An alternative liquid chromatography-mass spectrometric method for the determination of azithromycin in human plasma and its application to pharmacokinetic study of patients with malaria

A simple, sensitive, selective and reproducible method based on high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS) was developed for the determination of a macrolide antibiotic azithromycin in human plasma. The internal standard (roxithromycin) was separated from azithromycin on a Hypersil Gold C18 column, with retention times of 10.71 and 13.67 minutes, respectively. The mobile phase consisted of a mixture of 20 mM ammonium acetate buffer (pH 5.2), acetonitrile and methanol (50:40:10, v/ v/v), running through the column at a flow rate of 0.3 ml/minute. Chromatographic analysis was carried out at 25 degrees C. Sample preparation was by liquid-liquid extraction with a mixture of 7:3 (v/v) diethylether:dichloromethane. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 5% (% coefficient of variations: % CV). Good accuracy was observed for both intra-day and inter-day assays. The limit of quantification was acceptable at 0.5 ng using 200 microl plasma samples. The mean recoveries for azithromycin and the internal standard were greater than 85%. The method was applied successfully to the investigation of the pharmacokinetics of azithromycin when given in combination with fosmidomycin as oral doses of 750 mg twelve hourly for 3 days in 5 Thai male patients with acute uncomplicated falciparum malaria.     

Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.     

Pharmacokinetics and bioavailability of oral and intramuscular artemether

Objective: The pharmacokinetics and bioavailability of artemether and dihydroartemisinin were investigated in eight Thai males following the administration of single oral and intramuscular doses of artemether (300 mg) in a randomized two-way cross-over study. Results: Both oral and intramuscular artemether were well-tolerated. In most cases, artemether and dihydroartemisinin were detected in plasma after 30 min and declined to levels below the limit of detection within 18–24 h. Compared with intramuscular administration, oral administration of artemether resulted in a relatively rapid but incomplete absorption [C_max: 474 vs 540 ng · ml⁻¹; t _max: 2.0 vs 3.9 h; AUC: 2.17 vs 5.20 μg · h · ml⁻¹]. Geographic means of lag-time and absorption half-life (t _1/2a) of oral vs intramuscular artemether were 0.28 and 1.1 h vs 0.30 and 2 h, respectively. t _1/2z was significantly shortened after the oral dose [2.8 vs 6.9 h]. Mean oral bioavailability relative to intramuscular administration was 43.2%. The ratio of the AUCs of artemether to dihydroartemisinin was significantly lower after the oral than after the intramuscular dose (geometric mean: 0.29 vs 0.60). Received: 18 October 1996 / Accepted in revised form: 28 January 1997     

Comparative assessment of the in vitro sensitivity of Brugia malayi infective larvae to albendazole, diethylcarbamazine and ivermectin alone and in combination

The antifilaricidal drugs ivermectin (IVM), diethylcarbamazine (DEC), and albendazole (ALB), used alone or in combinations against infective third-stage larvae (L3) of nocturnally subperiodic (NSP) Brugia malayi (Narathiwat strain), were tested in vitro for sensitivity, for 7 days. IVM alone reduced larval motility at concentrations of 10(-7), 10(-6), and 10(-5) M on day 3. DEC alone also had this effect at concentrations of 10(-6). 10(-5), and 10(-4) M on day 2. ALB alone did not have this effect throughout the experiment, at various concentrations. However, it had greater effect when used in combination with either DEC or IVM. The result also indicated that DEC or IVM, when used in combination with ALB at concentrations of 10(-6) M/10(-6) M, and 10(-5) M/10(-5) M was effectively better than each drug used alone at those concentrations. When both drug combinations were compared, ALB/DEC seemed to be more effective than ALB/IVM at a concentration of 10(-6) M/10(-6) M on day 3. Although IVM and DEC can reduce larval motility when used alone or in combination with ALB, they cannot kill these larvae in an in vitro cultivation, even at a high concentration (10(-5) M).     

