Chapter 18 The Anti-Infective and Anti-Cancer Properties of Artemisinin and its Derivatives

CHAPT ER

18 The Anti-Infective and Anti-Cancer Properties of Artemisinin and its Derivatives Christopher Paul Hencken*, Alvin Solomon Kalinda* and John Gaetano D’Angelo**

Contents

1. Introduction 2. Antiparasitic Uses of Artemisinin 2.1 Antimalarial activity 2.2 Anti-toxoplasmosis activity 2.3 Anti-leishmaniasis activity 2.4 Anti-shistosomiasis activity 2.5 Anti-trypanosomiasis activity 3. Other Therapeutic Uses of Artemisinin 3.1 Antiviral activity 3.2 Antifungal activity 3.3 Anticancer activity 4. Toxicity 4.1 Evidence in favor of safety 4.2 Evidence of neurotoxicity 5. Conclusion References

359 360 360 365 367 368 369 370 370 371 372 373 374 374 375 375

1. INTRODUCTION Artemisinin, a 1,2,4-trioxane sesquiterpene lactone with an atypical endoperoxide moiety, was isolated as the active component from the * Johns Hopkins University, 3400N. Charles Street, Baltimore, MD 21218, USA ** Alfred University, 1 Saxon Drive, Alfred, NY 14802, USA Annual Reports in Medicinal Chemistry, Volume 44 ISSN: 0065-7743, DOI 10.1016/S0065-7743(09)04418-2

r 2009 Elsevier Inc. All rights reserved.

359

360

Christopher Paul Hencken et al.

Chinese medicinal plant commonly known as Qinghaosu (Artemisia annua) [1,2] and was found to be extremely active against the deadly cerebral form of malaria. While it has become the new cornerstone of antimalarial treatment, it has biological applications that reach far beyond this one disease. These applications include activity against toxoplasmosis, leishmaniasis, viral infections, certain bacterial infections, shistosomiasis, and cancer. The use of artemisinin and its derivatives as a broad-spectrum anti-infective agents was recently patented [3]. It has been shown that the active pharmacophore is the 1,2,4-trioxane, specifically (and perhaps strangely), the peroxide unit [4]. In this chapter, we have paid particular attention to the activity of artemisinin-based compounds beyond the well-documented area of malaria, while providing a cursory overview of their antimalarial properties. For the interested reader, we suggest some recent reviews on the antimalarial activity of artemisinin and its derivatives [5–7]. For the purposes of saving space, we will use the following short hand for the structural core of artemisinin (1) throughout the chapter. H O H

O O O C-10

O C-10

O

O

1

1

2. ANTIPARASITIC USES OF ARTEMISININ The use of herbal teas brewed from A. annua against fever and chills dates to AD 340; however, the first mention of its use dates as far back as 168 BC [8]. Since that time, artemisinin has been identified as the active constituent of A. annua in 1972 [4]. More recently, the uses of artemisinin have been shown to be quite vast. The wide therapeutic applications of artemisinin make it difficult to attribute artemisinin’s effectiveness to a common mechanism. Since intra-cellular/parasitic metabolic degradation of artemisinin is known to induce the formation of oxygen and carbon-centered radicals, radical-induced cell death is certainly one attractive possibility. Despite the increasing known breadth of activity, the most popular uses

Anti-Infective and Anti-Cancer Properties of Artemisinin

361

remain the antiparasitic uses. These uses, though, are not necessarily limited to antimalarial (Section 2.1 for which artemisinin has been made famous), anti-toxoplasmosis (Section 2.2), anti-leishmaniasis (Section 2.3) anti-shistosomiasis (Section 2.4), and anti-trypanosomiasis (Section 2.5).

2.1 Antimalarial activity Malaria (from Italian origin, ‘‘aria male,’’ meaning bad air) is caused by an erythrocytic protozoan parasite first identified in 1880 by Alfonse Laveran [7]. Every year, between 300 and 500 million people are infected with malaria in the endemic areas (Africa, India, southeast Asia, the Middle East, Oceania, and Central/South America) with 1–2 million of these infected people dying each year and a child dying of malaria approximately every 30 s. Human malaria is caused by four species of the genus Plasmodium; vivax, ovale, malariae, and falciparum. The falciparum species is responsible for the majority of human deaths from malaria. Humans contract malaria when bitten by the female of any one of the 60 species of Anopheles mosquito [9]. The life cycle of the parasite from mosquito to human blood, to the human liver, back to the blood, and back to another mosquito is well-known [9,10]. The antimalarial mechanism of action for the artemisinin class of compounds is the most extensively studied of its mechanisms of action. It is possible, though unproven, that all of the potential mechanisms, two prevailing hypotheses presented below, are at work. It is now widely believed that the 1,2,4-trioxane class of antimalarials, including artemisinin, exert their activity on the erythrocytic stage of the parasite life cycle. Additionally, the fact that the endoperoxide is vital for artemisinin’s antimalarial activity has been well-established [4]. The most widely agreed-upon view is that liberated heme acts as the source of FeII responsible for the activation of the endoperoxide bridge of artemisinin (1) to produce cytotoxic radical species. The liberated heme results from the breakdown of hemoglobin that Plasmodium uses as a food source. The hemoglobin is transported to the parasitic food vacuole where it is digested becoming amino acids essential to parasite life and the aforementioned free heme that is toxic to the parasite. The parasite then detoxifies the free heme by converting it to hemazoin, a polymeric form of heme. It is believed that this potentiated heme in the form of hemazoin is what activates the artemisinin family of compounds. The activation cascade is initiated by reduction of the peroxide bond with heme FeII to give an oxy radical and oxidized heme FeIII that can lead to a number of subsequent radical entities, all of which may be lethal to the parasite. This mechanism has been called into question by others who believe artemisinins disrupt parasitic calcium homeostasis by targeting sarco/endoplasmic reticulum Ca+2-ATPases (SERCAs) [11,12]. Artemisinin

