5-HT3 receptor antagonist [(3)H]granisetron. Kinetic measurements (kon = 4.0 × 10(4) M(-1) s(-1) ; koff = 0.23 s(-1)) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 µM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 µM, respectively), Schild plot (Kb = 2.97 and 11.4 µM), and radioligand competition (Ki = 4.89 and 0.41 µM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 µM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

Quantitative structure-activity relationships for cycloguanil analogs as PfDHFR inhibitors using mathematical molecular descriptors.

Computed molecular descriptors were used to develop quantitative structure-activity relationships (QSARs) for binding affinities (K(i)) for a set of 58 cycloguanil (2,4-diamino-1,6-dihydro-1,3,5-triazine) analogues for dihydrofolate reductase (DHFR) enzyme extracted from wild and A16V+S108T mutant type (a double mutation) malaria parasite Plasmodium falciparum (Pf). High-quality models were obtained in both cases. The results of statistical analyses show that ridge regression (RR) outperformed the two other modelling methods, principal component regression (PCR) and partial least squares (PLS). For both enzymes, recognition of the inhibitors was based on four broad categories of descriptors encoding information on: (1) the electronic character of the various atoms in the molecule, (2) the size and shape of the structure, (3) the degree of branching in the molecular skeleton, and (4) two to five atom molecular fragments with aliphatic carbon at one end and aliphatic or aromatic carbon or nitrogen at the other end. The subsets of influential descriptors underlying the QSARs for the wild versus the mutant DHFR are quite non-overlapping. This indicates that the two enzymes recognize the inhibitor molecules on the basis of mutually distinct structural attributes. Such differential QSARs can be useful in the design of novel drugs active against malaria parasites which are growing in resistant to existing chemotherapeutic agents.

Do 5-fluorouracil therapies alter CYP2C19 metaboliser status?

PURPOSE: To report a case of altered CYP2C19 metaboliser status following 5-fluorouracil treatment. METHODS: A 78-year-old male with stage III colorectal adenocarcinoma was prescribed with weekly iv 5-fluorouracil and folinic acid (FU/FA). Fourteen weeks after starting FU/FA, the patient was enrolled in a clinical study to investigate the role of tumour burden on drug metabolising enzyme activity. CYP2C19 genotype was determined and the activity of CYP2C19 was measured using proguanil (PG) as a probe substrate. A metabolic ratio (PG/CG) for CYP2C19 activity was determined on three separate occasions, 7 days apart. RESULTS: The patient was homozygous wild type (CYP2C19*1/*1), and on the first test, the metabolic ratio was concordant with the extensive metaboliser genotype. However, at day 14 and day 21, the metabolic capacity of this enzyme had decreased, and the subject had become a poor metaboliser (PG/CG > 30). The patient developed grade III hand and foot syndrome at day 10 of the study during the period of null CYP2C19 activity. CONCLUSIONS: Although 5FU is not a substrate for hepatic drug metabolising CYP enzymes, it may interfere with the synthesis of CYP2C19. Decreased activity of a related enzyme, CYP2C9, following 5FU has been reported previously. Down regulation of CYP2C9 and CYP2C19 synthesis by 5FU therapies may explain the adverse effect of 5FU on the clinical disposition of warfarin and phenytoin.

Interactions between cycloguanil derivatives and wild type and resistance-associated mutant Plasmodium falciparum dihydrofolate reductases.

Comparative molecular field analysis (CoMFA) and quantum chemical calculations were performed on cycloguanil (Cyc) derivatives of the wild type and the quadruple mutant (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) of Plasmodium falciparum dihydrofolate reductase (PfDHFR). The represented CoMFA models of wild type (r(2) = 0.727 and r(2) = 0.985) and mutant type (r(2) = 0.786 and r(2) = 0.979) can describe the differences of the Cyc structural requirements for the two types of PfDHFR enzymes and can be useful to guide the design of new inhibitors. Moreover, the obtained particular interaction energies between the Cyc and the surrounding residues in the binding pocket indicated that Asn108 of mutant enzyme was the cause of Cyc resistance by producing steric clash with p-Cl of Cyc. Consequently, comparing the energy contributions with the potent flexible WR99210 inhibitor, it was found that the key mutant residue, Asn108, demonstrates attractive interaction with this inhibitor and some residues, Leu46, Ile112, Pro113, Phe116, and Leu119, seem to perform as second binding site with WR99210. Therefore, quantum chemical calculations can be useful for investigating residue interactions to clarify the cause of drug resistance.

