Atractylodin-loaded PLGA nanoparticles:formulation and characterization / 中国药理学与毒理学杂志

OBJECTIVE To formulate atractylodin-loaded poly (lactic- co- glycolic acid) (PLGA) nanoparticles and characterize the prepared nanoparticle formulation.METHODS The nanoparticle formu-lation was developed using solvent displacement method. The encapsulation and loading efficiency were characterized and particle size, and zeta potential were determined by dynamic light scattering technique.Drug release was assessed in vitro.RESULTS The size(mean±SD of diameter)of the prepared atractylodin-loaded PLGA nanoparticles were (161.27 ± 1.87)nm with narrow size distribution (mean PDI: 0.068±0.015)and zeta potential(28.83±0.35)mV.The encapsulation and loading efficiency were (48.31±0.83)% and(2.15±0.04)%,respectively.Drug release from atractylodin-loaded PLGA nanoparticles was observed up to (87.70 ± 0.47)% in 72 h with biphasic manner. Moreover, the nanoparticles were found to be freely dispersible in water without aggregation. CONCLUSION Results suggest that PLGA nanoparticles may be used as an effective drug delivery system for atractylodin.The anti-cholangiocar-cinoma activity of this nanoparticle formulation is required.

A brief review on biomarkers and proteomic approach for malaria research

Malaria remains as one of the significant health threat to people living in countries throughout tropical and subtropical zones. Proteomic studies of Plasmodium, the protozoan causing malaria, is essential for understanding its cellular structure, growth stage-specific expression of protein metabolites and complex interaction with host. In-depth knowledge of the pathogen is required for identification of novel biomarkers that can be utilized to develop diagnostic tests and therapeutic antimalarial drugs. The alarming rise in drug-resistant strains of Plasmodium has created an urgent need to identify new targets for drug development that can act by obstructing life cycle of this parasite. In the present review, we briefly discuss on role of various biomarkers including Plasmodium-associated aldolase, histidine-rich proteins and lactate dehydrogenase for diagnosis of malaria. Here we also summarize the present and future prospects of currently used techniques in proteomic approaches such as two dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) for diagnosis and potential identification of novel proteins for malaria research.

Proteomics analysis of antimalarial targets of Garcinia mangostana Linn

Objective: To investigate possible protein targets for antimalarial activity of Garcinia mangostana Linn. (G. mangostana) (pericarp) in 3D7 Plasmodium falciparum clone using 2-dimensional electrophoresis and liquid chromatography mass-spectrometry (LC/MS/MS). Methods: 3D7 Plasmodium falciparum was exposed to the crude ethanolic extract of G.mangostana Linn. (pericarp) at the concentrations of 12μg/mL (IC50 level: concentration that inhibits parasite growth by 50%) and 30 μg/mL (IC90 level: concentration that inhibits parasite growth by 90%) for 12 h. Parasite proteins were separated by 2-dimensional electrophoresis and identified by LC/MS/MS.Results:At the IC50 concentration, about 82% of the expressed parasite proteins were matched with the control (non-exposed), while at the IC90 concentration, only 15% matched proteins were found. The selected protein spots from parasite exposed to the plant extract at the concentration of 12 μg/mL were identified as enzymes that play role in glycolysis pathway, i.e., phosphoglycerate mutase putative, L-lactate dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase/phosphoglycerate kinase. The proteosome was found in parasite exposed to 30 μg/mL of the extract.Conclusions:Results suggest that proteins involved in the glycolysis pathway may be the targets for antimalarial activity of G. mangostana Linn. (pericarp).

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

Objective: To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border. Methods: Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. Results:Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

Objective: To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE). Methods: Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software. Results:Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference. Conclusions: These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99 percent). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5 percent), followed by the wild-type A383/A553 (17.5 percent) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective: To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210. Methods: The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine.In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays. Results: A total of 30 out of 64 blood samples collected from patients withP. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210. Conclusions: On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.

Cadmium exposure in Thai populations from central, northern and northeastern Thailand and the effects of food consumption on cadmium levels.