362

Christopher Paul Hencken et al.

and thapsigargin, a sesquiterpene lactone with structural similarities to artemisinin and known SERCA inhibitor, were shown to be similarly potent as inhibitors of a P. falciparum SERCA ortholog (PfATP6) in Xenopus oocytes. However, this has been contested by the Posner lab with their demonstration that enantiomers of a fully synthetic trioxane had the same level of activity [13]. Were the SERCA enzymes the primary target, one would expect only one of the enantiomers to be active as is seen in other enzyme-based mechanisms. Additionally, the epimers of artemether (3) showed nearly equal in vivo activity (1.02 mg/kg of body weight for the a epimer vs. 1.42 mg/kg of body weight for the b epimer) [14] furthering the heme-based mechanism over the SERCA-based mechanism where a greater disparity in the activities would be expected. Some final points regarding the mechanism bear mentioning here. First, it was recently shown [15] that there exists a strong correlation between a set of fully synthetic trioxolanes’ ability to alkylate heme in the presence of FeII and their respective in vitro antimalarial activity. Curiously, artemisinin-derived derivatives were less potent heme-alkylators, suggesting possible alternative modes of action for the more structurally complex artemisinin derivatives. Another study [16] found that endoperoxides tagged with a fluorescent label localized within the parasitic digestive vacuole. It was proposed that heme-iron activation then followed, allowing parasite membrane damage to occur. Despite this promising progress, more work must be done to fully elucidate the mechanism of these compounds. For a more detailed discussion, we direct the interested reader to work done by the Posner lab and others [13,16], which supports this accepted mechanism, especially evidence of the alkylation of heme by artemisinin [17], even in malaria-infected mice [18], and those that argue against it, especially the work by the Haynes lab [5,6,19–21]. Despite the debate that still surrounds the mechanism of action, this family of compounds has displayed and continues to display an exceptional safety profile and very rapid, high levels of activity. However, artemisinin and dihydroartemisinin (DHA) (2) do suffer drawbacks such as low bioavailability and recrudescence of the disease. Even with these drawbacks, they are especially effective against severe cerebral malaria, one of the most lethal forms of the disease. Arteether (4), the ethyl ether of DHA, was designed with the intent on optimizing the lipophilicity of artemisinin to improve passive permeability through cell membranes and improve bioavailability. This is especially important in the treatment of cerebral malaria where the treatment needs to permeate the bloodbrain barrier (BBB). The BBB requires compounds to be fairly lipophilic with an octanol-water partition coefficient, or logP, value of W2. That is to say that the compound in question is two times more soluble in octanol than it is in water. Arteether, logP of 3.99, is more lipophilic than the parent artemisinin, logP of 2.94, and therefore is expected to be more effective against cerebral malaria. Arteether is also fast-acting and effective

Anti-Infective and Anti-Cancer Properties of Artemisinin

363

against drug-resistant strains of Plasmodium. Notably, the World Health Organization selected arteether as an emergency treatment for individuals infected with malaria. Sodium artesunate, the sodium salt of artesunic acid (5), is well-tolerated and less toxic than (1) and along with (5) represents an example of one of the few water-soluble derivatives [22].

O C-10 O

O C-10

O OR 2 R=H 3 R = CH3 4 R = CH2CH3

HO O 5

To combat the shortcomings of first generation endoperoxides such as metabolic instability, short plasma half-life, poor bioavailability, and chemical instability, a new generation of molecules has been designed. One new generation of molecules, the C-10 carba analog class, replaces the exocyclic C-10 carbon–oxygen bond with a carbon–carbon bond, resulting in derivatives with no exocyclic oxygen group at C-10. The range of C-10 analogs is large and varies from fluorinated substituents [23] to amino sulfones [24] to differing-length linked dimers [25]. Begue’s lab sought to improve the activity of artemisinin by the addition of fluorinated alkyl groups to a variety of artemsinins. The most active of these derivatives, (6), substitutes a trifluoromethyl group for the C-10 hydrogen of (2) creating a trifluoromethyl alcohol moiety. When tested in vitro against the D6 and W2 strains of falciparum malaria, (6) was active at very low levels, 2.6 and 0.9 nM, respectively. When (6) was given subcutaneously to P. bergei-infected mice, ED50 was 0.7 mg/kg and ED90 was 1.8 mg/kg, but when given orally, the ED50 and ED90 values were 4.3 and 13.0 mg/kg respectively. The oral values were not significantly better than those for sodium artesunate (5.4 mg/kg for ED50 and 15.3 mg/kg for ED90) while those for the subcutaneous route of administration were appreciably better than those for sodium artesunate (2.8 mg/kg for ED50 and 10.4 mg/kg for ED90) [23].

O C-10

OH

F3C 6

The Haynes lab prepared and tested several C-10 nitrogen derivatives against P. bergei of which (7) demonstrated the best mix of lipophilicity

364

Christopher Paul Hencken et al.

(logP of 2.49), ease of preparation (crystalline compound that is easily purified), safety parameters (considered ‘‘nontoxic’’ by the author), and activity. The activity of (7) was tested against chloroquine-sensitive P. bergei-infected and chloroquine-resistant P. yoelii-infected mice. When administered subcutaneously, the ED90 values were 1.5 and 3.9 mg/kg, respectively. When given orally to the mice the values were 3.1 and 5.0 mg/kg, respectively [24].

