Antimalarial activity of new floxacrine-related acridinedione derivatives: studies on blood schizontocidal action of potential candidates againstP. berghei in mice andP. falciparum in vivo and in vitro

Deoxyfloxacrine derivatives (1-hydrazone: S 83 0083; 1-imine: S 84 7277) and floxacrine derivatives (10-methoxy-floxacrine: L 84 7667; 1-imine: L 84 7693) selected from a series of newly synthesized 3-aryl-7-chloro-3,4-dihydro-1,9(2H, 10H)-acridinediones were evaluated for blood schizontocidal activities in mice infected with asexual stages of various drug-resistant lines ofP. berghei and in New World monkeys infected with blood schizonts of different chloroquine-resistant strains ofP. falciparum. All compounds tested showed high activity against drug-resistant lines ofP. berghei (ED₅₀: 1.0–4.4 mg/kg×5, per os) and were distinctly superior in their antimalarial potency to floxacrine. Compounds L 84 7667 and L 84 7693 proved to be highly active against the FCBR strain ofP. falciparum in vitro (IC₅₀: 0.73–1.78 nmol); they effected temporary clearance of parasitemias due to the Palo Alto strain ofP. falciparum in squirrel monkeys at oral doses of 15 mg/kg given daily for 5 consecutive days. Compounds S 83 0083 and S 84 7277, showing moderate in vitro effects (12.9–24.8 nmol), cleared parasitemias of the FCBR strain ofP. falciparum in owl monkeys at oral doses of 20 mg/kg (S 84 7277) given daily for 5 or 7 consecutive days (follow-up period, 17 and 30 days, respectively) or at doses of 20 mg/kg (×4) (S 83 0083) followed by doses of 40 mg/kg (×3) within a follow-up period of 30 days. These observations suggest that the range of doses required for the cure of establishedP. falciparum infections is probably too large to cover infections with strains of the least susceptibility and might evoke toxic reactions by the potential candidates tested.     

Efficacy and safety of artemether against a natural <Emphasis Type="Italic">Fasciola hepatica</Emphasis> infection in sheep

Triclabendazole is the current drug of choice against Fasciola spp. infections in livestock, but resistance has become a major problem. In this study, we assessed the efficacy and safety of artemether, a derivative of artemisinin, in sheep with a low natural Fasciola hepatica infection. Artemether was administered orally or intramuscularly; sheep were monitored for 8 h posttreatment and then once daily for adverse events, and drug efficacy was estimated by fecal egg count reductions and worm burden reductions. Single 40- and 80-mg/kg oral doses of artemether showed no effect on F. hepatica egg and worm burden. Treatment with a single 160-mg/kg intramuscular dose of artemether significantly reduced the egg burden (64.9%) and worm burden (91.3%). At half this dose, a worm burden reduction of 65.3% was obtained, which was still statistically significant (P < 0.05). The lowest intramuscular dose of artemether investigated (40 mg/kg) yielded no effect on egg counts and worm burden. There were no adverse events due to artemether; however, two abortions were observed 7 days posttreatment. In conclusion, artemether shows interesting fasciocidal properties in sheep, but embryotoxicity is of concern. Further studies are warranted to assess the potential of additional artemisinin derivatives and other peroxidic compounds for the treatment of Fasciola spp. infections in different ruminants.     

Artemether and tribendimidine lack activity in experimental treatment of Paragonimus westermani in the dog

Artemether and tribendimidine are active against several trematode species, but no data are available regarding the lung fluke Paragonimus westermani. We infected six dogs with 100 P. westermani metacercariae each. At day 103 post-infection, four dogs were treated orally for 3 days with either artemether (total dose, 66.7 and 75 mg/kg) or tribendimidine (total dose, 100 mg/kg). The remaining dogs were left untreated and served as control. Sixteen days after the final dosing, dogs were killed, and P. westermani flukes were recovered from the lungs and counted. Neither artemether nor tribendimidine showed activity against P. westermani at this dose regimen in dogs.     