Pharmacokinetics of mefloquine, when given alone and in combination with artemether, in patients with uncomplicated falciparum malaria

Summary— The pharmacokinetics of mefloquine at a single oral dose of 750 mg, when given alone or 24 hours after a single oral dose of artemether. (300 mg) was investigated in 27 Thai patients with acute uncomplicated falciparum malaria (17 with mefloquine alone, 10 with the combination). The oral bioavailability of mefloquine was significantly decreased when administered 24 hours after an oral dose of artemether. This was evident by the significantly lower values of C_max, AUC[0–24 h], AUC[0–48 h], AUC[0–72 h], as well as total AUC[C_max: 1,290 (827-2,619) vs 1,820 (1,283-2,531) ngml⁻¹; AUC[0–24 h]: 0.99 (0.64-1.41) vs 1.33 (1.07–1.95) μgdayml⁻¹; AUC[0–48 h]: 1.78(1.23-2.58) vs 2.67 (2.09-3.84) μgdayml⁻¹; AUC[0–72 h]: 2.74 (1.63-3.6) vs 4.54 (2.88-5.38) μgdayml⁻¹; AUC: 11.11 (6–20.96) vs 15.29 (9.3–36.71) μgdayml⁻¹]. T_max was also delayed with the combination regimen [14 (5–24) vs 6 (4–16) h). Terminal elimination half-lives were comparable [t_1/2z: 11.1 (6.8–14.3) vs 13.4 (10.5–19.1) h].     

Genetic and environmental influences on therapeutic and toxicity outcomes: studies with CYP2A6

The idea that the liver enzyme cytochrome P450 2A6 (CYP2A6), known also as nicotine C-oxidase, is one of the determinants of smoking addiction and smoking behavior is primarily based on its role in nicotine metabolism and disposition. The results of studies linking the CYP2A6 genetic polymorphism with smoking dependence and smoking behavior however remain controversial. The most likely causes of the controversies appeared to be consideration given to a few allelic variants coupled with the uses of the CYP2A6 alleles lacking in vivo phenotypic validation. In the present review, we summarize research findings on biological significance of CYP2A6 and gene polymorphisms together with a discussion on CYP2A6 inhibitors that hold the promise of uses in smoking cessation. In addition, we provide the phenotype/genotype information derived from our systematic investigation on the relationship between CYP2A6 genotypes, smoking habits and coumarin metabolism phenotypes in a group of 393 normal adults (197 women and 196 men), 16 to 60 years of age, whose exposure to cadmium and lead were also determined, enabling us to assess the CYP2A6 phenotypic variability associated with CYP2A6 genotypes and environmental exposure. The results indicate that the phenotype of CYP2A6 enzyme in liver is an outcome of interactions between the CYP2A6 gene, cadmium, nicotine and possibly its metabolites.     

Clinical-parasitological response and in-vitro sensitivity of Plasmodium vivax to chloroquine and quinine on the western border of Thailand

This study was conducted during 2002-2004 at Mae Sot District, on the Thai-Myanmar border, an area of multidrug-resistant Plasmodium falciparum malaria. Sixty-two patients with P. vivax malaria were included in the study. All were randomized into two groups to receive a 3-day regimen of chloroquine or a 3-day regimen of quinine. Primaquine was given to patients in both groups for the elimination of hepatic stages. Results from the present study suggest that the standard regimen of chloroquine and a 3-day course of quinine at the dose regimens under investigation were very effective and well tolerated for the treatment of P. vivax malaria in this area. All patients responded well to both drug regimens; the cure rates with chloroquine or quinine, when given concurrently with the tissue schizontocidal drug primaquine, were virtually 100% within 28 days of follow-up. No significant correlations between parasite clearance time (PCT) or fever clearance time (FCT) and inhibitory concentration 50 (IC50) were found. Patients who had PCT < or = 24 h and those with PCT >24 h had comparable IC50 to chloroquine (alone and plus primaquine) and quinine, as well as similar concentrations of chloroquine/desethylchloroquine (in blood) or quinine (in plasma) at the investigated time points.     

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002-2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt-pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai-Laos border and no cases of QN resistance were found in this study.     

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.