O

C-10

N

S O

O 7

The in vivo mouse activity results for several C-10 isobutylene-linked dimers prepared by the Posner lab showed these analogs to be orally ‘‘curative’’ 30 days post-infection at doses of 3  30 mg/kg. Eight derivatives were shown to double the lifetime of the animals relative to the control animals at 3  10-mg/kg doses. Shown below are the most active derivatives prepared and tested by the Posner lab to date. Both (8, 9) were curative at 3  30 mg/kg, while 3  10 mg/kg dosing of (8) displayed 17.7 days average survival post-infection and 3  10 mg/kg dosing of (9) displayed 16.3 days average survival post-infection. Control animals (drug-delivery vehicle only) survived 6–7 days in both dosing groups. Animals given (1) survived 7.2 days at 30 mg/kg and 6.5 days at 10 mg/kg [25].

O C-10

O C-10

O C-10

O C-10

O O O

N O

N H N

8

9

365

Anti-Infective and Anti-Cancer Properties of Artemisinin

Curiously, a family of hybrid analogs related to (10), which incorporate a traditional antimalarial drug into a hybrid molecule with (1), showed moderate increases in activity compared to (1), but less activity than (3) [26]. This is somewhat surprising when it is considered that these drugs should have the potential for multiple mechanisms of action. It is possible that the parasite was able to pump the compound out of the cell due to the presence of the chloroquine unit based on the accepted mechanism of malaria resistance to chloroquine.

O C-10 O O N O C-10

Cl

HN

H N

H N

HO

N 3

3

N

O N

O

10

Cl

N chloroquine

11

In another recent study, an artemisinin-quinine hybrid was found to have potent antimalarial activity with IC50 values ranging from 8.95 nm to 10.4 nM against various strains of P. falciparum [27]. Notably, hybrid analog (11) was found to have more potent activity than quinine or artemisinin alone and, even more importantly, more potent activity than a 1:1 molar concentration of artemisinin and quinine combination treatment. Together, these studies make the important point that artemisininbased compounds can be chemically combined with other drugs leading to increased biological activity compared to combined treatment with two separate drugs. Due to the fact that combination therapy has become the recommended form of treatment for malaria, HIV, cancer, and many other diseases, artemisinin’s tolerance of such incorporation is highly important. Furthermore, with the fact that combination therapy often involves a complicated pill-taking regimen, a molecule that incorporates both drugs is an important simplification of treatment that will lead to less drug failure due to poor patient compliance. As an added benefit, as with (11), these hybrid analogs have been shown to possess better activity than even a 1:1 molar concentration treatment of the two drugs alone.

366

Christopher Paul Hencken et al.

2.2 Anti-toxoplasmosis activity Toxoplasma gondii is an apicomplexan protozoan parasite that infects humans and has been linked to chronic neuropsychiatric diseases and behavioral abnormalities [28]. Toxoplasmosis is most commonly transmitted by consumption of undercooked, contaminated meats; contaminated water; or contact with feces from an infected cat. In most immuno-competent humans, the disease is asymptomatic; however, there are serious fetal complications if acquisition of the disease occurs near conception. In immunocompromised individuals, such as HIV patients or organ transplant patients, systemic infection and widespread organ damage can occur [29]. The first reported activity of artemisinin against T. gondii was in the early 1990s by Ou-Yang and co-workers [30]. More recently, new C-10 carba derivatives such as (12) have been prepared by the Posner lab demonstrating an IC50 ¼ 1.2 mM [31]. An additional study by the same lab revealed multiple potential mechanisms of action. In this work [32], derivatives of artemisinin (13, 14) were found to not only inhibit growth (IC50 ¼ 1.0 and 1.7 mM, respectively) of the parasite but also result in parasite death and prevent entry of the parasite into the cells. Of particular note, a derivative lacking the endoperoxide (15) was found to be more effective at inhibiting entry into the cell by the parasite than its peroxide-containing analog. This result suggests, especially when it is considered that this same nonperoxide derivative showed no growth inhibition and no parasite death, that entry inhibition may not be dependent on the peroxide while growth inhibition clearly is. H no peroxide O

O C-10

H

O O

Br

O C-10

O C-10

O C-10 O S

N

S

N

O

12

13

14

15 inactive

At this time, no mechanism of action for the observed T. gondii activity has been proposed. However, recent studies have implicated calcium homeostasis as a possible mechanism of action of artemisinin against apicomplexa through its interaction with SERCA-type Ca2+

Anti-Infective and Anti-Cancer Properties of Artemisinin

367

ATPase [33], similar to what has been proposed for malaria. However, it is also believed that activation of the peroxide by ferrous iron is essential and that the likely source of this ferrous iron is heme. Given the close proximity of the molecule to heme upon activation, it has also been argued that heme alkylation may be the mechanism of action, especially with respect to malarial activity, as described earlier. This is a less likely mechanism in the case of T. gondii, which perhaps is explained by the SERCA-based mechanism. To date, no study has been done that investigates the relative effectiveness of two enantiomers for T. gondii like was done for malaria disallowing for the same counterargument made with malaria. More work must be done to fully elucidate the mechanism of action against this target. It is possible, though thus far unproven, that these two similar parasites share a biological target for artemisinin. It is appropriate to point out here that artemisinin is not the only peroxide-containing molecule that has shown activity against T. gondii or malaria. For example, see the work by Chang and co-workers [34] and the work of Vennerstrom [15] who demonstrated that fully synthetic peroxides (16, 17) also possess activity against Toxoplasma and malaria, respectively. The results of Chang’s study [34] suggest that 1,2,4-trioxanes were able to block nucleotide synthesis of intracellular parasites. While it is possible that artemisinin-derived analogs have the same mode of action; there is currently no proof this is the case. O

Ph

Ph

O

O

Ph

O

O

Ph

O

O

16

17

IC50 = 5.98 µM

IC50 = 71.7 µM

2.3 Anti-leishmaniasis activity Leishmaniasis is a widespread disease that takes three major forms in humans: cutaneous leishmaniasis, mucocutaneous leishmaniasis, and the potentially lethal visceral leishmaniasis. All of these forms are caused by various protozoan parasites of the genus Leishmania and are transmitted by the female sand fly. Most of the visceral leishmanaiasis cases have been related to HIV infections [35]. Similar to the antimalarial activity, a wide variety of derivatives of artemisinin have been prepared to target

368

Christopher Paul Hencken et al.

leishmaniasis, with substitutions on different regions of the artemisinin parent molecule. All of the derivatives presented here are known to also possess antimalarial activity. CF3 Cl

O C-10

O C-10

CF3

Cl O

19

18

CF3

O C-10

O 20

Several derivatives (18–20) have been reported to have quite potent activity against Leishmania, notably (19), whose IC50 is a very impressive 0.3 mM [36].