Efficacy of artesunate and artemether against <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> in rabbits

Artesunate and artemether display promising clonorchicidal properties in rats. In this study, we assessed the efficacy of artesunate and artemether against Clonorchis sinensis in rabbits. Rabbits were each fed with 300 C. sinensis metacercariae. At day 28 postinfection, the rabbits were administered oral artesunate at doses of 7.5–120 mg/kg and oral artemether of 15–120 mg/kg. Two groups of rabbits were treated with single oral praziquantel at 75 and 150 mg/kg. Untreated rabbits served as controls. Fourteen days after treatment, all rabbits were sacrificed, and C. sinensis adults were collected from the bile ducts and counted. At the highest doses tested (120 mg/kg) artesunate and artemether achieved statistical significant worm burden reductions of 88.8% and 67.2%, respectively. These rates were lower than worm burden reductions observed with praziquantel (88% and 100%, respectively). It is suggested that artesunate and artemether have moderate anthelminthic efficacy against C. sinensis in rabbits.     

Effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum

Artemether and artesunate, derivatives of the antimalarial artemisinin, as well as their main metabolite, dihydroartemisinin, all exhibit antischistosomal activities. The purpose of the current study was to compare the effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum. We carried out experiments with mice, infected with 40 cercariae of S. japonicum, and treated with artemether, aretesunate and dihydroartemisinin (all at a single dose of 300 mg/kg, and the dose of the mixed three drugs is also 300 mg/kg) at multiple doses or combination therapy on days 6–8 or 34–36 post-infection. Administration with artemether, artesunate or dihydroartemisinin for 3 successive days reduced total worm burdens by 79.5−86% (30.86 ± 4.98 of mean total worm burden in control), female worm burdens by 79.4−86.7% (11.29 ± 2.63 of mean female worm burden in control) (all P values <0.01 vs. control), depending on different treatment protocols given on days 6–8 post-infection. However, no differences were seen between each treatment group (all P > 0.05). While the same treatment was given on days 34–36 post-infection, total worm burden reductions of 73.8−75.8% were achieved (29.44 ± 3.36 of mean total worm burden in control), which were significant when compared with the untreated control group (all P values <0.01). In all different treatment groups, female worm reductions (ranging from 88.7% to 93.1%, while the mean female worm burden in control is 10.33 ± 1.80) were consistently higher than the total worm reductions, resulting always in significantly lower female worm burdens when compared to the corresponding control (all P values < 0.01). However, there were no significant differences found between each treatment group (all P values >0.05). It is concluded that artemether, artesunate and dihydroartemisinin can be used to control schistosomiasis japonica, as a strategy to prevent S. japonicum infection. Administration with artemether, artesunate and dihydroartemisinin at multiple doses or in combined treatment damages both juvenile and adult S. japonicum, without statistically significant differences among the three drugs at the same dose.     

Artemether shows promising female schistosomicidal and ovicidal effects on the Egyptian strain of <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> after maturity of infection

Artemether is an artemisinin derivative that is used as an antimalarial drug, especially in situations where chloroquine resistance is suspected. This compound has proved to be a good prophylactic agent against schistosomiasis japonica in China. In the present study, the therapeutic efficacies of different artemether-dosing protocols were evaluated in experimentally infected mice harbouring adult Schistosoma mansoni (Egyptian strain). Mice were treated on day 46 onwards with three dosing protocols (400 mg/kg/day for 2 days; 200 mg/kg/day for 4 days; 100 mg/kg/day for 6 days) after being infected. A number of parasitological and histopathological criteria were employed in the assessment of drug efficacies compared to infected untreated control 2 weeks post-treatment. The results of the present study suggest that artemether is efficacious against the Egyptian strain of S. mansoni with total worm reductions ranging from 40.7% to 59.7% and female worm reductions ranging from 69.3% to >90%. In addition, artemether induced significant reductions, ranging from 75.2% to 82.6%, in the liver tissue egg loads as well as significant reductions, ranging from 68.8% to 78.9% in the intestinal wall egg loads. It also induced significant alterations in the oogram pattern in the intestinal mucosa of infected mice with cessation of oviposition and increased rates of dead eggs. Antipathologic activities were also evident in the amelioration of granulomas in the liver with increased ratios of healed to active ones. In conclusion, artemether could be a promising agent in the control of schistosomiasis mansoni due to its schistosomicidal effects on female worms and to its ovicidal power as well as its potentiality in the improvement of hepatic lesions.     