The aim of the present study was to investigate the association between cadmium body burden and the areas of exposure in Thailand, as well as blood pressure levels, the types and frequencies of foods, and alcohol consumption. A total of 182 healthy adult Thai subjects of both genders (89 males, 93 females) ages 18 to 57 years old weighing 40-95 kg were included in this study. Participants were residents from three main areas of Thailand: Pathum Thani Province (central Thailand; n=50), Khon Kaen Province (northeastern Thailand; n=43) and Mae Sot District, Tak Province (northern Thailand; n=89). The total amount of cadmium excreted in urine over 2 hours (microg/g creatinine) was used as an indicator of long-term cadmium exposure. Quantitation of cadmium was performed using electrothermal (graphite furnace) atomic absorption spectrometry (GFAAS). The urinary cadmium excreted displayed a normal frequency of distribution. Significantly higher mean cadmium levels were observed in subjects residing in Mae Sot, Tak Province (0.63 +/- 1.41 microg/g creatinine) and Khon Kaen (0.51 +/- 0.76 microg/g creatinine) compared to Pathum Thani Province (0.23 +/- 0.35 microg/g creatinine). The proportion of subjects with elevated blood pressure was significantly higher in the group exposed to higher (n=39) as opposed to lower (n=5) levels of cadmium. There were no significant differences in the mean total amounts of cadmium excreted in the 2-hour urine samples from subjects who consumed different types of meat and offal, or from those who consumed them at different frequencies.

Identification of HLA-A24 restricted pre-erythrocytic stage specific T-cell epitopes using Plasmodium falciparum synthetic peptides: a preliminary study.

Fourteen (9 amino acids) peptides of Plasmodium falciparum pre-erythrocytic stage antigens, namely, TRAP, CTRP, LSA-1, STARP and MSP-1, restricted to HLA-A24 and specific to T-cell response were identified. The antigen-specific IFN-gamma responses of these synthetic peptides in malaria exposed and non-malaria exposed healthy adult volunteers were detected using the ex vivo ELISPOT assay. Five peptides from TRAP and CTRP antigens significantly increased IFN-y responses of 1/9 in malaria-exposed volunteers. There is no statistically significant difference in positive T-cell response induced by any peptides in malaria exposed volunteers when evaluated as a group. The frequency of expressed HLA-A24 in malaria-exposed and non-malaria-exposed healthy adults living in northwest and central Thailand was 90% (27/30) and 100% (12/12), respectively. Although no association between positive T-cell response and HLA-A24 was found, due to the low number of positive responders achieved, one positive responder in malaria- exposed group was presented as HLA-24.

Development and validation of a liquid chromatography-mass spectrometry method for the simultaneous quantification of artesunate and dihydroartemisinin in human plasma.

The present study describes the development and validation of a simple, sensitive, and specific liquid chromatography-mass spectrometry (LC-MS) analytical method used for the co-quantification of artesunate (ARS) and its active metabolite, dihydroartemisinin (DHA), in human plasma, using artemisinin (ARN) as an internal standard. The liquid-liquid extraction of samples was carried out using dichloromethane and tert.-methyl butyl ether (at a ratio of 8:2 v/v) and then evaporated to dryness by a stream of nitrogen gas at room temperature. Chromatographic separation and mass analysis were performed on the Agilent 1100 Series Liquid Chromatography/Mass Spectrometer Detector Trap system, using electrospray ionization as an interface. The stationary phase was an Elipse XDB-C18 column. The mobile phase contained acetonitrile and 0.003 M glacial acetic acid at a ratio of 62:38 (v/v) delivered at a flow rate of 0.5 ml per minute. Positive ion mode was selected to detect extracted ions at m/z 407 and 261 for ARS, at m/z 307 and 261 for DHA, and at m/z 305 for ARN. The retention times for alpha-DHA, ARS, beta-DHA, and ARN were 6.6, 8.0, 9.2, and 10.8 minutes, respectively, and the total chromatography run time was 12 minutes. The limit of detection (LOD) was 2 ng/ml while the limit of quantification (LOQ) was 10 ng/ml for both ARS and DHA. In order to address any complications caused by the spontaneous non-catalytic breakdown of ARS to DHA, two calibration curves were prepared separately for both analytes. These graphs were found to be linear over the range of 10 to 3,200 ng/ml (r2 > 0.99). The recoveries at concentrations of 100, 200, 400, and 800 ng/ml were 108, 106, 91, and 89%, respectively, for ARS and were 112, 95, 80, and 86%, respectively, for DHA. For ARN, the recoveries were 119, 119, and 90% for concentrations of 200, 400, and 800 ng/ml, respectively. ARS working solutions were not stable after two months of storage at 4 degrees C or after 21 days at room temperature. This newly developed LC-MS method was then applied for measuring of ARS and DHA concentrations in a healthy volunteer having received oral ARS at 200 mg once daily for 5 consecutive days. There was no decline in ARS concentration after repeated doses and the C(ss-max-average) for DHA was found to be 703 +/- 94 ng/ml at t(ss-max) of 2 h.