2.4 Anti-shistosomiasis activity Schistosoma mansoni and other schistosoma flatworms are the causative agents for schistosomiasis. The schistosomas are ordinarily located in the blood vessels of the human host. Although S. mansoni is the most widespread schistosoma, others such as Schistosoma japonicum and Schistosoma haematobia are also known. The parasite is contracted by humans through contact with infected water by way of direct penetration of the human skin. After in vivo maturation, severe cases give rise to fibrosis of the liver and hepatosplenomegaly. Artemisinin derivatives, especially (3), are known to have activity against all of the schistosomas [37]. A recent clinical study in Sudan, where artesunate–sulfamethoxypyrazine–pyrimethamine or artemether-lumefantrine combinations were administered to patients co-infected with P. falciparum and S. mansoni, showed complete clearance of both parasites [38]. Curiously,

Anti-Infective and Anti-Cancer Properties of Artemisinin

369

a separate study found a lower cure rate for artesunate–sulfadoxide– pyrimethamine combinations compared to praziquantel alone [39]. In another study where schoolchildren infected with Schistosoma haematobium were treated with artesunate or praziquantel, both drugs demonstrated reduced egg counts in patients at a single dose of 40 mg/kg of praziquantel and 20 mg/kg artesunate. However, praziquantel was found to perform better [40], consistent with the aforementioned results [39].

2.5 Anti-trypanosomiasis activity The two most common human-afflicting trypanosomal infections are American and African trypanosomiasis. The American type, Trypanosoma cruzi, more commonly known as Chagas disease (named for Carlos Chagas, its discoverer in 1909), is passed from the feces of triatomine bugs to humans and affects 8–11 million people in South and Central America and Mexico. Many infected individuals are unaware of their infection and if left untreated will be lifelong and possibly fatal. The triatomine bugs are commonly found in earthen-made structures common in the rural regions of the affected countries. The African type, more commonly known as African sleeping sickness, is further distinguished based on the region of Africa where the infection was contracted. Trypanosoma brucei rhodesiense is responsible for East African sleeping sickness and Trypanosoma brucei gambiense is responsible for the West African form. Both types of African sleeping sickness are contracted from the painful bite of the honeybee-sized tsetse fly with 50,000–70,000 cases reported annually. T. b. gambiense is responsible for the majority of the African cases [41,42]. A recent report demonstrated the efficacy of (1, 2, 7, 21) against T. b. rhodesiense, T. cruzi, and L. donovani in vitro.

O

C-10

O

C-10

N

S O

O

7

F

21

370

Christopher Paul Hencken et al.

Both (7) and (21) exhibited IC50 values similar to that of (1) and (2) against both trypanosomes tested. The demonstrated IC50 values for (1), (2), (7), and (21) against T. cruzi were 13.4, 12.8, 23.3, and 17.9 mM, respectively. Likewise, the values against T. b. rhodesiense were 20.4, 24.6, 22.5, and 15.7 mM, respectively [43]. Recently, hybrid derivatives have been reported that incorporate either a moiety that permits targeted delivery of the drug (22, 23) or a second active unit with an ideally different mechanism of biological activity (24, 10). MeO NH2 N

O

N

O C-10

O N

N H2N

N

N H

O

X

22 X = O 23 X = NH

O

N

C-10 O

24 Cl

Chollet et al. recently demonstrated the superior activity of (22) and (23), compared to artesunate against all evaluated strains of Trypanosoma brucei [44]. T. brucei is known to take up diamidine, thus the superior biological activity of the diamidine-containing (22) and (23) was speculated to be due to targeted delivery of the artemisinin-based drug. However, no direct evidence of such an increase in delivery was provided.

3. OTHER THERAPEUTIC USES OF ARTEMISININ Although the antiparasitic uses of artemisinin are much more common, there are other uses that are becoming more prevalent. For example, antiviral (Section 3.1), antifungal (Section 3.2), and anticancer (Section 3.3) properties are discussed here. Although the anticancer activity is mentioned rather briefly here, the anticancer properties of this family of compounds are at least as well-documented as each of the antiinfective properties, with the only exception being malaria.