The action of salinomycin-Na and lasalocid-Na on chloroquine- and mepacrine-resistant line ofPlasmodium berghei K 173-strain in Wistar rats

Salinomycin-Na and lasalocid-Na, two ionophorous antibiotics known for their anticoccidial activity, exhibit in vivo blood schizontocidal action on thePlasmodium berghei Keyberg 173 RC/M line that has a high level of resistance to chloroquine and mepacrine. Salinomycin was found to have a greater effect than lasalocid on asexual stages of this line. Trophozoites and schizonts were no longer found after a single dose of 20 mg/kg or five doses of 1.25 mg/kg of salinomycin whereas a single dose of 40 mg/kg or five doses of 20 mg/kg of lasalocid showed no marked effect on parasitaemia within 96 h of starting treatment in rats. Some toxicological data show that lasalocid, however, is better tolerated in domestic animals than salinomycin. Early morphological changes in asexual blood stages were membrane-coiling in the cytoplasm followed by vacuolization and disruption of the cell membrane or pellicle after treatment with both compounds. In particular mature schizonts were totally destroyed showing enormously large vacuoles. Toxicological data and blood schizontocidal activity indicate the narrow safety margin inP. berghei infected rats, and place salinomycin in the markedly toxic group of antimalarial compounds.Dedicated to Professor Dr. G. Piekarski, former editor of theZeitschrift für Parasitenkunde, on his 75th birthday     

Effect of mefloquine administered orally at single, multiple, or combined with artemether, artesunate, or praziquantel in treatment of mice infected with Schistosoma japonicum

The purpose of the study is to better understand the antischistosomal properties of artemether, praziquantel, and ozonide (OZ) compounds (synthetic trioxolanes, secondary ozonides) in hamster (Mesocricetus auratus) model. A total of 230 male hamsters infected each with 100 Schistosoma japonicum cercariae were used in the study. Groups of five to ten hamsters were treated orally with artemether, praziquantel, and OZ78 or OZ277 7–35 days post-infection at single doses of 50, 100, 150, or 200 mg/kg. Untreated but infected hamsters in each batch of test served as the control. All treated hamsters were sacrificed 4 weeks post-treatment for collection of residual worms using perfusion technique. Nonparametric method (Mann–Whitney test) was used to analyze the data. In groups of five hamsters treated with artemether 7, 14, 21, 28, and 35 days post-infection at single doses of 150 and 200 mg/kg, the difference of mean worm burden between each treated group and control group was statistically significant (P < 0.01). Apart from individual group, no difference in mean worm burden between each two groups of them was seen (P > 0.05). Further test with various single doses of 50–200 mg/kg confirmed the similar susceptibility of 7-day-old juvenile and 35-day-old adult schistosomes to artemether. After administration of praziquantel 100 mg/kg to groups of five hamsters 7, 21, and 35 days post-infection, higher worm burden reduction of 95.5% was seen in the group with 35-day-old adult schistosomes while in the groups with 7- and 21-day-old juvenile schistosomes, poor efficacy was seen with mean worm burden reductions of 36.6% and 35.6%. In the same batch of hamster treated with praziquantel 200 mg/kg, the moderate effect of the drug against 7- and 21-day-old worms was seen, but their mean worm burden was significantly higher than that of the group with adult schistosomes. In comparison of artemether and praziquantel against various stages of schistosomes, the results further demonstrated that artemether possessed similar effect against juvenile and adult schistosomes in hamsters, while praziquantel was more effective against adult schistosomes than the juvenile ones in the same host. Finally, after administration of OZ78 and OZ277 to the groups of four to six hamsters with 14- and 35-day-old schistosomes at a single dose of 200 mg/kg, promising effect against juvenile and adult schistosome was observed with the mean worm burden and female worm burden reductions of 69.6–94.2% and 64.2–100% as well as 73.3–80.7% and 68.3–81.1%, respectively. The results indicate that in hamster model, praziquantel exhibits higher effect against adult schistosomes than the juvenile ones, while artemether and OZ compound display similar effect against both juvenile and adult schistosomes.     

