First study on efficacy and tolerability of a new alkylphosphocholine molecule (oleylphosphocholine-OlPC) in the treatment of canine leishmaniosis due to Leishmania infantum.

The alkylphosphocholine oleylphosphocholine (OlPC) represents a potential new therapy for the treatment of canine leishmaniosis caused by Leishmania infantum. The aim of the present study was to evaluate the efficacy and safety of OlPC in a small cohort of dogs naturally infected with L. infantum and defined as clinically sick (LeishVet stages II and III). A total of eight dogs were included in the study and were treated orally with 4 mg/kg OlPC for 14 days. Dogs were assessed at the clinical and parasitological level at four time points during a total follow-up period of 90 days (before treatment and at 15, 30, and 90 days post-treatment onset). Ln-PCR, real-time quantitative PCR, antibody testing (IFAT), and culture of bone marrow aspirates were evaluated at the four time points. OlPC treatment induced a rapid and satisfactory clinical recovery in terms of clinical score reduction and weight gain, and treatment efficacy was found to be associated with a decrease in bone marrow parasitic load. Serological titers measured by IFAT were stable in any of the treated dogs at any time point after treatment. OlPC was well tolerated and no severe adverse events were noted in any of the treated dogs; even some dogs showed slight intestinal disorders. This proof-of-principle study is the first to show that short oral treatment with OlPC improves clinical signs of canine L. infantum leishmaniosis, highlighting the need to perform additional studies to optimize the dosing regimen and to assess long-term treatment efficacy of this drug.

Novel artemisinin derivatives with potential usefulness against liver/colon cancer and viral hepatitis.

Antitumor and antiviral properties of the antimalaria drug artemisinin from Artemisia annua have been reported. Novel artemisinin derivatives (AD1-AD8) have been synthesized and evaluated using in vitro models of liver/colon cancer and viral hepatitis B and C. Cell viability assays after treating human cell lines from hepatoblastoma (HepG2), hepatocarcinoma (SK-HEP-1), and colon adenocarcinoma (LS174T) with AD1-AD8 for a short (6h) and long (72h) period revealed that AD5 combined low acute toxicity together with high antiproliferative effect (IC50=1-5µM). Since iron-mediated activation of peroxide bond is involved in artemisinin antimalarial activity, the effect of iron(II)-glycine sulfate (ferrosanol) and iron(III)-containing protoporphyrin IX (hemin) was investigated. Ferrosanol, but not hemin, enhanced antiproliferative activity of AD5 if the cells were preloaded with AD5, but not if both compouds were added together. Five derivatives (AD1>AD2>AD7>AD3>AD8) were able to inhibit the cytopathic effect of bovine viral diarrhoea virus (BVDV), a surrogate in vitro model of hepatitis C virus (HCV), used here to evaluate the anti-Flaviviridae activity. Moreover, AD1 and AD2 inhibited the release of BVDV-RNA to the culture medium. Co-treatment with hemin or ferrosanol resulted in enhanced anti-Flaviviridae activity of AD1. In HepG2 cells permanently infected with hepatitis B virus (HBV), AD1 and AD4, at non-toxic concentrations for the host cells were able to reduce the release of HBV-DNA to the medium. In conclusion, high pharmacological interest deserving further evaluation in animal models has been identified for novel artemisinin-related drugs potentially useful for the treatment of liver cancer and viral hepatitis B and C.

Efficacy and tolerability of oleylphosphocholine (OlPC) in a laboratory model of visceral leishmaniasis.

OBJECTIVES: The alkylphospholipid oleylphosphocholine (OlPC) is a structural analogue of miltefosine and may represent a potential therapeutic backup for the treatment of visceral leishmaniasis (VL). This laboratory study compared the in vitro and in vivo activity profile of both OlPC and miltefosine. METHODS: The in vitro potency of OlPC was compared with that of miltefosine, amphotericin B, paromomycin and pentavalent antimony (Sb(V)) using the intracellular amastigote assay on different Old World and New World Leishmania species. The in vivo efficacy was dose titrated in the Leishmania infantum hamster model after infection with 2 × 10(7) amastigotes (day 0) and oral treatment at day 21 using an aqueous (OlPC/H(2)O) and liposomal formulation of OlPC in single and repeated (5 day) oral dosing regimens. The amastigote reductions in the liver, spleen and bone marrow were assessed (day 35). RESULTS: The in vitro activity of OlPC against Leishmania donovani, L. infantum, Leishmania tropica, Leishmania mexicana and Leishmania panamensis showed mean IC(50) values <5 µM, while the IC(50) values for Leishmania major and Leishmania braziliensis were 7.7 and 13.5 µM, respectively. These results are fairly similar to those obtained for miltefosine. In the hamster model, treatment with 20 and 40 mg/kg for 5 days proved that both OlPC formulations were equipotent and showed a markedly higher efficacy compared with miltefosine. A single dosing of 100 mg/kg of OlPC/H(2)O or OlPC liposomes reduced the parasite burdens by 96.2% and 99.3% in liver, 99.8% and 99.9% in spleen, and 87.6% and 96.9% in bone marrow, respectively. No signs of toxicity or adverse drug-related effects were noted. CONCLUSIONS: These data suggest that OlPC may become a promising candidate to improve and simplify current case management of VL. Additional pharmacological and pharmacokinetic studies are ongoing to assess the full potential of OlPC as a 'drug candidate'.

