Artemisinin A Versatile Weapon from Traditional Chinese Medicine

Thomas Efferth

Abstract Traditional Chinese medicine (TCM) commands a unique position among all traditional medicines because of its 5000 years of tradition. Our own interest in natural products from TCM was triggered in the 1990s by sesquiterpene lactones of the artemisinin type from Artemisia annua L. The first description of the Chinese herb Artemisia annua L. (qinghao, Sweet wormwood) dates back to 168 B.C.E. Artemisinin (qinghaosu) was identified in 1972 as the active antimalarial constituent of Artemisia annua L. Artemisinin and its derivatives are used for the treatment of malaria. As shown in recent years, this class of compounds also shows activity against cancer cells, schistosomiasis, and certain viruses, i.e., human cytomegalovirus, hepatitis B anc C virus, and bovine viral diarrhea virus. Interestingly, the bioactivity of artemisinin seems to be even broader and also includes the inhibition of other protozaons such as Leishmania, Trypanosoma, and Toxoplasma gondii, as well as some trematodes, fungi, yeast, and bacteria. The analysis of its complete profile of pharmacological activities, as well as the elucidation of molecular modes of action and the performance of clinical trials, will further elucidate the full potential of this versatile weapon from nature against diseases.

Keywords Artemisinin • Cancer • Chemotherapy • Malaria • Pharmacognosy • Schistosomiasis • Traditional Chinese medicine • Viral infections

Abbreviation

TCM Traditional Chinese medicine

German Cancer Research Center, Pharmaceutical Biology (C015), Im Neuenheimer Fedl 280, 69120 Heidelberg, Germany, e-mail: [email protected]

K.G. Ramawat (ed.), Herbal Drugs: Ethnomedicine to Modern Medicine, DOI 10.1007/978-3-540-79116-4_11, © Springer-Verlag Berlin Heidelberg 2009

11.1 Introduction

Traditional Chinese medicine (TCM) comprises medicinal products from plants, animals and minerals, acupuncture, moxibustion, and other practices. Herbal prescriptions consist of a varying number of different medicinal plants and are used as extracts, decoctions, concoctions, and teas. Among all traditional medicines, TCM commands a unique position because of its 5000 years of tradition. Hence, it can be assumed that many ineffective prescriptions have disappeared over time. Until recently, TCM has been frequently regarded with some skepticism by Western academic medicine. On the other hand, prominent examples of isolated therapeutics derived from Chinese plants are established in modern medicine without being treated with the same reluctance as traditional herbal products. Among them are the ion channel blocker tetrandrine (Stephania tetrandra), the CNS stimulator ephedrine (Ephedra sinica), and the well-known anticancer agents camptothecin from Camptotheca acuminata and paclitaxel from Taxus chinensis. Since natural products represent a valuable source of drug discovery and development, there has been a recently thriving interest in chemically characterized compounds derived from TCM [1-3].

Our own interest in natural products from TCM was triggered in the 1990s by sesquiterpene lactones of the artemisinin type from Artemisia annua L. [4]. Apart from artemisinin, which is the focus of this chapter, we analyzed phytochemical and molecular biological aspects of natural products derived from TCM. The modes of action were studied on known compounds with still unknown cellular and molecular mechanisms such as arsenic trioxide, homoharringtonine, cephalotaxine, berberine, cantharidin, curcumin, luteolin, scopoletin, isoscopoletin, ascaridin, the quinolones 1-methyl-2-undecyl-4-quinolone, 1-methyl-2-trideca-dienyl-4-quinolone and evo-carpine, the indoloquinazoline alkaloids rutaecarpine and evodiamine, and four ger-anylated furocoumarines [5-19]. Furthermore, novel natural products were isolated and identified from plants derived from TCM, some of which showed growth inhibitory activity against cancer cells, i.e., tetracentronsine, a new indole alkaloid (3-(2-hydroxyethyl)-1H-indole-5-O-beta-D-glucopyranoside), and two new phenol derivatives, 3-{2-[(beta-glucopyranosyl) oxy]-4,5-(methylenedioxy)phenyl} propanoic acid and methyl 3-{2-[(beta-glucopyranosyl)oxy]-4,5-(methylene-dioxy) phenyl}propanoate, two new alpha-tetralone (= 3,4-dihydronaphthalen-1(2H)-one) derivatives, berchemiaside A and B, a new flavonoid, quercetin-3-O-(2-acetyl-alpha-L-arabinofuranoside), and a diprenylated indole, (E)-3-(3-hydroxy-methyl-2-butenyl)-7-(3-methyl-2-butenyl)-1H-indole [20-22].