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites

Three monoclonal antibodies generated by immunization of mice withPlasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and crythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365–681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites ofP. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity ofP. berghei sporozoites and to inhibit the development of liver stages ofP. yoelii.     

Identification and expression analysis of ABC genes in<Emphasis Type="Italic"> Plasmodium yoelii</Emphasis> and<Emphasis Type="Italic"> P. berghei</Emphasis>

The ATP-binding cassette (ABC) proteins are one of the largest evolutionarily conserved families. They are characterized by the presence of highly conserved nucleotide-binding sites (NBS). In the present study, we identified ABC genes in rodent Plasmodia. We queried the Plasmodium yoelii genome with the ABC signature motif and retrieved 15 contigs. Sequences were classified into seven ABC families by BLAST comparison. Conservation of the five signature ABC motifs in the P. yoelii contigs was examined by multi-alignment of the NBS. Expression of the ABC genes was examined during the blood stages of P. yoelii and P. berghei and the hepatocytic stages of P. yoelii. Our results with RT-PCR on total RNA from blood stages demonstrated the expression of 14 ABC genes in P. yoelii and ten in P. berghei. In P. yoelii hepatocytic stages, the expression of four ABC genes was detected. A.C. Szeto and J. Pérez-Rosado contributed equally to this work     

Effect of single-dose oral artemether and tribendimidine on the tegument of adult <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> in rats

The tegument of trematodes plays a key role in nutrient absorption, exerts secretory functions, protects the parasite against the immune system of the host, and is a target for anti-trematocidal drugs. We performed a temporal examination of tegumental changes following artemether and tribendimidine administration on adult Clonorchis sinensis in rats using scanning electron microscopy. Rats infected with C. sinensis for 6 weeks were treated orally with a single dose of artemether (150 mg/kg) or tribendimidine (300 mg/kg). Worms were collected between 8 h and 7 days (artemether) and between 4 h and 2 days post-treatment (tribendimidine). Worms recovered from untreated rats served as controls. Eight hours after artemether administration, the tegument of C. sinensis was extensively disrupted, including severe swelling, fusion and vacuolization, and the suckers were damaged. Four hours after administration of tribendimidine, C. sinensis worms showed extensive tegumental alterations, characterized by massive sloughing, and the suckers were damaged. Interestingly, the severity of tegumental changes did not progress further with time. Our results show that both artemether and tribendimidine rapidly disrupt the tegument and damage the suckers of adult C. sinensis. The subtle differences in tegumental changes induced by artemether and tribendimidine might indicate different mechanisms of action of these drugs against C. sinensis.     

Enhanced antimalarial effects of chloroquine by aqueous Vernonia amygdalina leaf extract in mice infected with chloroquine resistant and sensitive Plasmodium berghei strains

Background

The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest antimalarial drug coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the need to optimize antimalarial actions of plant extracts and restore chloroquine efficacy.

Objective

The present study determines the ability of Vernonia amygdalina leaf extract to enhance the prophylactic and therapeutic efficacy of chloroquine against Plasmodium berghei malaria in mice.

Methods

Chloroquine sensitive (P. berghei^S) and resistant (P.berghei^R) ANKA clones of Plasmodium berghei maintained by serial passage in mice were used to develop respective experimental rodent malaria models based on intraperitoneal injection of 10⁶ parasitize erythrocyte suspension in PBS (pH 7.2) and subsequent development of parasitaemia. These models were then used to investigate the prophylactic enhancement of chloroquine (CQ) at 5 mg/kg via combination with selected doses (31.25, 62.5, 125mg/kbw) of Vernonia amygdalina leaf extracts using a 4-day suppression test. Effect of these combinations on the therapeutic efficacy of CQ at 30mg/kg over 3 days were evaluated. Treatment outcomes including parasite clearance (PCT) and rescrudescent time (RT) were compared with CQ-chlorpheniramine combination. The acute toxicity of the extract-CQ combinations was also determined enzymatically.