Design of novel artemisinin-like derivatives with cytotoxic and anti-angiogenic properties.

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti-angiogenic, anti-tumorigenic and anti-viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin-like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti-angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin-like compounds that are easily synthesized, stable at room temperature, may overcome drug-resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.

Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells.

Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives have been shown to exert profound anti-cancer activity. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. Here, we analyzed the cytotoxicity and the ability of artemisinin, five of its derivatives, and two other endoperoxides to inhibit generation of nitric oxide (NO). In the RAW 264.7 mouse macrophage cell line, the well-established model cell line to analyze NO generation, artesunate revealed the highest ability to inhibit NO production among all compounds tested. In cytotoxicity assays (XTT assay), the IC(50) value of RAW 264.7 cells for artesunate was determined to be 3.1+/-0.7 microM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO metabolism and signaling, whole genome mRNA microarray analyses were conducted. RAW 264.7 cells were treated with artesunate using DMSO as vehicle control followed by microarray analysis. A total of 36 genes related to NO metabolism and signaling were found to be differentially expressed upon exposure to artesunate. Apart from NO-related genes, the expression of genes associated with other functional groups was also analyzed. Out of 24 functional groups, differential expression was most prominent in genes involved in cell-to-cell signaling and interactions. Further refinement of this analysis showed that the pathways for cAMP-mediated signaling and Wnt/beta-catenin signaling were most closely related to changes in mRNA expression. In conclusion, NO generation and signaling play a role in exhibiting cytotoxic activity of artesunate. In addition, other signaling pathways also contribute to the inhibitory effect of artesunate towards RAW 264.7 cells pointing to a multi-factorial mode of action of artesunate.

A randomized trial of artesunate-sulfamethoxypyrazine-pyrimethamine versus artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Mali.

The choice of artemisinin-based combination that is being adopted for malaria treatment in sub-Saharan Africa may depend on several factors, including cost, efficacy, side effects, and simplicity of administration. We tested the hypothesis that artesunate-sulfamethoxypyrazine-pyrimethamine is as efficacious as the four-dose regimen of artemether-lumefantrine for treatment of Plasmodium falciparum malaria. The study was carried out during two transmission seasons (2003 and 2004) in Sotuba, Mali. Participants at least 6 months of age with uncomplicated P. falciparum malaria were randomly assigned to receive artesunate-sulfamethoxypyrazine-pyrimethamine or artemether-lumefantrine. Treatment efficacy was assessed using the World Health Organization 28-day protocol. A total of 606 (303 in each arm) patients were enrolled. The cure rate was higher for artesunate-sulfamethoxypyrazine-pyrimethamine than for artemether-lumefantrine (98.7% versus 89.6%; P < 0.0001). After correction for cases of re-infection, the cure rates were 100% and 99.0%, respectively (P = 0.08). No serious adverse events occurred. Artesunate-sulfamethoxypyrazine-pyrimethamine is well-tolerated and effective against P. falciparum malaria. It showed an additional benefit of preventing new infections.

A Randomised, Double Blind, Placebo-Controlled Pilot Study of Oral Artesunate Therapy for Colorectal Cancer.