3.1 Antiviral activity One of the earliest reports of artemisinin’s antiviral activity came in the early 1980s [45]. Sodium artesunate has been shown to inhibit the human

Anti-Infective and Anti-Cancer Properties of Artemisinin

371

cytomegalovirus (HCMV) (IC50 ¼ 5.9 mM against the AD169 strain with 91% inhibition at 15 mM) [34] and the Herpes simplex virus type 1 (93% inhibition at 15 mM for a clinical isolate, no IC50 reported) [46]. It was found that sodium artesunate inhibited central regulatory processes of HCMV-infected cells such as activation pathways dependent on NF-kB or Sp1 [46]. This was suggested to interfere with critical host-cell-type interactions and metabolism requirements for viral replication. However, since other sesquiterpene lactones have shown similar anti-HCMV activity [47,48], this suggests it may not be the endoperoxide that is responsible for the activity. It should be stated, however, that no mechanism of action has been elucidated to date and that although artemisinin does contain a lactone, sodium artesunate does not. Sodium artesunate has also been found to inhibit the Epstein–Barr virus [49]. Furthermore, sodium artesunate was found to be active at 600 nM concentration against both the M-tropic and the T-tropic HIV-1 strains [46]. Although this concentration is quite high, given the relative safety of the artemisinin family of drugs, even treatment at this high concentration may be well-tolerated. Considering that this is thus far an unused method of HIV treatment, the possibility becomes more attractive. Curiously, a separate study investigating patients with HIV/malaria co-infection not only showed delayed clearance of P. falciparum but no report of anti-HIV activity for artemisinin [50]. It was also found that hepatitis B virus (HBV) DNA release was inhibited by sodium artesunate at an IC50 of 0.5 mM with host cell viability being reduced at 20 mM, and the compound also showed activity against HBV at W10 mM. Meanwhile, artemisinin was found to inhibit hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two HCV subgenomic replicon constructs at concentrations that did not affect the host cells. The reader is referred to recent broad reviews [47–49] and a recent patent [51], describing the antiviral activity of artemisinin and its semisynthetic derivatives, for more information and additional references.

3.2 Antifungal activity There have been several accounts of artemisinin derivatives possessing antifungal activity. For example [52], it has been shown that a-arteether (4) inhibits various genotypes of the EG-1-103 and F-400 strains of Saccharomyces cerevisiae at minimal inhibitory concentrations of 2.0 and 1.0 mg/mL respectively. Furthermore in another study [53], a wide variety of artemisinin-derived compounds (25–27) displayed activity against the yeasts Canadida albicans and Cryptococcus neoformans at IC50 values ranging from 0.045 mg/mL to 30 mg/mL. Curiously, a deoxo derivative (27) related to 15 also displayed marginal activity, suggesting

372

Christopher Paul Hencken et al.

the peroxide may not be the active pharmacophore in this application. Generally, the activity against C. albicans was far weaker than C. neoformans and in some cases was completely absent.

O

H

O

O

O

O

H

O H

O

HO HO 25

26

27

It was also recently discovered [54] that artemisinin displayed growth inhibitory properties against a set of isogenic S. cerevisiae strains that carried disruptions of the major multidrug ABC transporter genes in the multidrug-resistant PDR1-3 background. In this study, a synergistic effect was observed between artemisinin and ketoconazole where genotypes (PDR1-3 and PDR1) that displayed minimum inhibitory concentrations (MICs) W200 mg/mL with artemisinin alone displayed MICs of 25 and 3 mg/mL, respectively, when artemisinin was employed to potentiate ketoconazole. Against two other strains (D5 and D1D2D5), artemisinin alone possessed an MIC of 50 mg/mL. To date, there is no clear explanation that accounts for the antifungal activity of the artemisinin family of compounds.

3.3 Anticancer activity Nearly 1.5 million Americans were diagnosed with cancer in 2008 with more than one-third of them dying from the disease. Cancer (sarcoma, carcinoma, leukemia, lymphoma/myeloma, and central nervous system cancer) treatments have come to include radiation therapy, chemotherapy, surgery, and other treatment methods including anti-angiogenesis therapy, gene therapy, and hyperthermia [55]. The anticancer properties of artemisinin have been under in vivo investigation since the 1980s. Anfosso et al. demonstrated a correlation between angiogenesis-related genes and the cellular response to artemisinins [56]. They found many angiogenesisregulating factors among their panel such as, vascular endothelial growth factor-C (VEGFC), fibroblast growth factor-1 (FGF1), matrix metalloproteinase-9 (MMP9), thrombospondin-1 (THBS1), hypoxia-inducing factor-a (HIF1A), and angiogenin (ANG) [57–59]. Artemisinin has also been found

Anti-Infective and Anti-Cancer Properties of Artemisinin

373

to inhibit proliferation, migration, and tube formation in human umbilical vein endothelial cells (HUVEC); inhibit VEGF binding to surface receptors on HUVEC; and reduce expression of VEGF receptors on HUVECs [60– 62]. Artesunate has been shown to play other roles in the control of cancer such as inducing apoptosis and oncogene deactivation/tumor suppressor gene activation [63]. The reader is encouraged to read the review by Krishna for further discussion of this topic [64].

O C-10

O C-10 O O

O P OR

28 R = Me 29 R = Ph

Two of a series of bis-trioxane dimers (28, 29) were shown to be more potent against cancer, in vitro, than doxorubicin, a currently used cancer chemotherapy agent. It was noted that all the cell lines sensitive to (28) and (29) all overexpressed transferrin receptors [65]. Based on that information, nuclear iron-dependent activation to free radical species that damage DNA was proposed to be involved in the tumoricidal mechanism of action [66]. To further the idea that transferrin receptors are a likely target, a series of artemisinin–transferrin conjugates were prepared and tested against the prostate cancer cell line DU 145. These conjugates were found to be cytotoxic to the cell line through a transferrin receptor–dependent induction of apoptosis [67]. Currently, it is unknown whether the anticancer properties of artemisinin derivatives are due to cytotoxicity against the cancer cells, antiangiogenesis properties, or some combination of both. Other recent work has shown that artemisinin-acridine hybrids such as the aforementioned (23) were found to have a cytotoxicity profile against several cancer cell lines, with IC50 values four times lower than DHA in several derivatives [68], demonstrating the widespread applicability of this concept of hybrid drug analogs.

4. TOXICITY Great debate has raged on the topic of toxicity for this compound class. While generally characterized as having excellent tolerability and

374

Christopher Paul Hencken et al.

safety [69], some adverse affects, particularly in non-human mammals, have been observed.