Results

Prophylatically, chloroquine (5mg/kg) in combination with vernonia extracts achieved a dose-dependent (57.2–72.7%) suppression of parasitaemia due to CQ sensitive and resistant P berghei strains in the experimental animals. Therapeutically, chloroquine (30mg/kg for 3 days) combined with vernonia to dose-dependently shorten the parasite clearance times (2.6–4.4 vs. 4.8 days; P < 0.05 for CQ-V62.5/125 combination), prolong the recrudescent times (8.9–18.9 vs. 7.2 days; P < 0.05) and improve day 14 cure rate (66.7–100 vs. 58.3%) in the treated P. berghei^S infected mice compared to CQ monotherapy. Whereas CQ monotherapy failed, resolution of parasitaemia due to the CQ resistant parasite with day 14 cure rates of 25 – 100% were also observed with these combinations. In therapeutic terms, the potencies of CQ-V125 combination were comparable to those of CQ-chlorpheniramine (0.25mg/kg, 12hourly, 7 days) in the infected animals. Toxicity testing indicates that these combinations elicited mild to - moderate increases in the liver enzymes measured when administered orally to mice for 7 days.

Conclusion

This study indicates that Vernonia amygdalina leaf extract dose - dependently restore the efficacy of CQ against CQ resistance P. berghei malaria in mice.     

Plasmodium falciparum ookinetes migrate intercellularly throughAnopheles stephensi midgut epithelium

The migration ofPlasmodium falciparum andP. berghei ookinetes through the midgut epithelium inAnopheles stephensi was studied by transmission electron microscopy. With ruthenium red (RR) staining, the results of previous studies were confirmed:P. falciparum ookinetes take an intercellular route through the midgut epithelium. In the same mosquito species, the rodent parasiteP. berghei appeared to take an intracellular position, as previously suggested by other authors. The intra- or intercellular ookinete migration ofP. berghei orP. falciparum, respectively, can perhaps be related to the higher mortality ofP. berghei-infected mosquitoes within the first 2 days of infection. Evidence is presented that oocyst capsule formation begins as early as during the migration of the ookinete. After localization between the epithelial cells and the midgut basal lamina, the rapidly expanding oocyst stretches the overlying layer of the latter at the haemocoelic surface while a new basal lamina is generated between the oocyst and epithelial cell.Abbreviations BL basal lamina - CR cristalloid - N nucleus - RER rough endoplasmic reticulum - Mp malarial pigment - M mitochondrion - MV microvillous border - OC oocyst capsule     

Oral administration ofl-arginine, a nitric oxide precursor, decreases nociceptive sensitivity in rats

Tail-flick test was used to evaluate the effect of orally administeredl-arginine on nociceptive sensitivity of albino rats, which produced a) analgesia at 30 min postadministration lasting about 1.5 h (100 mg/kg); b) short-term analgesia (50 mg/kg); and c) no analgesic effect (250 mg/kg).d-Arginine (100 mg/kg) did not affect the nociceptive sensitivity. A significant NO increase took place in cerebral cortex at 30 min postadministration ofl-arginine in the given doses. At 1 h postadministration ofl-arginine in doses of 50 and 250 mg/kg, cortical NO content was lower than that in control animals. Analgesic effect ofl-arginine is presumably related to additional synthesis of NO. This effect seems to be not directly produced by NO, but is realized via other transmitter systems. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 498–501, November, 1997     

Duffy antigen is important for the lethal effect of the lethal strain of<Emphasis Type="Italic"> Plasmodium yoelii</Emphasis> 17XL

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.     