11.2 Use of Artemisinin in Traditional Chinese Medicine

The first description of the Chinese herb Artemisia annua L. (qinghao, sweet wormwood) dates back to 168 B.CE. The plant was mentioned in the prescriptions for 52 diseases in the Mawangdui tomb of the Han dynasty. The next historical tradition is from the year 1086, written by Shen Gua. In the "Handbook of Prescriptions for

Joachim Stadler
Fig. 11.1 Artemisia annua seedlings for the sustainable production of artemisinin (with permission of Dr. Joachim Stadler, GE Healthcare, Bio-Sciences, Freiburg, Germany)

Emergency Treatment" Ge Hong (281-340 C.E.) recommended tea-brewed leaves to treat fever and chills. The "Compendium of Materia Medica" published by Li Shizen in 1596 cited Ge Hong's prescription. In the course of the Vietnam War, the Chinese government started an antimalarial research program to systematically search for antimalarial TCM plants to support the Vietnamese army. This task was certainly not easy to fulfill during the Cultural Revolution in China [23]. As a result, artemisinin (qinghaosu) was identified in 1972 as the active antimalarial constituent of Artemisia annua L. [24,25]. Today, artemisinin is widely used around the world to combat otherwise drug-resistant Plasmodium strains, cerebral malaria, and malaria in children [26]. Since harvesting of Artemisia annua plants in the wild does not meet the requirements for a sustainable production of artemisinin, the cultivars are bred in plantations and greenhouses (Fig. 11.1). While Artemisia annua and artemisinin were evaluated by the World Health Organization (WHO) with much reluctance fora long time, the full potential has recently been recognized.

11.3 Mode of Action of Artemisinin

In malaria parasites, artemisinin acts by a two-step mechanism. It is first activated by intraparasitic heme-iron, which catalyzes the cleavage of the endoperoxide. The Plasmodium trophozoites and schizonts live within red blood cells. Hemoglobin serves as an amino acid source, being taken up by the parasites into food vacuoles where enzymatic degradation takes place [27, 28]. The release of heme-iron during hemoglobin digestion facilitates the cleavage of the endoperoxide moiety by an Fe(II) Fenton reaction. The breaking of the endoperoxide bridge results in the generation of typical reactive oxygen species such as hydroxyl radicals and superoxide anions. These damage the membranes of food vacuoles and lead to autodigestion [29, 30]. In addition, the heme-iron(II)-mediated decomposition of artemisinin generates carbon-centered radical species [31-33]. The cleavage of the endoperoxide bond of artemisinin and its derivatives leads to the alkylation of heme and some Plasmodium-specific proteins, including the P. falciparum translationally controlled tumor protein (TCTP).