Influence of the genetic polymorphisms in the 5' flanking and exonic regions of CYP2C19 on proguanil oxidation.

CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including proguanil. Variation in the 5' regulatory region may influence CYP2C19 activity. This study evaluates the relationship between proguanil metabolic ratio and genetic variations of CYP2C19 in a South Indian population. Fifty unrelated healthy subjects were genotyped for CYP2C19 (*)2 and (*)3 alleles and the 5' flanking region of CYP2C19 was sequenced. Plasma concentrations of proguanil and cycloguanil were estimated by reverse phase HPLC after single oral doses (200 mg) of proguanil. In silico docking analysis of transcription factors binding to its sites in CYP2C19 5' regulatory region was performed. The mean metabolic ratios (proguanil/cycloguanil) were highest in (*)1/(*)2 or (*)1/(*)3 subjects and in (*)2/(*)2 or (*)2/(*)3 as compared to (*)1/(*)1 subjects. Subjects with promoter region variation -98T>C showed decrease in the metabolic ratios irrespective of other variation, which may explain the deviation from the genotype-phenotype association of CYP2C19. In silico analysis predicted alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The results of this study would be useful in predicting interindividual differences in the metabolism of substrates of CYP2C19.

Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

Effect of honey on CYP3A4, CYP2D6 and CYP2C19 enzyme activity in healthy human volunteers.

Honey is a common food supplement but not many studies have studied honey and drug interaction. This study investigates the influence of 7 days of honey administration on the activity of CYP3A4, CYP2D6 and CYP2C19 drug-metabolizing enzymes in healthy volunteers by using appropriate biomarker and probe drugs. A within-group pharmacokinetic study was done in 12 healthy volunteers. Urine samples (0-8 hr) were collected after administration of 30 mg of oral dextromethorphan (probe drug for CYP2D6) for analysis of dextromethorphan and dextrorphan. A plasma sample (4 hr) was collected after administration of 200 mg of oral proguanil (probe drug for CYP2C19) for the analysis of proguanil and cycloguanil. Urine samples (0-24 hr) were collected for the analysis of 6beta-hydroxycortisol (biomarker for CYP3A4). The volunteers were administered honey for 7 days. Subsequently blood and urine samples were collected after drug dosing as before. These samples were analysed for drug and metabolite concentrations in urine and plasma using high performance liquid chromatography method. Seven days of honey administration resulted in statistically significant increase in 24-hr urinary excretion of 6beta-hydroxycortisol. However, the metabolic ratios of dextromethorphan and proguanil were not significantly altered after 7 days of honey administration. Honey obtained from Western Ghats of southern India may induce CYP3A4 enzyme activity but not CYP2D6 and CYP2C19 enzyme activities.

[Cytochrome P-450 and the response to antimalarial drugs]. / El citocromo P-450 y la respuesta terapéutica a los antimaláricos.