Screening of antiplasmodial efficacy of <Emphasis Type="Italic">Ajuga bracteosa</Emphasis> Wall ex. Benth

The rising problem of Plasmodium resistance to the classical antimalarial drugs stresses the need to look for newer antiplasmodial components with effective and new mode of action. In the present study, the traditional medicinal plant Ajuga bracteosa has been screened for its antiplasmodial efficacy. The extract was found to possess significant in vitro antiplasmodial efficacy with an IC₅₀ of 10.0 μg/ml. Thus, the extract was further evaluated for its in vivo schizontocidal activity and efficacy in terms of survival time in Plasmodium berghei infected BALB/c mice. The extract at 250, 500, and 750 mg/kg/day exhibited significant (p < 0.0001) blood schizontocidal activity during established infection with enhanced mean survival time comparable to that of standard drug chloroquine, 5 mg/kg/day. The significant schizontocidal activity and enhanced mean survival time of mice stress the need to identify and characterize active antiplasmodial principle from this plant.     

Antimalarial properties of ebselen

The seleno-organic compound ebselen showed anti-malarial activity in vitro against the murinePlasmodium berghei and the humanP. falciparum. InP. berghei, the uptake and incorporation of [³H]-methionine and [³H]-adenosine was inhibited and the infectivity of plasmodia was reduced. Ebselen affects the development of asexual stages of chloroquine-resistant and-sensitiveP. falciparum strains. Its IC₅₀ forP. falciparum was about 14 mol/l and that forP. berghei, about 10 mol/l. The growth ofP falciparum was blocked by ebselen at all stages, including the invasion of erythrocytes by merozoites. In a human hepatoma cell line and in mouse peritoneal macrophages, no cytostatic or cytotoxic effects were found, indicating selective inhibition of plasmodia by ebselen. Its in vitro inhibitory effect is discussed in relation to its possible reactivity with thiol groups and its lack of an anti-malarial effect in infected mice.Abbreviations BSA bovine serum albumin - FCS fetal calf serum - GSH reduced glutathione - GSSG oxidised glutathione - HBSS Hanks' blanced salt solution - PBS phosphate-buffered saline - PE parasitised erythrocytes - Se selenium - TCA trichloroacetic acid     

Schistosoma japonicum-infected hamsters (Mesocricetus auratus) used as a model in experimental chemotherapy with praziquantel, artemether, and OZ compounds

The purpose of the study is to better understand the antischistosomal properties of artemether, praziquantel, and ozonide (OZ) compounds (synthetic trioxolanes, secondary ozonides) in hamster (Mesocricetus auratus) model. A total of 230 male hamsters infected each with 100 Schistosoma japonicum cercariae were used in the study. Groups of five to ten hamsters were treated orally with artemether, praziquantel, and OZ78 or OZ277 7-35 days post-infection at single doses of 50, 100, 150, or 200 mg/kg. Untreated but infected hamsters in each batch of test served as the control. All treated hamsters were sacrificed 4 weeks post-treatment for collection of residual worms using perfusion technique. Nonparametric method (Mann-Whitney test) was used to analyze the data. In groups of five hamsters treated with artemether 7, 14, 21, 28, and 35 days post-infection at single doses of 150 and 200 mg/kg, the difference of mean worm burden between each treated group and control group was statistically significant (P<0.01). Apart from individual group, no difference in mean worm burden between each two groups of them was seen (P>0.05). Further test with various single doses of 50-200 mg/kg confirmed the similar susceptibility of 7-day-old juvenile and 35-day-old adult schistosomes to artemether. After administration of praziquantel 100 mg/kg to groups of five hamsters 7, 21, and 35 days post-infection, higher worm burden reduction of 95.5% was seen in the group with 35-day-old adult schistosomes while in the groups with 7- and 21-day-old juvenile schistosomes, poor efficacy was seen with mean worm burden reductions of 36.6% and 35.6%. In the same batch of hamster treated with praziquantel 200 mg/kg, the moderate effect of the drug against 7- and 21-day-old worms was seen, but their mean worm burden was significantly higher than that of the group with adult schistosomes. In comparison of artemether and praziquantel against various stages of schistosomes, the results further demonstrated that artemether possessed similar effect against juvenile and adult schistosomes in hamsters, while praziquantel was more effective against adult schistosomes than the juvenile ones in the same host. Finally, after administration of OZ78 and OZ277 to the groups of four to six hamsters with 14- and 35-day-old schistosomes at a single dose of 200 mg/kg, promising effect against juvenile and adult schistosome was observed with the mean worm burden and female worm burden reductions of 69.6-94.2% and 64.2-100% as well as 73.3-80.7% and 68.3-81.1%, respectively. The results indicate that in hamster model, praziquantel exhibits higher effect against adult schistosomes than the juvenile ones, while artemether and OZ compound display similar effect against both juvenile and adult schistosomes.     