4.1 Evidence in favor of safety A review of clinical trials [70] showed that together, 9% of patients showed adverse drug reactions. These reactions included neutropenia, reduced reticulocyte count, anemia, eosinophilia, acute haemolysis, elevated aspartate aminotransferase, ECG abnormalities (w/o clinical effect), transient bradycardia, prolongation of the QTc interval, prolonged PR interval (first-degree atrioventricular block), atrial extrasystoles, and non-specific T-wave changes. In all cases, the effects were independent of the artemisinin derivative and route of administration. An additional study [71] showed that when treating uncomplicated falciparum malaria with artemisinin derivatives alone, substantially fewer side effects were observed than with mefloquine-containing combination therapies. Notably, there was significantly less nausea, vomiting, anorexia, and dizziness. Furthermore, studies have shown no adverse effects on mother or fetus if artesunate or artemether are used to treat acute falciparum at various stages of gestation [72] or during breast feeding [69]. However, this matter can hardly be considered closed. Although several studies have been done and no evidence of adverse effects have been observed, the sample size has thus far been small [73], and complications (most notably, a high percentage of post-implantation losses) have been observed at doses of 35 and 75 mg/kg in Wistar rats, especially when administered early in the pregnancy [74]. Meanwhile, another study using Wistar rats and considerably lower doses of artemether (3.5 and 7 mg/kg) during blastogenesis, organogenesis and fetal period had no adverse effects other than reduction in fetal body weight and pre-term delivery in 3/10 rats at the 7-mg/kg dose. Some fetal growth retardation without incidence of malformations was also noted in the study [75].

4.2 Evidence of neurotoxicity It is important to note that dogs [76], mice, rats, and Rhesus monkeys [77–79] have all demonstrated evidence of neurotoxicity apparently caused by artemisinin derivatives. Specifically, gait disturbances, loss of spinal and pain response reflexes, cardio respiratory depression, and even death have been documented. In mice, balance was damaged irreversibly and death also occurred. Importantly, it was found that intramuscular administration was found to be more toxic than oral [69]. In humans, even when adverse neurological effects have been documented, the effects resolved with time after the treatment was

Anti-Infective and Anti-Cancer Properties of Artemisinin

375

stopped [71]. These effects included problems with co-ordination, fine finger dexterity, hearing, nystagmus, and balance. For more information concerning the toxicity of this important class of compounds, we direct the interested reader to a relatively recent review [80]. An explanation for the apparent lack of a correlation between animal toxicity and human toxicity is still lacking and is urgently needed.

5. CONCLUSION The artemisinin family of compounds has become more important in recent years. The very diverse biological activity and relative safety of this family of drugs make it important in the global fight against many diseases. In the coming years, it is anticipated that this exotic natural product will become even more important to the chemotherapy of various diseases, perhaps even above and beyond those mentioned here.

REFERENCES [1] J. M. Liu, M. Y. Ni, J. F. Fen, Y. Y. Tu, Z. H. Wu, Y. L. Wu and W. S. Zhou, Huaxue Xueabo, 1979, 37, 129. [2] D. L. Klayman, A. J. Lin, W. Acton, J. P. Scovill, J. M. Hoch, W. K. Milhous and A. D. Theoharides, J. Nat. Prod., 1984, 47, 715. [3] M. A. Avery and K. M. Muraleedharan, International patent 03/095444A1 2003. [4] D. L. Klayman, Science, 1985, 228, 1049. [5] R. K. Haynes and S. Krishna, Microbes Infect., 2004, 6, 1339. [6] S. Krishna, L. Bustamante, R. K. Haynes and H. M. Staines, Trends Pharmacol. Sci., 2008, 29, 520. [7] N. J. White, Science, 2008, 320, 330. [8] T. Efferth, Planta Med., 2007, 73, 299. [9] www3.niaid.nih.gov/topics/Malaria/understandingMalaria/facts.htm [10] www.cdc.gov/malaria/faq.htm [11] R. K. Haynes, D. Monti, D. Taramelli, N. Basilico, S. Parapini and P. Olliaro, Antimicrob. Ag. Chemother, 2003, 47, 1175. [12] U. Eckstein-Ludwig, R. J. Webb, I. D. A. vanGoethem, J. M. East, A. G. Lee, M. Kimura, P. M. O’Neill, P. G. Bray, S. A. Ward and S. Krishna, Nature, 2003, 424, 957. [13] P. M. O’Neill, S. L. Rawe, K. Borstnik, A. Miller, S. A. Ward, P. G. Bray, J. Davies, C. H. Oh and G. H. Posner, ChemBioChem, 2005, 6, 2048. [14] Chinese-Cooperative-Research-Group, J. Trad. Chin. Med., 1982, 2, 31. [15] D. J. Creek, W. M. Charman, F. C. K. Chiu, R. J. Prankerd, Y. Dong, J. L. Vennerstrom and S. A. Charman, Antimicrob. Ag. Chem., 2008, 52, 1291. [16] C. L. Hartwig, A. S. Rosenthal, J. D’Angelo, C. F. Griffin, G. H. Posner and R. A. Cooper, Biochem. Pharmacol., 2009, 77, 322. [17] P. L. Olliaro, R. K. Haynes, B. Meunier and Y. Yuthavong, Trends Parasitol., 2001, 17, 122. [18] F. B.-E. Garah, C. Claparols, F. Benoit-Vical, B. Meunier and A. Robert, Antimicrob. Ag. Chem., 2008, 52, 2966. [19] R. K. Haynes, W. C. Chan, C.-M. Lung, A.-C. Uhlemann, U. Eckstein, D. Taramelli, S. Parapini, D. Monti and S. Krishna, ChemMedChem, 2007, 2, 1480.

376

Christopher Paul Hencken et al.

