Assessment of Thermal and Hydrolytic Stabilities and Aqueous Solubility of Artesunate for Formulation Studies.

For the purpose of establishing the optimum processing parameters and storage conditions associated with nanolipid formulations of the artemisinin derivative artesunate, it was necessary to evaluate the thermal stability and solubility profiles of artesunate in aqueous solutions at various temperatures and pH. The effect of increased temperature and humidity on artesunate was determined by storing samples of the raw material in a climate chamber for 3 months and analyzing these by an established HPLC method. Artesunate remained relatively stable during storage up to 40°C ± 0.5°C and 75% relative humidity for 3 months, wherein it undergoes approximately 9% decomposition. At higher temperatures, substantially greater decomposition supervenes, with formation of dihydroartemisinin (DHA) and other products. In solution, artesunate is relatively stable at 15°C with less than 10% degradation over 24 h. The aqueous solubility of artesunate at different pH values after 60 min are pH 1.2 (0.1 M HCl) 0.26 mg/mL, pH 4.5 (acetate buffer) 0.92 mg/mL, distilled water 1.40 mg/mL, and pH 6.8 (phosphate buffer) 6.59 mg/mL, thus relating to the amount of ionized drug present. Overall, for optimal preparation and storage of the designated formulations of artesunate, relatively low temperatures will have to be maintained throughout.

The Artemisinin Derivative Artemisone Is a Potent Inhibitor of Human Cytomegalovirus Replication.

Human cytomegalovirus (HCMV) is a major cause of disease in immunocompromised individuals and the most common cause of congenital infection and neurosensorial disease. The expanding target populations for HCMV antiviral treatment along with the limitations of the currently available HCMV DNA polymerase inhibitors underscore the need for new antiviral agents with alternative modes of action. The antimalarial artemisinin derivative artesunate was shown to inhibit HCMV in vitro yet has demonstrated limited antiviral efficacy in vivo, prompting our search for more potent anti-HCMV artemisinin derivatives. Here we show that the innovative artemisinin derivative artemisone, which has been screened for its activity against malaria parasites in human clinical studies, is a potent and noncytotoxic inhibitor of HCMV. Artemisone exhibited an antiviral efficacy comparable to that of ganciclovir (50% effective concentration, 1.20 ± 0.46 µM) in human foreskin fibroblasts, with enhanced relative potency in lung fibroblasts and epithelial cells. Significantly, the antiviral efficacy of artemisone was consistently ≥10-fold superior to that of artesunate in all cells. Artemisone effectively inhibited both laboratory-adapted and low-passage-number clinical strains, as well as drug-resistant HCMV strains. By using quantitative viral kinetics and gene expression studies, we show that artemisone is a reversible inhibitor targeting an earlier phase of the viral replication cycle than ganciclovir. Importantly, artemisone most effectively inhibited HCMV infection ex vivo in a clinically relevant multicellular model of integral human placental tissues maintained in organ culture. Our promising findings encourage preclinical and clinical studies of artemisone as a new inhibitor against HCMV.

Antimalarial efficacy and drug interactions of the novel semi-synthetic endoperoxide artemisone in vitro and in vivo.

OBJECTIVES: The in vitro and in vivo efficacy and drug-drug interactions of the novel semi-synthetic endoperoxide artemisone with standard antimalarials were investigated in order to provide the basis for the selection of the best partner drug. METHODS: Antimalarial activity and drug interactions were evaluated in vitro against Plasmodium falciparum by the incorporation of [(3)H]hypoxanthine. In vivo efficacy and drug interactions were assessed using the standard 4-day Peters' test. RESULTS: Artemisone was 10 times more potent than artesunate in vitro against a panel of 12 P. falciparum strains, independent of their susceptibility profile to antimalarial drugs, and consistently 4 to 10 times more potent than artesunate in rodent models against drug-susceptible and primaquine- or sulfadoxine/pyrimethamine-resistant Plasmodium berghei lines and chloroquine- or artemisinin-resistant lines of Plasmodium yoelii. Slight antagonistic trends were found between artemisone and chloroquine, amodiaquine, tafenoquine, atovaquone or pyrimethamine and additive to slight synergistic trends with artemisone and mefloquine, lumefantrine or quinine. Various degrees of synergy were observed in vivo between artemisone and mefloquine, chloroquine or clindamycin. CONCLUSIONS: These results confirm the increased efficacy of artemisone over artesunate against multidrug-resistant P. falciparum and provide the basis for the selection of potential partner drugs for future deployment in areas of multidrug-resistant malaria. Artemisone represents an important addition to the repertoire of artemisinin combination therapies currently in use, as it has enhanced antimalarial activity, improved bioavailability and stability over current endoperoxides.

Artemisinins.

Artemisinins were discovered to be highly effective antimalarial drugs shortly after the isolation of the parent artemisinin in 1971 in China. These compounds combine potent, rapid antimalarial activity with a wide therapeutic index and an absence of clinically important resistance. Artemisinin containing regimens meet the urgent need to find effective treatments for multidrug resistant malaria and have recently been advocated for widespread deployment. Comparative trials of artesunate and quinine for severe malaria are in progress to see if the persistently high mortality of this condition can be reduced.