K‐Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

K‐Ras mutant fraction (MF) was measured to examine the default assumption of low‐dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of 10 male A/J mice (7‐ to 9‐weeks old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P and were sacrificed 28 days after treatment. K‐Ras codon 12 TGT and GAT MFs in lung DNAs were measured using Allele‐specific Competitive Blocker‐PCR (ACB‐PCR). The K‐Ras codon 12 TGT geometric mean MF was 3.88 × 10^?4 in controls, indicating an average of 1 mutation in every ～1,288 lung cells. The K‐Ras codon 12 TGT geometric mean MFs were as follows: 3.56 × 10^?4; 6.19 × 10^?4; 2.02 × 10^?3, and 3.50 × 10^?3 for the 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The 5 and 50 mg/kg dose groups had TGT MFs significantly higher than did controls. Although 10^?5 is considered as the limit of accurate ACB‐PCR quantitation, K‐Ras codon 12 GAT geometric mean MFs were as follows: 8.38 × 10^?7, 1.47 × 10^?6, 2.19 × 10^?6, 5.71 × 10^?6, and 8.99 × 10^?6 for the 0, 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The K‐Ras TGT and GAT MFs increased in a B[a]P‐dose‐dependent manner, with response approximately linear over the 0.05 to 5 mg/kg dose range. K‐Ras MF increased with B[a]P adduct burden measured for identical doses in a separate study. Thus, ACB‐PCR may be useful in characterizing the shape of a dose‐response curve at low doses and establishing relationships between DNA adducts and tumor‐associated mutations. Environ. Mol. Mutagen. 2010. Published 2009 Wiley‐Liss, Inc.     

Praziquantel efficacy in mice infected with PZQ non‐susceptible S. mansoni isolate treated with artemether: parasitological,biochemical and immunohistochemical assessment

Botros SS, Hammam O, Mahmoud M, Bergquist R. Praziquantel efficacy in mice infected with PZQ non‐susceptible S. mansoni isolate treated with artemether: parasitological, biochemical and immunohistochemical assessment. APMIS 2010; 118: 692–702. Based on the fact that artemether (ART) affects immature schistosomes and that the effect of praziquantel (PZQ) mainly targets mature schistosomes, this work investigates the possible enhanced efficacy of PZQ in combination with ART in mice harboring a PZQ non‐susceptible Schistosoma mansoni isolate. Associated schistosomal, inflammatory, hepatic histopathological changes have been investigated by examining the tissue markers expressing apoptosis using FAS (CD95), anti‐apoptosis (Bcl2) and angiogenesis [vascular endothelial growth factor (VEGF)]. A batch of Swiss albino mice infected with a PZQ non‐susceptible (EE10) S. mansoni isolate was divided into 12 groups. Animals of the first group were left without treatment as infected controls, while groups 2–6 received PZQ in increasing doses. The animals of group 7 received ART in double doses. Those comprising groups 8–12 received combined therapy of PZQ and ART in the same doses and at the same timings postinfection (PI) as those belonging to groups 2–6. Parasitological parameters, liver function, and histopathological and immunohistochemical studies of FAS, Bcl2 and VEGF antibodies were assessed. Combined administration of ART and PZQ reduced the ED₅₀ (the dose at which the worm burden was decreased by 50%) of PZQ. Typical granulomas were not seen in animals treated with ART alone and combined with PZQ, with least expression of FAS and VEGF and increased expression of Bcl2. The minimal histopathological changes recorded in mice treated with both ART and PZQ could be related to a synergistic/additive effect of ART, markedly reducing the intensity of infection. Improved liver function tests support the less severe histopathological changes under the influence of this treatment protocol. This study encourages human trials especially in areas where malaria is not endemic, and differing combination doses should be investigated in view of the antagonistic effect noticed with some dose regimens.