OBJECTIVES: To assess the relationship between the genetic and phenotypic factors linked to the cytochrome P-450 enzyme system and the response to the antimalarial drugs chloroquine, amodiaquine, mefloquine, and proguanil, as well as to determine how certain biological and social factors of the host influence the behavior of this enzymatic complex. METHODS: We performed a systematic review of the medical bibliographic databases PubMed, Excerpta Medica, LILACS, and SciELO by using the following Spanish and English descriptors: "CYP-450" and "citocromo P-450" in combination with "proguanil" (and with "mefloquina," "cloroquina," and "amodiaquina"), "farmacocinética de proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "resistencia a proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "metabolismo," "farmacogenética," "enfermedad," "inflamación," "infección," "enfermedad hepática," "malaria," "nutrición," and "desnutrición." The same terms were used in English. The search included only articles published in Spanish, English, and Portuguese on or before 30 June 2005 that dealt with only four antimalarial drugs: amodiaquine, chloroquine, mefloquine, and proguanil. RESULTS: Some genetic factors linked to human cytochrome P-450 (mainly its polymorphism), as well as other biological and social factors (the presence of disease itself, or of inflammation and infection, the use of antimalarials in their various combinations, and the patient's nutritional status) influence the behavior of this complex enzymatic system. It has only been in the last decade that the genetics of the cytochromes has been explored and that the mechanisms underlying some therapeutic interactions and aspects of drug metabolism have been uncovered, making it possible to characterize the biotransformation pathway of amodiaquine and chloroquine. Hopefully new research will help answer the questions that still remain, some of which pertain to the metabolism of other antimalarial drugs, the distribution in the population of the genetic alleles linked to the enzymes involved in their metabolism, the contribution of these genetic mutations to therapeutic failure, and the possibility of predicting the response to antimalarial therapy. CONCLUSIONS: The therapeutic response to antimalarial drugs is a multifactorial process that is poorly understood, so that it is not possible to ascribe to a specific phenotype or genotype a role in the response to antimalarial therapy. Attention should be given to biological and social factors, such as diet, nutritional status, and inflammatory and infectious processes that are often present in areas where malaria is endemic.

El citocromo P-450 y la respuesta terapéutica a los antimaláricos / Cytochrome P-450 and the response to antimalarial drugs

OBJETIVOS: Evaluar la relación entre los factores genéticos y fenotípicos del sistema enzimático del citocromo P-450 y la respuesta terapéutica antimalárica a la cloroquina, la amodiaquina, la mefloquina y el proguanil, así como determinar la influencia de algunos factores biológicos y sociales del hospedero en el comportamiento de este complejo enzimático. MÉTODOS: Revisión sistemática de las bases de literatura biomédica PubMed, Excerpta Medica, LILACS y SciELO mediante descriptores en español e inglés. Se usaron los siguientes descriptores: "CYP-450" y "citocromo P-450" y sus combinaciones con "proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina"), "farmacocinética de proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina"), "resistencia a proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina"), "metabolismo", "farmacogenética", "enfermedad", "inflamación", "infección", "enfermedad hepática", "malaria", "nutrición" y "desnutrición". Estos mismos términos se usaron en inglés. La búsqueda se limitó a los artículos publicados en español, inglés y portugués hasta el 30 de junio de 2005 y a cuatro medicamentos antimaláricos: amodiaquina, cloroquina, mefloquina y proguanil. RESULTADOS: Algunos factores genéticos del citocromo P-450 humano (principalmente su polimorfismo), así como otros de tipo biológico y social (la propia presencia de enfermedad, inflamación o infección, la administración de medicamentos antimaláricos y su combinación, y el estado nutricional del paciente), influyen en la actividad de ese complejo enzimático. Solo en la última década se ha abordado el estudio de las bases genéticas de los citocromos y se han podido dilucidar los mecanismos de algunas interacciones entre fármacos y del metabolismo de estos, lo que ha permitido caracterizar el proceso de biotransformación de la amodiaquina y de la cloroquina. Se espera que nuevas investigaciones permitan responder a las interrogantes que aún subsisten, entre ellas cuál es la ruta metabólica de otros medicamentos antimaláricos, la distribución en la población de los alelos de las enzimas que participan en su metabolismo, y la contribución de tales mutaciones al fracaso terapéutico, y predecir la respuesta a los tratamientos antimaláricos...

Analysis of Sepik populations of Papua New Guinea suggests an increase of CYP2C19 null allele frequencies during the colonization of Melanesia.