As the iron storage of tumor cells is generally much less than that of erythrocytes, but is greater in tumor cells compared to normal cells [34], the question arises as to whether iron may also play a role in the inhibitory action of artemisinins toward tumor cells [35]. The growth rate of a tumor was significantly retarded by daily oral administration of ferrous sulfate followed by dihy-droartemisinin. No significant tumor growth retardation effect was observed in rats treated with either dihydroartemisinin or ferrous sulfate alone. The drug treatment did not significantly affect body weight compared with untreated tumor-implanted animals, and no apparent toxic effect was observed after drug treatment [36]. Iron(II) glycine sulfate (Ferrosanol) and transferrin increased the cytotoxicity of free arte-sunate, artesunate microencapsulated in maltosyl-P-cyclodextrin, and artemisinin toward CCRF-CEM leukemia and U373 astrocytoma cells compared with that of artemisinins applied without iron [37]. Growth inhibition by artesunate and ferrous iron correlated with induction of apoptosis. The effect of ferrous iron and trans-ferrin was reversed by monoclonal antibody RVS10 against the transferrin receptor, which competes with transferrin in binding to the receptor. The IC50 values for eight different artemisinin derivatives in the NCI cell line panel were correlated with the microarray mRNA expression of 12 genes involved in iron uptake and metabolism to identify iron-responsive cellular factors enhancing the activity of artemisinins. This analysis pointed to mitochondrial aconitase and ceruloplasmin (ferroxidase). Interestingly, exposure of artemisinins produces no or only marginal cytotoxicity to normal peripheral blood mononuclear cells (PBMC). The absorption of iron increases in growing cells and tissues, and the uptake of transferrin is related to the rate of tumor cell proliferation [38]. Cellular iron uptake and inter-nalization are mediated by the binding of transferrin-iron complexes to the trans-ferrin receptor (CD71), expressed on the cell surface membrane, and subsequent endocytosis. CD71 expression in normal tissues is limited, e.g., to the basal epidermis, endocrine pancreas, hepatocytes, Kupfer cells, testes, and pituitary, while most other tissues are CD71-negative [39]. In contrast, CD71 is expressed in much larger amounts in proliferating and malignant cells [40-42] and is widely distributed among clinical tumors [39]. We found that CD71 expression was much higher in CCRF-CEM and U373 tumor cells (48-95%) than in peripheral mononuclear blood cells of healthy donors (< 2%) [37]. This raises the attractive possibility that tumors that express more CD71 than normal cells are preferentially affected by combination treatments of transferrin or Ferrosanol plus artemisimn derivatives. The finding that iron(II) glycine sulfate increased the action of artemisinins is interesting, since Ferrosanol has been in clinical use for many years. Hence, artemisinins might be safely applied in combination with Ferrosanol in a clinical setting.

Whether oxidative stress and iron ions also play a role for artemisinins' activity against other diseases such as viral infections or schistosomiasis remains unknown.

11.4 Activity Against Malaria

Antimalarial drug resistance has spread and intensified in recent decades and represents a severe global challenge. It is estimated that 300 to 500 million human beings are infected each year and that 1.5 to 2.5 million individuals die of malaria annually [43,44]. The development of novel drugs has not kept pace even worsening the problem. Artemisinin and its derivates are, therefore, promising new drugs on the horizon that are expected to ease the malaria burden worldwide.

Drug combinations based on artemisinins offer an effective possibility to counteract drug resistance [45]. Combination regimes prolong the useful therapeutic life of existing antimalarial drugs. The probability that a mutant strain of Plasmodium simultaneously would exert resistance to two drugs with different modes of action and different therapeutic targets is low. Combinations of drugs are generally accepted to improve treatment efficacy and to delay the selection of drug-resistant parasites [46].

Despite the recommendation of WHO to use artemisinin-based combination therapies, in order to avoid the emergence of artemisinin resistance, the overall deployment of such combination regimes was still unsatisfactory [47]. For this reason, WHO banned artemisinin monotherapy in 2006.

Artemisinins proved to be valuable in drug combinations since they are able to reduce the number of parasites by approximately 104 per asexual cycle [26, 48]. Artemisinins are active within 48 to 72 h [49, 50]. This considerably reduces the number of parasites to be killed by a partner drug in a combination regimen. Since they inhibit the production of gametocytes, artemisinins are able to reduce transmission [51].

Another favorable feature of artemisinins is that they are active in uncomplicated as well as severe forms of malaria. Severe malaria does not stop with clearing parasitemia. Even if parasites are cleared, the clinical symptoms associated with cerebral malaria may get worse. Besides the brain, other organs such as kidneys or lungs can also be injured in severe malaria. Artemisinin has been proven as an effective antimalarial drug for the treatment of cerebral malaria [52]. Artemisinin derivatives reveal a very good tolerability [53, 54]. Mild and reversible hemato-logical and electrocardiographic abnormalities, such as neutropenia and first-degree heart block, have been observed infrequently. Neurotoxicity, e.g., ataxia, slurred speech, and hearing loss have been reported in few patients [55]. Due to their lack of severe side effects, artemisinins are also well suited for the treatment of malaria in children [56].