Assessment of in vitro antimalarial interactions between dihydroartemisinin and fosmidomycin.

Malaria remains one of the leading causes of morbidity and mortality in the tropics with an annual estimate of 500 million clinical cases and 2 million deaths. The treatment and control of malaria is becoming increasingly difficult due to Plasmodium falciparum resistance to commonly used antimalarials. Combination therapy is currently the strategy for combating multi-drug resistant falciparum malaria, through exploiting pharmacodynamic synergistic effects and delaying the emergence of drug resistance. The combination of artemisinin derivatives with fosmidomycin, which have different modes of action, appears to be one of the most promising combinations. The objective of the present study was to investigate the antimalarial interactions between dihydroartemisinin and fosmidomycin in vitro, against chloroquine-resistant (K1) and chloroquine-sensitive (G112) P. falciparum strains. Concentration-response analysis was performed based on an in vitro schizont maturation inhibition test. The fixed concentration ratios of dihydroartemisinin: fosmidomycin used were 0:5,000, 2:4,500, 6:3,500, 10:2,500, 14:1,500, 18:500 and 20:0 nM. The highest final concentrations of dihydroartemisinin and fosmidomycin were 20 and 5,000 nM, respectively. Results showed IC50 (drug concentration which produced 50% schizont maturation inhibition) medians (range) for dihydroartemisinin against K1 and G112 strains to be 1.6 (1.2-2.0) and 2.5 (2.4-2.6) nM, respectively. The IC50 medians (range) for fosmidomycin against K1 and G112 strains were 1,347 (1,068-1,625) and 786 (737-834) nM, respectively. An isobologram revealed an increasing trend for the fraction IC50 (FIC), which indicates marked antagonism of this drug combination against both chloroquine resistant and chloroquine sensitive strains.

Pharmacokinetics of mefloquine with dihydroartemisinin as 2-day regimens in patients with uncomplicated falciparum malaria.

The objective of this study was to investigate the pharmacokinetics of mefloquine (MQ) when given as 750 mg at two different times in combination regimens with dihydroartemisinin (DHA) in patients with acute uncomplicated falciparum malaria. A total of 12 Vietnamese patients (6 in each group) were randomized to receive two MQ-DHA regimens as follows: regimen-A: an initial oral dose of 300 mg DHA, followed by 750 mg MQ and 300 DHA 6 and 24 hours later; regimen-B: an initial dose of 300 mg DHA, followed by 300 mg DHA and 750 mg MQ at 24 hours. Both combination regimens were well tolerated. All patients responded well to treatment with no recrudescence during a 42 day follow-up period. The pharmacokinetics of MQ following both regimens were similar but pooled data from both groups suggest that the kinetics of MQ was different from that observed in Vietnamese healthy subjects reported in a previous study. The median (95% CI) time period for maintenance of whole blood MQ concentrations above 500 ng/ml was 16 (0-24) days. It was concluded that since no pharmacokinetic drug interaction was observed, MQ dose given 24 hours after an initial dose of DHA is a preferable combination treatment regimen with regard to patient compliance.

Comparison of two in vitro sensitivity tests for Plasmodium falciparum.