The cytochrome P450 (CYP) isozyme CYP2C19 metabolizes clinically important drugs, including the anti-malarial proguanil currently used for multi-drug resistant Plasmodium falciparum malaria. CYP2C19 activity varies among geographical regions due to high frequencies of two null alleles (CYP2C19*2/*3) in Asian and especially Pacific populations. Previously, we reported an unprecedentedly high frequency of CYP2C19 poor metabolizers (PM) within populations of Vanuatu, which suggested even higher PM frequencies in Papua New Guinea. We examined CYP2C19 allele frequencies of three malarious populations from inland East Sepik Province, Papua New Guinea to evaluate this prediction and the use of proguanil in malaria treatment programs. These Papua New Guinean populations have PM frequencies intermediate between island South-east Asia and Vanuatu, most likely resulting from genetic drift during the settlement of the Pacific. This study highlights the medical consequences of population origins and the need for a better understanding of the genetic diversity of our global species.

Concordance between proguanil phenotype and CYP2C19 genotype in Chinese.

OBJECTIVE: To investigate whether urinary proguanil (chlorguanide) metabolite ratios incorporating its minor metabolite, 4-chlorophenylbiguanide, define individuals as extensive metabolisers (EMs) or poor metabolisers (PMs) of CYP2C19 more reliably than the standard phenotyping ratio [proguanil/cycloguanil (PG/CG)]. METHODS: Thirty-eight ethnic Chinese subjects ingested 100 mg proguanil, collected urine for 8 h and were genotyped for CYP2C19*1, *2 and *3 alleles. Proguanil metabolite ratios (PG/CG; proguanil/4-chlorophenylbiguanide (PG/CPB); proguanil/(cycloguanil+4-chlorophenylbiguanide) [PG/(CG+CPB)] were determined from the urinary recoveries of proguanil, cycloguanil and 4-chlorophenylbiguanide. Proguanil phenotypes were determined from the ratios using frequency distribution histograms, probit and normal test variable plots. RESULTS: Data from 35 subjects were suitable for analysis. Of subjects, 5 were CYP2C19*2/*2, 1 was *2/*3, 21 were *1/*2 and 8 were *1/*1. A rank order of proguanil metabolic ratios was observed, with *1/*1 subjects having the lowest, *1/*2 intermediate and *2/*2, *2/*3 having the highest ratios (P<0.0001). All subjects with PM genotypes were classified as PMs of proguanil by probit analysis of PG/CPB and PG/(CG+CPB) ratios, but not PG/CG. CONCLUSION: A gene-dose effect of CYP2C19 genotype on the conversion of proguanil to cycloguanil and 4-chlorophenylbiguanide has been demonstrated in ethnic Chinese subjects. Complete concordance between PM CYP2C19 genotype and PM phenotype was only achieved with probit analysis of proguanil metabolite ratios that incorporated 4-chlorophenylbiguanide.

Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo: rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker.

BACKGROUND: Resistance of Plasmodium falciparum to atovaquone in vitro and in vivo has been associated to mutations in the parasite cytochrome b gene. METHODS: Cultures were sequentially subjected to increasing doses of atovaquone alone or in combination with cycloguanil and the cytochrome b gene was sequenced. Additionally, we investigated the parasite cytochrome b gene of a patient returning from Mali with Malarone treatment failure in vivo. RESULTS: All strains that survived atovaquone concentrations in vitro of 2 x 10(-8) to 2 x 10(7) M showed the M133I mutation and one strain with the highest atovaquone concentration the additional mutation L171F. Sequencing of the in vivo treatment failure revealed a point mutation at codon 268 resulting in an amino acid change from tyrosine to serine. Based on the repeated emergence of mutations at codon 268, but no detection of alterations at codon 133 in vivo, we developed a detection method for the diagnostic of codon 268 polymorphisms as a potential atovaquone/proguanil resistance marker. A nested PCR with 3 different pairs of primers for the second round was designed. Each product was digested with restriction enzymes, capable to distinguish the wild type from the two reported mutations at codon 268. CONCLUSION: Mutations at codon 268 of the parasite cytochrome bc1 gene are associated with atovaquone/proguanil treatment failure in vivo and can be used as potential resistance marker This method provides a novel and robust tool to investigate the relevance of codon 268 polymorphisms as resistance marker and to monitor the further emergence of atovaquone/proguanil resistance.