The efficacy of artemisinin and its derivatives in combination with standard antimalarial drugs has been shown in numerous clinical studies. Recent meta-analyses provide convincing evidence for the success of artemisinin-containing regimens for uncomplicated Plasmodium falciparum malaria in terms of both treatment response and beneficial profile of side effects.

A meta-analysis by Bakshi et al. [57] investigated a total of 1869 patients treated with artemether and lumefantrine (A-L). The most commonly reported adverse effects involved the gastrointestinal (abdominal pain, anorexia, nausea, vomiting, diarrhea) and central nervous (headache, dizziness) systems. Pruritus and rash were reported by more than 2% of patients. There were no serious or persistent neurological side effects and no adverse clinical cardiac events. More than 90% of the reported adverse events, many of which overlapped considerably with the clinical symptomatology or evolution of acute malaria, were rated mild to moderate in intensity.

Omari et al. [58] reported on nine trials (n = 4547 patients) that tested the six-dose regimen of A-L. Total failure at day 28 for A-L was lower when compared with amodiaquine plus sulfadoxine-pyrimethamine, but not with chloroquine plus sulfadoxine-pyrimethamine. In comparisons with artemisinin derivative combinations, A-L performed better than amodiaquine plus artesunate, worse than meflo-quine plus artesunate, and similar to dihydroartemisinin-napthoquine-trimethoprim. The authors conclude that the six-dose regimen of A-L appears more effective than antimalarial regimens not containing artemisinin derivatives.

Another meta-analysis of 16 randomized trials (n = 5948 patients) studied the effects of the addition of artesunate to standard treatment [54]. Parasitological failure was lower with 3 d of artesunate at day 14 and at day 28. Parasite clearance was significantly faster. Recrudescence and gametocyte carriage was substantially reduced. The occurrence of serious adverse events did not differ significantly between artesunate and placebo.

The meta-analysis of Bukirwa and Critchley [59] included four trials with 775 participants and compared sulfadoxine-pyrimethamine plus amodiaquine (SP plus AQ) with sulfadoxine-pyrimethamine plus artesunate (SP plus AS) for treating uncomplicated Plasmodium falciparum malaria. SP plus AQ performed better at controlling treatment failure at day 28, but was not as good as SP plus AS at reducing gametocyte carriage at day 7.

Seven randomized trials of 4472 children were included in the meta-analysis of Obonyo et al [60]. The authors compared the efficacy of amodiaquine plus sulfa-doxine/pyrimethamine (AQ + SP) versus artemisinin-based combination therapies (ACT) in the treatment of uncomplicated malaria. Treatment failure of AQ + SP was significantly reduced compared with AS + SP, but increased compared with AL. All treatment regimens were safe and well tolerated.

11.5 Activity Against Cancer

During the past dozen years, our own efforts have been focused on the activity of artemisinin and its derivatives with respect to cancer cells. Our results suggest oxidative stress as a mechanism of artesunate against cancer cells [4], [61-67]. We found that thioredoxin reductase and catalase expression correlated significantly with the IC50 values for artesunate. WEHI7.2 mouse thymoma cells selected for resistance to hydrogen peroxide or transfected with thioredoxin, manganese superoxide dis-mutase or catalase showed resistance to artesunate as compared to the parental cell line [68]. The microarray-based mRNA expression of dihydrodiol dehydrogenase, y-glutamylcysteine synthase (y-GCS; GLCLR), glutathione S-transferases GSTM4, GSTT2, GSTZ1, and microsomal glutathione S-transferase MGST3 correlated significantly with resistance to artesunate in the NCI cell line panel. A tendency for correlation (0.05 < p < 0.1) was observed for GSTA1, GSTA2, GSTP1, and MGST1. MSC-HL13 cells transfected with cDNAs for heavy and light subunits of y-GCS were more resistant to ART than mock transfected MSV-PC4 cells [69]. L-buthionine sulfoximine, a y-GCS inhibitor that depletes cellular glutathione pools, completely reversed ART resistance in MSV-HL13 cells [70].