BACKGROUND: Artesunate is an antimalarial agent with broad anti-cancer activity in in vitro and animal experiments and case reports. Artesunate has not been studied in rigorous clinical trials for anticancer effects. AIM: To determine the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC). METHODS: This was a single centre, randomised, double-blind, placebo-controlled trial. Patients planned for curative resection of biopsy confirmed single primary site CRC were randomised (n = 23) by computer-generated code supplied in opaque envelopes to receive preoperatively either 14 daily doses of oral artesunate (200 mg; n = 12) or placebo (n = 11). The primary outcome measure was the proportion of tumour cells undergoing apoptosis (significant if > 7% showed Tunel staining). Secondary immunohistochemical outcomes assessed these tumour markers: VEGF, EGFR, c-MYC, CD31, Ki67 and p53, and clinical responses. FINDINGS: 20 patients (artesunate = 9, placebo = 11) completed the trial per protocol. Randomization groups were comparable clinically and for tumour characteristics. Apoptosis in > 7% of cells was seen in 67% and 55% of patients in artesunate and placebo groups, respectively. Using Bayesian analysis, the probabilities of an artesunate treatment effect reducing Ki67 and increasing CD31 expression were 0.89 and 0.79, respectively. During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group developed recurrent CRC. INTERPRETATION: Artesunate has anti-proliferative properties in CRC and is generally well tolerated.

LC-UV/MS quality analytics of paediatric artemether formulations.

A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for ß-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5 µm), flow rate set at 1.5 mL/min combined with a linear gradient (where A=25 mM phosphate buffer (pH 2.5), and B=acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile-water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of ß-artemether paediatric formulations. Furthermore, LC-MS on references as well as on stability samples was performed allowing identity confirmation of the ß-artemether related impurities. MS-fragmentation scheme of ß-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.

Relative response factor determination of ß-artemether degradants by a dry heat stress approach.

During the stability evaluation of ß-artemether containing finished drug products, a consistent and disproportional increase in the UV-peak areas of ß-artemether degradation products, when compared to the peak area decline of ß-artemether itself, was observed. This suggested that the response factors of the formed ß-artemether degradants were significantly higher than ß-artemether. Dry heat stressing of ß-artemether powder, as a single compound, using different temperatures (125-150 °C), times (10-90 min) and environmental conditions (neutral, KMnO(4) and zinc), resulted in the formation of 17 degradants. The vast majority of degradants seen during the long-term and accelerated ICH stability study of the drug product, were also observed here. The obtained stress results allowed the calculation of the overall average relative response factor (RRF) of ß-artemether degradants, i.e. 21.2, whereas the individual RRF values of the 9 most prominent selected degradants ranged from 4.9 to 42.4. Finally, Ames tests were performed on ß-artemether as well as a representative stressed sample mixture, experimentally assessing their mutagenic properties. Both were found to be negative, suggesting no mutagenicity problems of the degradants at high concentrations. Our general approach and specific results solve the developmental quality issue of mass balance during stability studies and the related genotoxicity concerns of the key antimalarial drug ß-artemether and its degradants.

Efficacy of artesunate + sulfamethoxypyrazine/pyrimethamine versus praziquantel in the treatment of Schistosoma haematobium in children.

BACKGROUND: This study was conducted to determine the efficacy of the antimalarial artemisinin-based combination therapy (ACT) artesunate +sulfamethoxypyrazine/pyrimethamine (As+SMP), administered in doses used for malaria, to treat Schistosoma haematobium in school aged children. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted in Djalakorodji, a peri-urban area of Bamako, Mali, using a double blind setup in which As+SMP was compared with praziquantel (PZQ). Urine samples were examined for Schistosoma haematobium on days -1, 0, 28 and 29. Detection of haematuria, and haematological and biochemical exams were conducted on day 0 and day 28. Clinical exams were performed on days 0, 1, 2, and 28. A total of 800 children were included in the trial. The cure rate obtained without viability testing was 43.9% in the As+SMP group versus 53% in the PZQ group (Chi(2) = 6.44, p = 0.011). Egg reduction rates were 95.6% with PZQ in comparison with 92.8% with As+SMP, p = 0.096. The proportion of participants who experienced adverse events related to the medication was 0.5% (2/400) in As+SMP treated children compared to 2.3% (9/399) in the PZQ group (p = 0.033). Abdominal pain and vomiting were the most frequent adverse events in both treatment arms. All adverse events were categorized as mild. CONCLUSIONS/SIGNIFICANCE: The study demonstrates that PZQ was more effective than As+SMP for treating Schistosoma haematobium. However, the safety and tolerability profile of As+SMP was similar to that seen with PZQ. Our findings suggest that further investigations seem justifiable to determine the dose/efficacy/safety pattern of As+SMP in the treatment of Schistosoma infections. TRIAL REGISTRATION: ClinicalTrials.gov NCT00510159.