The main purpose of the study was to compare the in vitro sensitivity results obtained from the two widely-used in vitro systems: (1) standard WHO micro-technique based on schizont maturation inhibition using fresh isolates (M-I), and (2) micro-technique based on incorporation of [3H]-hypoxanthine using culture-adapted isolates (M-II). The study was conducted during 1998 and 2002. A total of 473 Plasmodium falciparum isolates were collected from five highly malaria endemic areas of Thailand, ie, Mae Sot district, Tak (north-western), Kanchanaburi (western), Ranong (south-western), Ratchaburi (south-western) and Chantaburi (eastern) Provinces. The antimalarials tested were: mefloquine, quinine, chloroquine, artemisinin and dihydroartemisinin. The sensitivity results for mefloquine obtained from the two methods were significantly different from each other. The IC50 values for M-II was less than M-I. The median (95%C.I.) IC50 value for mefloquine using the M-II method was significantly lower [696.47 (393.11-1,233.2) nM] than for M-I [3,955.4 (1,035.61-5,108.9) nM]. The in vitro sensitivity results for quinine were significantly different from each other. The median (95% C.I.) IC50 value for M-II [161 (42-351) nM] was 2.5-fold that of M-I [66 (24-450) nM].

Life-saving rectal artesunate for complicated malaria in children.

We report the effectiveness of two regimens of rectal artesunate formulation in treating 13 Thai children with cerebral/complicated falciparum malaria. The drug was given at an initial dose of 40 mg/kg bodyweight, in 3 or 4 divided doses in the first 24 hours, followed by 10 mg/kg bodyweight once daily for three consecutive days. Mefloquine, at a dose of 15 mg/kg bodyweight was given orally at 72 hours after the initial dose of artesunate, followed by 10 mg/kg bodyweight 6 hours later. Three cases with cerebral malaria gained consciousness within 20 hours of artesunate administration. The median time required for reduction of parasitemia by 90% of the initial value (P90) in 13 children was 11.2 hours. No recrudescence was observed in any of the patients during the 28-day follow-up period. Plasma concentrations of artesunate and dihydroartemisinin (active plasma metabolite of artesunate) measured in two patients who received the high initial dose regimen (20 mg/ kg bodyweight) suggested rapid absorption and adequate plasma concentrations of both compounds following the administration of artesunate via the rectal route. Further studies for the optimized regimen of rectal artesunate in the treatment of cerebral/complicated childhood falciparum malaria in areas of multidrug resistance are warranted.

Pharmacokinetics of the four combination regimens of dihydroartemisinin/mefloquine in acute uncomplicated falciparum malaria.

The pharmacokinetics of oral dihydroartemisinin and mefloquine were investigated in 40 patients (aged 16-30 y, weighing 45-60 kg) with acute uncomplicated falciparum malaria following the four combination regimens of dihydroartemisinin/ mefloquine [regimen-I: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-0); regimen-II: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-24); regimen-III: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-24 and 30); regimen-IV: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-0, 24)]. The four combination regimens were well tolerated. Patients in all treatment groups had a rapid initial response. However, 9 patients (4, 4, and 1 cases in regimens-I, II, and IV) had reappearance of parasitemia during the follow-up period. Significant changes in the pharmacokinetic parameters of both mefloquine and dihydroartemisinin were observed in patients with malaria compared with healthy subjects reported in a paralleled study. For mefloquine, Cmax (mg per dose), AUC0-day1 (mg per dose), and AUC0-day7 (mg per dose) were significantly higher in patients. Furthermore, tmax, was prolonged while V/F contracted and tl/2,z, MRT shortened in patients with malaria. For dihydroartemisinin, Cmax AUC, tmax and Vz/F were changed in the same direction as mefloquine, whereas t1/2z and MRT were prolonged. CL/F was also significantly reduced in patients with malaria. Absorption/disposition kinetics of oral dihydroartemisinin were similar among the various regimens. On the other hand, AUC0-day1 (mg per dose) of mefloquine after regimen-III was significantly higher than the other three regimens. Combination regimens with two divided doses of mefloquine (regimens-III and IV) resulted in a significantly delayed tmax (especially regimens-IV) compared with those with single dose regimens (regimens-I and II).

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).