As tumor cells contain much less iron than erythrocytes, but more than other normal tissues [34], the question arises as to whether iron may be critical for artemisinin's action with respect to tumor cells. Cellular iron uptake and internalization are mediated by binding of transferrin-iron complexes to the transferrin receptor (CD71) expressed on the cell surface membrane and subse-quent endo-cytosis. While most normal tissues are CD71-negative, CD71 is highly expressed in clinical tumors and is widely distributed among clinical tumors [39, 42, 71]. We found that CD71 expression was much higher in CCRF-CEM and U373 tumor cells (48 to 95%) than in peripheral mononuclear blood cells of healthy donors (< 2%) [37]. Iron(II) glycine sulfate (Ferrosanol) and transferrin increased the cytotoxicity of free artesunate, artesunate microencapsulated in maltosyl-P-cyclodextrin, and artemisinin towards CCRF-CEM leukemia and U373 astrocytoma cells compared with artemisinins applied without iron [37, 72]. This effect was reversed by monoclonal antibody RVS10 against the transferrin receptor, which competes with transferrin for binding to the receptor. These results are in accordance with data from other authors

The outgrowth of new blood vessels from preexisting ones is crucial for tumors to gain access to sufficient amounts of oxygen and nutrients [75]. If tumors reach a size where diffusion alone cannot supply enough oxygen and nutrients, a process termed angiogenesis is promoted by numerous proangiogenic or antiangiogenic factors. As a consequence, inhibitors of angiogenesis has raised considerable interest for cancer treatment [76]. Artemisinin and its derivatives inhibit angiogenesis, as shown by several groups including our own [77-82].

In 1996, we showed for the first time that artesunate induces apoptosis in cancer cells [4]. This result was subsequently confirmed by others [83-87]. Recently, we analyzed the precise apoptotic pathways induced by artesunate [88]. Using leukemic

T-cells as a model system, we showed that artesunate induces malignant T-cells to undergo apoptosis through the mitochondria pathway. This was demonstrated by inducing the release of cytochrome c from the mitochondria upon ART treatment and followed by activation of caspase-9, the main caspase involved in the intrinsic pathway. In contrast, no activation of caspase-8, the main caspase for the extrinsic pathway, was seen. Furthermore, cells deficient in either the death adapter molecule FADD or caspase-8 were at least as sensitive to ART as the parental cells. Further investigation of the molecular mechanisms by which ART triggers apoptosis revealed that ART induces the intrinsic death pathway by generation of reactive oxygen species (ROSs). This was confirmed by the fact that the antioxidant NAC could completely block ROS generation and, consequently, inhibited ART-induced apoptosis.

The activity of artemisinin and its derivatives in vivo has been demonstrated by several authors. Moore et al. [36] found that the growth of fibrosarcoma in Fisher 344 rats was significantly delayed by the daily application of the active metabolite of artemisinin, dihydroartemisinin, plus ferrous sulfate compared to untreated control animals. Chen et al. [79] applied a chorioallantoic membrane (CAM) assay in chicken eggs. This represents a well-established assay to analyze the development of blood vessels in vivo. In particular, the CAM assay is suited for the screening of angiogenesis inhibitors. Dihydroartemisinin significantly suppressed neoangiogen-esis by means of this test system. These results are conceivable with results of our own investigations [80]. We soaked Matrigel plugs with vascular epithelial growth factor (VEGF), tumor necrosis factor-a (TNF-a), and heparin, which act as strong stimuli for angiogenesis. The Matrigel plugs were subcutaneously injected into nude mice. In control animals without artesunate treatment, a strong vascularization blood filling of the plugs took place after 4 d. In contrast, a statistically significant reduction of Matrigel vascularization was observed in mice fed with artesunate in the drinking water. To determine the in vivo effects of artesunate on tumor growth, we subcutaneously injected KS-IMM Kaposi sarcoma cells to nude mice [80]. Whereas a strong tumor growth was found in untreated mice, it was strongly suppressed in artesunate-treated animals. These results were subsequently confirmed by other authors. Disbrow et al. [89] found that dihydroartemisinin inhibited the virus-induced tumor formation in vivo. Dogs infected with canine oral papillomavirus developed tumors in the oral mucosa. The tumor development was, however, significantly inhibited by topical application of dihydroartemisinin. Lai and Singh [90] induced breast cancer in rats by application of 7,12-dimethylbenzo[a]anthracene (DMBA). In comparison to untreated control animals, rats fed with artemisinin showed a delayed tumor development and the tumor size was smaller. Furthermore, fewer rats showed multiple breast tumors and fewer rats developed tumors at all.