[20] S. Krishna, A.-C. Uhlemann and R. K. Haynes, Drug Resist. Updat., 2004, 7, 233. [21] A.-C. Uhlemann, A. Cameron, U. Eckstein-Ludwig, J. Fischbarg, P. Iserovich, F. A. Zuniga, M. East, A. Lee, L. Brady, R. K. Haynes and S. Krishna, Nat. Struct. Mol. Biol., 2005, 12, 628. [22] A. Ryde´n and O. Kayser, Top. Heterocycl. Chem., 2007, 9, 1. [23] J. Be´gue´ and D. Bonnet-Delpon, ChemMedChem, 2007, 2, 608. [24] R. K. Haynes, B. Fugmann, J. Stetter, K. Rieckmann, H.-D. Heilmann, H.-W. Chan, M.-K. Cheung, W.-L. Lam, H.-N. Wong, S. L. Croft, L. Vivas, L. Rattray, L. Stewart, W. Peters, B. L. Robinson, M. D. Edstein, B. Kotecka, D. E. Kyle, B. Beckermann, M. Gerisch, M. Radtke, G. Schmuch, W. Steinke, U. Wollborn, K. Schmeer and A. Ro¨mer, Angew. Chem. Int. Ed., 2006, 45, 2082. [25] G. H. Posner, W. Chang, L. Hess, L. Woodard, S. Sinishtaj, A. R. Usera, W. Maio, A. S. Rosenthal, A. S. Kalinda, J. G. D’Angelo, K. S. Peterson, R. Stohler, J. Chollet, J. Santo-Tomas, C. Snyder, M. Rottmann, S. Wittlin, R. Brun and T. A. Shapiro, J. Med. Chem., 2008, 51, 1035. [26] N. C. P. Arauju, V. Barton, M. Jones, P. A. Stocks, S. A. Ward, J. Davies, P. G. Brat, A. E. Shone, M. L. S. Cristiano and P. M. O’Neill, Bioorg. Med. Chem. Lett., 2009, 19, 2038. [27] J. J. Walsh, D. Coughlan, N. Hehghan, G. Gaynor and A. Bell, Bioorg. Med. Chem. Lett., 2007, 17, 3599. [28] S. Bachmann, J. Schro¨der, C. Bottmer, E. F. Torrey and R. H. Yolken, Psychopathology, 2005, 38, 87. [29] A. M. Tenter, A. R. Keckeroth and L. M. Weiss, Int. J. Parasitol., 2000, 30, 1217. [30] K. Ou-Yang, E. C. Krug, J. J. Marr and R. L. Berens, Antimicrob. Ag. Chem., 1990, 34, 1961. [31] L. Jones-Brando, J. D’Angelo, G. H. Posner and R. Yolken, Antimicrob. Ag. Chem., 2006, 50, 4206. [32] J. G. D’Angelo, C. Bordo´n, G. H. Posner, R. Yolken and L. Jones-Brondo, J. Antimicrob. Chem., 2009, 63, 146. [33] K. Nagamune, W. L. Beatty and L. D. Sibley, Eukaryot. Cell, 2007, 6, 2147. [34] H. R. Chang, C. W. Jefford and J.-C. Peche`re, Antimicrob. Ag. Chem., 1989, 33, 1748. [35] J. Moreno, C. Caravate, C. Chamizo, F. Laguna and J. Alvar, Tran. R. Soc. Trop. Med. Hyg., 2000, 94, 328. [36] M. A. Avery, K. M. Muraleedharan, P. V. Desai, A. K. Bandyopadhyaya, M. M. Furtado and B. L. Tekwani, J. Med. Chem., 2003, 46, 4244. [37] X. Shuhua, M. Tanner, E. K. N’Goran, J. Utzinger, J. Chollet, R. Benquist, C. Minggang and Z. Jiang, Acta Trop., 2002, 82, 175. [38] I. Adam, O. A. Elhardello, M. O. Elhadi, E. Abdalla, K. A. Elmandi and F. H. Jansen, Ann. Trop. Med. Parasitol., 2008, 102, 39. [39] A. A. Mohamed, H. M. Mahgoub, M. Magzoub, G. I. Gasim, W. N. Eldein, A. Ahmed and I. Adam, Trans. Roy. Soc. Trop. Med. Hyg., 2009, in press. [40] D. DeClercq, J. Vercruysse, A. Kongs, P. Verlei, J. P. Dompnier and P. C. Faye, Acta Trop., 2002, 82, 61. [41] http://wwwn.cdc.gov/travel/yellowbookCh4-AfricanSleepingSickness.aspx [42] http://wwwn.cdc.gov/travel/yellowBookCh4-Chagas.aspx [43] Y. V. Mishina, S. Krishna, R. K. Haynes and J. C. Meade, Antimicrob. Ag. Chem., 2007, 51, 1852. [44] C. Chollet, A. Baliani, P. E. Wong, M. P. Barrett and I. H. Gilbert, Bioorg. Med. Chem., 2009, 17, 2512. [45] R. S. Qian, Z. L. Li, J. L. Yu and D. J. Ma, J. Tradit. Chin. Med., 1982, 2, 271. [46] T. Efferth, M. Marschall, X. Wang, S.-M. Huong, I. Hauber, A. Olbrich, M. Kronschnabl, S. Stamminger and E.-S. Huang, J. Mol. Med., 2002, 80, 233. [47] P. M. Bork, M. L. Schmitz, M. Kuhnt, C. Escher and M. Heinrich, FEBS Lett., 1997, 402, 85.