Polymorphic oxidative metabolism of proguanil in a Nigerian population.

OBJECTIVE: The genetic polymorphic metabolic oxidation of proguanil was investigated in 126 healthy, unrelated Nigerian subjects as an indication of the phenotypic status of CYP2C19 in Nigerians. METHODS: The proguanil oxidation capacity was determined using the 8-h urinary metabolic ratio of the parent drug and its metabolite (cycloguanil) after a single oral dose of 200 mg proguanil. RESULTS: The frequency distribution of the proguanil metabolic ratio ranged from 0.01 to 39.64 with a median of 1.38 in the 126 Nigerians. On the basis of the antimode value of 10 for the proguanil/cycloguanil ratio, the prevalence of poor metabolisers in this Nigerian population was estimated to be 4.8% (6 of 126), which is very similar to that of S-mephenytoin (4.3%) found in a previous study in Nigerians. The data also demonstrated enormous inter-individual differences in the urinary proguanil/cycloguanil ratios with poor metabolisers excreting, on average, only about 8% of the quantity of cycloguanil excreted by extensive metabolisers. CONCLUSION: The incidence of phenotypically poor metabolisers of proguanil in this Nigerian population is similar to those reported for Caucasian and other African populations but is much lower than those reported for Orientals. The study further supports previous studies that proguanil can be used as an alternative probe to phenotype for CYP2C19 activity.

Slow chloroguanide metabolism in Tanzanians compared with white subjects and Asian subjects confirms a decreased CYP2C19 activity in relation to genotype.

BACKGROUND: We have previously found decreased CYP2C19 activity in Tanzanians tested with mephenytoin and omeprazole in relation to genotype when compared with white and Asian subjects. OBJECTIVE: We investigated the impact of CYP2C19 genotype and phenotype on chloroguanide (INN, proguanil) metabolism to its metabolites cycloguanil and 4-chlorophenylbiguanide. METHODS: A single oral chloroguanide dose was given to 25 healthy Tanzanian subjects with CYP2C19 genotypes (CYP2C19*1, CYP2C19*2, and CYP2C19*3). Homozygous wild-type and mutated genotype groups were chosen randomly, but the heterozygous genotype group was chosen with a range in phenotype. We used a novel HPLC method for drug determination. RESULTS: Pharmacokinetics of chloroguanide did not differ between groups. Maximum plasma concentration (Cmax) and area under the plasma concentration versus time [AUC(0-infinity)] for cycloguanil was significantly lower (t test P < .05) in the homozygously mutated group compared with the homozygously wild-type group. There were similar significant group differences of median urinary excretion. The chloroguanide/cycloguanil ratio closely correlated (r(s) = .87) with omeprazole metabolic ratio, confirming that Tanzanian subjects are generally slower CYP2C19 metabolizers. It also confirms that CYP2C19 genotype and phenotype predicts cycloguanil formation. In addition, a 3-hour plasma sample metabolic ratio also seems to be a proper time for omeprazole phenotyping in Tanzanian subjects. Because the plasma concentrations of cycloguanil and 4-chlorophenylbiguanide covary (r(s) = .89), it is now suggested that their formation be catalyzed by the same enzyme (ie, CYP2C19) through a common intermediate, the structure of which is also presented. CONCLUSIONS: As shown in an earlier study, also with a third substrate, Tanzanians have a lower capacity to form cycloguanil than white and Asian subjects. Individuals with two mutated alleles have lower metabolic capacity than individuals with two wild-type alleles or individuals in the heterozygous group, which may lead to chloroguanide therapeutic failure. This knowledge should be important when selecting appropriate patients and doses of chloroguanide in different populations.