The successful treatment of human xenograft tumors in nude mice with arte-sunate [80] inspired us to apply artesunate in a clinical setting. We have treated two patients suffering from uveal melanoma on a compassionate-use basis after standard chemotherapy alone was ineffective in stopping tumor growth [91]. Generally, this tumor entity has a median survival of 2 to 5 months. We did not observe additional side effects that exceed those seen with standard chemotherapy, indicating that artesunate was well tolerated. One patient experienced a temporary response after the addition of artesunate while the disease was progressing under standard therapy with fotemustine alone. The patient died after 24 months. The second patient first experienced a stabilization of the disease after the addition of artesunate plus iron to the standard drug dacarbazine followed by objective regressions of splenic and lung metastases. This patient is still alive 47 months after first diagnosis of stage IV uveal melanoma. This promising result indicates that artesunate might be a valuable adjuvant drug for the treatment of melanoma and other tumors in combination with standard chemotherapy. The treatment of single cases of a laryngeal squamous cell carcinoma with artesunate [92] and a pituitary macroadenoma with artemether has been reported recently [93]. Larger clinical trails are required to establish artesunate for cancer therapy in the clinical setting.

11.6 Activity Against Schistosomiasis

It is estimated that more than 200 million people are affected with schistosomiasis caused by blood flukes (Schistosoma haematobium, S. intercalatum, S. japon-icum, S. mansoni, and S. mekongi) [94-96]. While in some countries Schistosomiasis seems to be under control, i.e., Brazil, China, and Egypt, other regions suffer from this disease, i.e., sub-Saharan Africa [97, 98]. Affected hosts develop immuno-logical reactions against the parasite eggs in the host tissues ranging from allergic reactions at early stages to considerable morbidity in chronic phases later ones. Typical symptoms of schistosomiasis are exercise intolerance, anemia, delayed cognitive development, and growth retardation. Praziquantel is a standard drug in the treatment of the disease that enables one to control morbidity in affected areas [98].

Again, Chinese scientists were the first to observe that artemisinin and its derivatives were active against Schistosoma infections [99] - a result that was subsequently confirmed by authors outside China (as reviewed by Utzinger et al. [100]).

In contrast to praziquantel, which shows highest activity against adult worms (and very young schistosomula), detailed studies in a mouse model revealed that 2-to 3-week-old schistosomula were killed more efficiently by artemether than adult worms [100]. Artemether is most active against juvenile Schistosoma parasites, and prevents morbidity associated with schistosomiasis, since the egg-laying worms do not develop [101]. In combination with praziquantel, which is most active against adult worms, all developmental parasite stages can be attacked [102].

As reviewed in a recent meta-analysis by Utzinger et al. [100], 16 randomized, placebo-controlled clinical trials were conducted with oral artesunate for the prevention of S. japonicum infection, which demonstrated convincingly the efficacy of the drug combating the disease. Eight randomized, placebo-controlled clinical trials were conducted with artemether. The meta-analysis of Utzinger et al. (2007) [100] also showed the utility of this drug to treat schistosomiasis. Artemisinins also revealed a good safety profile when used to prevent Schistosoma infections [103, 104].

