Drug selection pressure and fitness cost for artemether-resistant Plasmodium berghei ANKA parasites in vivo.

BACKGROUND: The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AMR) parasites from Plasmodium berghei ANKA (AMS), and determine their fitness cost. METHODS: Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED50 and ED90) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I50 and I90) were determined using the ratio of either ED50 or ED90 of AMR to AMS, respectively. The stability of the AMR parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AMR and AMS with 95% confidence intervals. RESULTS: ED50 and ED90 of AMS were 0.61 and 3.43 mg/kg/day respectively. I50 and I90 after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AMR (24.70% ± 3.60) relative to AMS (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%. CONCLUSION: A moderately stable AMRP. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended.

Antimalarial activity assay of artesunate-3-chloro-4(4-chlorophenoxy) aniline in vitro and in mice models.

The global spread of multi-drug resistant P. falciparum, P. vivax, and P. malariae strains and absence of long-term effective vaccine makes chemotherapy the mainstay of malaria control strategies in endemic settings. The Mossman's assay and the Organization for Economic Co-operation and Development (OECD), 2001 guideline 423, were used to determine the cytotoxicity and acute oral toxicity of a novel hybrid drug, artesunate-3-Chloro-4(4-chlorophenoxy) aniline (ATSA), in vitro and in vivo, respectively. A modified Desjardins method was used to screen for antiplasmodial activity using P. falciparum (3D7 and W2) strains in vitro. The Peter's 4-day suppressive tests (4DTs) was used to evaluate the in vivo antimalaria activity using P. berghei ANKA strain, lumefantrine resistant (LuR), and piperaquine resistant (PQR) P. berghei lines. In silico prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles was assayed using PreADMET online prediction tool. The reference drug in all experiments was artesunate (ATS). Statistical significance between ATSA's activities in treated and control mice was evaluated by one-way analysis of variance (ANOVA). Results show that inhibitory concentrations-50 (IC50) of ATSA is 11.47 ± 1.3 (3D7) and 1.45 ± 0.26 (W2) against 4.66 ± 0.93 (3D7) and 0.60 ± 0.15 (W2) ng/ml of ATS with a selective index of 2180.91(3D7) and a therapeutic index (TI) of > 71). No mortalities were observed in acute oral toxicity assays and mean weight differences for test and controls were statistically insignificant (P > 0.05). The in vivo activity of ATSA was above 40% with effective dosage-50 (ED50) of 4.211, 2.601, and 3.875 mg/kg body weight against P. berghei ANKA, LuR, and PQR lines, respectively. The difference between treated and control mice was statistically significant (P < 0.05). ATSA has high intestinal absorption (HIA) > 95% and has medium human ether-a-go-go related gene (hERG) K+ channel inhibition risks. Preclinical and clinical studies on ATSA are recommended to evaluate its value in developing novel drugs for future management of multi-drug resistant malaria parasites.

Simultaneous electrical online estimation of changes in blood hematocrit and temperature in cardiopulmonary bypass.

Two equations have been developed from multi-frequency measurements of blood impedance Zb for a simultaneous electrical online estimation of changes in blood hematocrit ΔH [%] and temperatures ΔT [K] in cardiopulmonary bypass (CPB). Zb of fixed blood volumes at varying H and T were measured by an impedance analyzer and changes in blood conductivity σb and relative permittivity εb computed. Correlation analysis were based on changes in σb with H or T at f = 1 MHz while H and T equations were developed by correlating changes in εb with H and T at dual frequencies of f = 1 MHz and f = 10 MHz which best capture blood plasma Zp and red blood cell cytoplasm Zcyt impedances respectively. Results show high correlations between σb and H (R2 = 0.987) or σb and T (R2 = 0.9959) indicating dependence of the electrical parameters of blood on its H and T. Based on computed εb, changes in blood hematocrit ΔH and temperature ΔT at a given time t are estimated as ΔH(t) = 1.7298Δεb (f = 1 MHz) - 1.0669Δεb (f = 10 MHz) and ΔT(t) = -2.186Δεb (f = 1 MHz) + 2.13Δεb (f = 10 MHz). When applied to a CPB during a canine mitral valve plasty, ΔH and ΔT had correlations of R2 = 0.9992 and R2 = 0.966 against H and T respectively as measured by conventional devices.