Possible modes of action of the artemisinin-type compounds.

Artemisinin-type compounds are used for the treatment of uncomplicated and severe forms of malaria. They reduce parasitaemia more rapidly than any other antimalarial compound known, and are effective against multidrug-resistant parasites. However, uncertainties remain as to how they act on the parasite and cause toxicity. In this review, we summarize current ideas.

Artemisinin and derivatives: the future for malaria treatment?

The isolation in 1972 of artemisinin by Chinese scientists, and their development of all the derivatives now used in the treatment of malaria today, were of outstanding importance. The results which have accumulated both from the Chinese work and from that subsequently conducted on a worldwide basis provide for a relatively comprehensive understanding of the chemistry, pharmacological profiles, toxicology, metabolism, and effects on the malaria parasite. The optimal regimens for use in the field are also apparent, particularly in combinations with longer half-life quinoline antimalarials. Thus the future use of the artemisinin class of drug appears assured. However, the mechanism of action needs to be clarified. More importantly from a clinical viewpoint, problems inherent in the current derivatives must be addressed, particularly that of neurotoxicity, if new artemisinin derivatives are to be introduced in a normal drug regulatory environment. The application of established principles of modern drug design should indeed allow for the first truly rationally designed, in so far as the target is still unknown, derivatives to come to hand.

Establishment of an in vitro screening model for neurodegeneration induced by antimalarial drugs of the artemisinin-type..

The establishment of an in vitro screening model for neurodegeneration inducing antimalarial drugs was conducted in stepwise fashion. Firstly, the in vivo selective neurotoxic potency of artemisinin was tested in neuronal cells in vitro in relation to the cytotoxic potency in other organ cell cultures such as liver and kidney or versus glial cells. Secondly, a comparison between different parts of the brain (cortex vs. brain stem) was performed and in the last step, a fast and sensitive screening endpoint was identified. In summary, non-neuronal cell lines such as hepatocytes (HEP-G2), liver epithelial cells (IAR), proximal tubular cells (LLC-PK(1)) and glial cells from the rat (C6) and human (GO-G-IJKT) displayed only moderate sensitivity to artemisinin and its derivatives. The same was found in undifferentiated neuronal cell lines from the mouse (N-18) and from human (Kelly), whereas during differentiation, these cells became much more sensitive. Primary astrocytes from the rat also were not specifically involved. In the comparison of primary neuronal cell cultures from the cortex and brain stem of the rat, the brain stem was found to be more sensitive than the cortex. The neurotoxic potential was determined by cytoskeleton elements (neurofilaments), which were degradated in vitro by diverse neurodegenerative compounds. In comparison of dog and rat primary brain stem cultures, the dog cells were found to be more sensitive to artemisinin than the rat cells. In addition to the primary brain stem cell cultures it was shown that the sprouting assay, which determines persistent delayed neurotoxic effects, is also useful for screening antimalarial drugs. To other compounds, artemether and artesunate, showed that use of the sprouting assay followed by primary brain stem cultures of the rat will be a good strategy to select candidate compounds.

Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatography-mass spectrometry-selected ion monitoring.

A sensitive, selective, and reproducible GC-MS-SIM method was developed for determination of artemether (ARM) and dihydroartemisinin (DHA) in plasma using artemisinin (ART) as internal standard. Solid phase extraction was performed using C18 Bond Elut cartridges. The analysis was carried out using a HP-5MS 5% phenylmethylsiloxane capillary column. The recoveries of ARM, DHA and ART were 94.9 +/- 1.6%, 92.2 +/- 4.1% and 81.3 +/- 1.2%, respectively. The limit of quantification in plasma was 5 ng/ml (C.V. < or = 17.4% for ARM and 15.2% for DHA). Calibration curves were linear with R2 > or = 0.988. Within day coefficients of variation were 3-10.4% for ARM and 7.7-14.5% for DHA. Between day coefficients of variations were 6.5-15.4% and 7.6-14.1% for ARM and DHA. The method is currently being used for pharmacokinetic studies. Preliminary data on pharmacokinetics showed Cmax of 245.2 and 35.6 ng/ml reached at 2 and 3 h and AUC0-8 h of 2463.6 and 111.8 ngh/ml for ARM and DHA, respectively.

Extraction of artemisinin and artemisinic acid: preparation of artemether and new analogues.

The preparation of artemether from artemisinin is reviewed. Firstly, the extraction of artemisinin from Artemisia annua is described and an estimation of the yield per hectare based on literature data is given. Artemisinin is reduced with sodium borohydride to produce dihydroartemisinin as a mixture of epimers. The mixture is treated with methanol and an acid catalyst to provide artemether. Increasing demand for use of artemether places pressure on the supply of artemisinin, and an alternative means of preparing the drug from artemisinic acid, an abundant constituent of A. annua, which could triple current yields, is described. In anticipation of problems of drug resistance emerging with the continued use of artemether and artesunate to treat malaria, development of new derivatives of artemisinin which have enhanced stability is required. Examples of such derivatives which have been prepared in our laboratories, or proposed, are described.