11.7 Activity Against Viral Infections 11.7.1 Human Cytomegalovirus

Although ART and other artemisinin derivatives have been described as antimalaria drugs [24], their antiproliferative activity is not restricted to protozoans [4, 65]. We investigated whether ART had antiviral activity and to identify possible underlying molecular mechanisms. We found that ART effected a strong inhibition of plaque formation of HCMV AD169 and HSV-1, a partial inhibition of HIV-1, but no inhibition of influenza A virus [105]. Concerning the inhibitory potential for HCMV, it was important to demonstrate that viruses with various phenotypes, i.e., natural isolates, resistant mutants, and laboratory strains and recombinant virus clones, were all highly sensitive to treatment with ART. A possible mechanism is provided by the finding that central regulatory processes of the infected cell were inhibited by ART, thus interfering with critical requirements of HCMV replication in terms of host cell type and metabolism.

Human cytomegalovirus (HCMV) infection can cause maldevelopment of the central nervous system of embryos and neonates [106]. HCMV can be fatal to im-munocompromised adults, for example, organ transplant recipients and patients with acquired immunodeficiency syndrome [107]. Furthermore, the virus is indirectly involved in the etiology of certain tumor types (e.g., by the synergistic interaction with tumor-inducing viruses), indicating its role in the coregulation of cellular proliferation [108]. HCMV infections are generally treated with the nucleoside/nucleotide analogs ganciclovir (GCV) and cidofovir (CDV) or the inorganic pyrophosphate analog foscarnet [109-111], all of which cause adverse side effects and reveal low oral bioavailability [112]. In addition, the therapeutic effectiveness is frequently compromised by the emergence of drug-resistant virus isolates. A variety of amino acid changes in the UL97 protein kinase and the viral DNA polymerase have been reported to cause drug resistance [113]. For this reason, the identification of novel drugs with activity toward drug-resistant HCMV variants with a low level of toxic side effects is urgently needed. A possible mechanism is provided by the finding that central regulatory processes of the infected cell were inhibited by ART, thus interfering with critical requirements of HCMV replication in terms of host cell type and metabolism. In particular, the replication of HCMV is tightly coregulated with cellular activation pathways, mediated by direct or indirect interaction with cellular DNA-binding factors such as NF-kB or Sp1. These factors provide a major determinant of the virus-host cell interaction. Moreover, there are several examples demonstrating that chemical compounds interfering with activation pathways of cellular transcription factors (e.g., the signal transduction pathway including mitogen-activated protein kinase p38) possess a strong inhibitory effect on the replication of HCMV. As ART and other artemisinin derivatives have captured the attention of scientists and physicians concerned with malaria treatment for its activity toward multidrug-resistant Plasmodium strains [51], we were interested in analyzing whether ART was active toward drug-resistant HCMV as well. Indeed, we observed that the GCV-resistant strain AD169-GFP314 was inhibited with similar efficacy as the drug-sensitive parental AD169-GFP virus. It is obvious from these experiments with GCV-resistant HCMV that the putative inhibitory mechanisms of ART must differ from those of the DNA polymerase inhibitor GCV.