Anti-Infective and Anti-Cancer Properties of Artemisinin

377

[48] B. Siedle, A. J. Garcia-Pineres, R. Murillo, J. Schulte-Mo¨nting, V. Castro, P. Ru¨ngeler, C. A. Klaas, F. B. DaCosta, W. Kisiel and I. Merfort, J. Med. Chem., 2004, 47, 6042. [49] T. Efferth, M. R. Romero, D. G. Wolf, T. Stamminger, J. J. G. Marin and M. Marschall, Clin. Infect. Dis., 2008, 47, 804. [50] Y. Binku, E. Mekonnen, A. Bjorkman and D. Wolday, Ethiop. Med. J., 2002, 40, 17. [51] J. Vandenkerckhov, E. B. Sas, E. Pets and J. VanHemel, WO International Patent 2004/ 041176A2, 2004. [52] S. Kumar, S. P. S. Khanuja, T. R. S. Kumar, D. C. Jain, S. Srivastava, A. K. Bhattacharya, D. Saikia, A. K. Shasany, M. P. Darokar and R. P. Sharma, US Patent 6,127,405, 2000. [53] A. M. Galal, S. A. Ross, M. Jacob and M. A. ElSohly, J. Nat. Prod., 2005, 68, 1274. [54] M. Kolaczkowski, A. Kolaczkowska and F. R. Stermitz, Microb. Drug Resist., 2009, 15, 11. [55] http://seer.cancer.gov/statfacts/html/all.html [56] L. Anfosso, T. Efferth, A. Albini and U. Pfeffer, Pharmacogenomics J., 2006, 6, 269. [57] J. Folkmann, J. Natl. Cancer Inst., 1990, 82, 4. [58] A. F. Karamysheva, Biochemistry (Moscow), 2008, 73, 751. [59] M. Shibuya, J. Biochem. Mol. Biol., 2006, 39, 469. [60] H.-H. Chen, H.-J. Zhou, W.-Q. Wang and G.-D. Wu, Cancer Chemother. Pharmacol., 2004, 53, 423. [61] H.-H. Chen, H.-J. Zhou, G.-D. Wu and X.-E. Lou, Pharmacology, 2004, 71, 1. [62] H.-H. Chen, H.-J. Zhou and X. Fang, Pharmacol. Res., 2003, 48, 231. [63] T. Efferth, G. Ruecker, M. Falkenberg, D. Manns, A. Olbrich and U. Fabry, Arzneimittelforschung, 1996, 46, 196. [64] S. Krishna, L. Bustamante, R. K. Haynes and H. M. Staines, Trends Pharmacol. Sci., 2008, 29, 520. [65] G. H. Posner, J. D’Angelo, P. M. O’Neill and A. Mercer, Expert Opin. Ther. Patents, 2006, 16, 1665. [66] J. P. Jeyadevan, P. G. Bray, J. Chadwick, A. E. Mercer, A. Byrne, S. A. Ward, B. K. Park, D. P. Williams, R. Cosstick, J. Davies, A. P. Higson, E. Irving, G. H. Posner and P. M. O’Neill, J. Med. Chem., 2004, 47, 1290. [67] I. Nakase, B. Gallis, T. Takatani-Nakase, S. Oh, E. Lacoste, N. P. Singh, D. R. Goodlett, S. Tanaka, S. Futaki, H. Lai and T. Sasaki, Cancer Lett., 2009, 274, 290. [68] M. Jones, A. E. Mercer, P. A. Stocks, L. J. I. LaPense´e, R. Cosstick, B. K. Park, M. E. Kennedy, I. Piantanida, S. A. Ward, J. Davis, P. G. Bray, S. L. Rawe, J. Baird, T. Charidza, O. Janneh and P. M. O’Neill, Bioorg. Med. Chem. Lett., 2009, 19, 2033. [69] W. R. J. Taylor and N. J. White, Drug Saf., 2004, 27, 25. [70] I. R. Ribeiro and P. Olliaro, Med. Trop. (Mars), 1998, 58(3 suppl.), 50. [71] R. Price, M. vanVugt, L. Phaipun, C. Luxemburger, J. Simpson, R. McGready, F. TerKuile, A. Kham, T. Chongsuphajaisiddhi, N. J. White and F. Nosten, Am. J. Trop. Med. Hyg., 1999, 60, 547. [72] R. McGready, T. Cho, N. K. Keo, K. L. Thwai, L. Villegas, S. Looareesuwan, N. J. White and F. Nosten, Clin. Infect. Dis., 2001, 33, 2009. [73] S. Dellicour, S. Hall, D. Chandramohan and B. Greenwood, Malar. J., 2007, 6, 15. [74] A. C. Boareto, J. C. Muller, A. C. Bufalo, G. G. K. Botelho, S. L. deAraujo, M. A. Foglio, R. N. deMorais and P. R. Dalsenter, Reprod. Toxicol., 2008, 25, 239. [75] M. H. El-Dakdoky, Food Chem. Toxicol., 2009, 47, 1437. [76] China-Cooperative-Research-Group-On-Qinghaosu-and-its-Derivatives-as-Antimalarials, J. Tradit. Chin. Med., 1982, 2, 45. [77] R. F. Genovese, D. B. Newman, J. M. Petras and T. G. Brewer, Pharmacol. Biochem. Behav., 1998, 60, 449.

378

Christopher Paul Hencken et al.

[78] J. M. Petras, G. D. Young, R. A. Bauman, D. E. Kyle, M. Gettayacamin, H. K. Webster, K. D. Corcoran, J. O. Peggins, M. A. Vane and T. G. Brewer, Anat. Embryol. (Berl), 2000, 201, 383. [79] A. Nontprasert, S. Pukrittayakamee, A. M. Dondorp, R. Clemens, S. Looareesuwan and N. J. White, Am. J. Trop. Med. Hyg., 2002, 67, 423. [80] T. Gordi and E.-I. Lepist, Toxicol. Lett., 2004, 147, 99.