We were further intrigued by the question of how the anti-HCMV effect is mediated at the cellular level. Upon the infection of cultured cells with HCMV, immediate early (e.g., IE1-72, IE2-86), early (e.g., UL84), and late genes (e.g., UL94) are expressed. IE gene products are essential for the subsequent expression of viral early and late genes, all of which are involved in viral replication. A reduction in IE2-86 expression is generally considered a critical limitation in viral replication since IE2 gene products are essential for HCMV infection [114]. It is also noteworthy that the viral immediate early promoter enhancer (in addition to other viral promoters) contains binding sites for both Sp1 and NF-kB and therefore is responsive to both factors [115]. Thus the activation pathways involving Sp1 and NF-kB are important factors in the initial onset of the viral replication cycle and might be critical in the mechanism of ART's inhibition of HCMV. In particular, the replication of HCMV is tightly coregulated with cellular activation pathways, mediated by the direct or indirect interaction with cellular DNA binding factors such as NF-kB or Sp1 [116,117]. These regulatory events are required for viral replication and provide a major determinant of the efficiency of virus multiplication. The present investigation demonstrated that ART inhibits the HCMV-induced DNA binding activities of both NF-kB and Sp1. For both NF-kB and Sp1 we observed a drastic reduction in HCMV-induced Sp1 protein synthesis under ART treatment, which explains (at least in most part) the reduced DNA binding activity. This is a clue that ART's anti-cytomegaloviral activity is mediated by inhibition of transactivation of NF-kB and Sp1 transcription factors. Although we have shown that NF-kB and Sp1 binding activities are molecular targets of ART, we cannot rule out the possibility that other mechanisms may also contribute to ART's antiviral action. Infection of quiescent human fibroblasts with HCMV was found to cause a rapid activation of cellular PI3-K. PI3-K regulates phosphorylation of a number of kinases in cellular signaling cascades, including Akt (also known as protein kinase B), cyclic AMP-dependent kinase (protein kinase A), some isoforms of protein kinase C, and the ribosomal S6 kinases p70 and p85 (p70S6K and p85S6 K, respectively). Akt and p70S6K are two major downstream effectors of PI3-K and are strongly activated upon HCMV infection, in a manner similar to the PI3-K-dependent activation of NF-kB and Sp1 [118]. Akt activation is needed for HCMV DNA replication. It is very likely that the ART inhibition of HCMV replication is targeted to the initial stage of viral signaling. In particular, those kinase cascades are suggestive to be involved that are already induced by the interaction of the viral ligands with the cellular receptor. Thus inhibition of PI3-K activity inhibits viral replication and virus-induced signaling [118].

Currently, a limited number of drugs is used for the treatment of a systemic or locally reactivated HCMV infection: (1) ganciclovir (GCV), (2) its prodrug valgan-ciclovir, (3) cidovovir (CDV), (4) foscarnet (FOS), and (5) fomivirsen. Nevertheless, each of these drugs has a number of disadvantages. First, these antiviral compounds are usually administered intravenously or intravitreally, except for valganciclovir, which possesses improved oral bioavailability. In addition, prolonged treatment with each one of these drugs is frequently accompanied by serious side effects. Moreover, GCV, valganciclovir, CDV, and FOS have similar mechanisms of action by targeting, either directly or indirectly, the viral DNA polymerase. Treatment with any of these antiviral agents may therefore ultimately result in the emergence of single or multiresistant HCMV mutants [119, 120]. These considerations have promoted an intense search for novel therapeutic agents that are safe, potent, and act as alternative antiviral targets.

We demonstrated that the antiviral activity of ART against CMV is not restricted to human strains, but also includes animal CMVs, in particular RCMV [121]. An important feature seems to be the finding that increased intracellular iron concentrations in the presence of ART significantly enhance its anti-CMV activity. This enhancing effect was demonstrated by several observations: (1) treatment of CMV-infected fibroblasts with ART combined with ferrous iron (Ferrosanol) and/or soluble transferrin resulted in enhanced suppression of viral replication; (2) the expression of a cell surface marker (Thy-1), which is associated with the proinflammatory effect of CMV infection and which is not affected by established antiviral drugs, is strongly influenced by ART treatment; (3) the antiviral activity of ART against CMV could also be demonstrated in vivo using the RCMV/rat model; and, finally, (4) the antiviral activity of ART is additive when applied in combination with conventional drugs such as GCV, CDV, and FOS. The last finding might be particularly helpful in the treatment of HCMV disease inflicted by mutant viruses that are resistant to conventional antiviral drugs [122]. GCV, CDV, and FOS are all directed at an identical target of viral replication (i.e., DNA synthesis mediated by the viral DNA polymerase) and, consequently, cross-resistance conferred by polymerase-related mutations has frequently been reported [113]. The combination of drugs with different modes of action may delay the development of drug resistance in a clinical setting. Therefore, using an antiviral drug that targets an alternative pathway and does not interfere with the activities of conventional antiviral drugs seems highly promising. Moreover, Ferrosanol is a clinically approved medication and has been in practical use for many years. It is a safe drug without severe toxicity and can, hence, be safely combined with ART.

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