Cancer combination therapies with artemisinin-type drugs

Accepted Manuscript Review Cancer combination therapies with artemisinin-type drugs Thomas Efferth PII: DOI: Reference:

S0006-2952(17)30181-8 http://dx.doi.org/10.1016/j.bcp.2017.03.019 BCP 12773

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

20 January 2017 28 March 2017

Please cite this article as: T. Efferth, Cancer combination therapies with artemisinin-type drugs, Biochemical Pharmacology (2017), doi: http://dx.doi.org/10.1016/j.bcp.2017.03.019

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Cancer combination therapies with artemisinin-type drugs Thomas Efferth1 1

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes

Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany Tel.: 49-6131-3925751; Fax: 49-6131-3923752; E-Mail: [email protected]

Running title: Artemisinin-based combination therapies Key words: Chemotherapy, Drug resistance, Natural products, Sesquiterpenoids, Toxicology

Abbreviations: ACE, angiotensin I-converting enzyme; ACT, artemisinin-based combination therapy; Akt, AKT seronine/threonine kinase, v-akt murine thymoma viral oncogene homologue; AMP, adenosine monophosphate; ARE, arteether; ARM, artemether; ARS, artemisinin; ART, artesunate; BCL- XL, break point cluster X, long; BCRP, breast cancer resistance protein; BMI1, B-lymphoma Mo-MLV insertion region 1 homologue (mouse); Ca, carcinoma; CDK2, cyclin-dependent kinase 2; CIP, cyclin-interacting protein; CRISPR, clustered regulatory interspaced short palindromic repeats; CYP, cytochrome P450 monooxygenase; DHA, dihydroartemisinin; DR5, death receptor 5; ERK1/2, extracellular signal-regulated kinase 1/2; FAS, Fas cell surface death receptor; 1

FDA, Food and Drug Administration; Fos, FBJ murine osteosarcoma viral (v-fos) oncogene homologue; G6PD, glucose-6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; HDAC, histone deacetylase; HIF-1α, hypoxia-induced factor 1-alpha HSP, heat shock protein; KIP, kinase-interacting protein; MAPK, mitogen-activated protein kinase; MDR, multidrug resistance; MMP, matrix metalloproteinase; MRP, multidrug resistance-related protein; NADP+, nicotinamide adenine dinucleotide phosphate; NF-κB, nuclear factor kappa-B; NHL, non-Hodgkin lymphoma; PARK7, Parkinonism-associated deglycase 7; P-gp, P-glycoprotein; PUVA, psoralen plus UVA light therapy; RAC-1, Ras-related C3 Botulinum toxin substrate 1 (Rho family); RAD51, RAD51 (radiation 51) recombinase; R-CHOP, rituximab, cyclophosphamide, vincristine, prednisone combination therapy; ROS, reactive oxygen species; Sp1, specificity protein 1 transcription factor; TCM, traditional Chinese medicine; TRAIL, tumor necrose factor receptor apoptosis-inducing ligand; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor; XIAP, X-linked inhibitor of apoptosis; YY1, yin yang 1 transcription factor.

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Abstract Artemisia annua L. is a Chinese medicinal plant, which is used throughout Asia and Africa as tea or press juice to treat malaria. The bioactivity of its chemical constituent, artemisinin is, however, much broader. We and others found that artemisinin and its derivatives also exert profound activity against tumor cells in vitro and in vivo. Should artemisinin-type drugs be applied routinely in clinical oncology in the future, then probably as part of combination therapy regimens rather than as monotherapy. In the present review, I give a comprehensive overview on synergistic and additive effects of artemisinin-type drugs in combination with different types of cytotoxic agents and treatment modalities: (a) standard chemotherapeutic drugs, (b) radiotherapy and photodynamic therapy, (c) established drugs for other indications than cancer, (d) novel synthetic compounds, (e) natural products and natural product derivatives, (f) therapeutic antibodies and recombinant proteins, and (g) RNA interference. I also summarize the activity of artemisinin-type drugs towards multidrug-resistant cells and tumor cells with other drug resistance phenomena. As synergistic interactions may not only occur in tumor cells, toxic reactions in normal cells (hepatotoxicity, drug interactions) were also considered. This review summarizes the scientific literature of more than 20 years until the end of 2016.

1 Introduction Artemisinin (ARS) from the Sweet wormwood (qinhao, Artemisia annua L., Asteraceae) is a medicinal plant used in Chinese medicine to treat chills and fever. In the 1970 and 1980s it turned out that the drug reveals surprisingly strong efficacy towards Plasmodia. Nowadays, artemisinin belongs to the standard treatment protocols to treat malaria. Because artemisinin saved millions of lives of malaria patients, Youyou Tu, who first identified the malaria activity of A. annua, was honored with the Nobel Prize for Medicine or Physiology 2015 [1]. Interestingly, A. annua preparations (decoctions, tea, press juice) are frequently used outside the official medical system in Asia and Africa to treat malaria [2,3]. During the past years, experimental evidence was accumulated that ARS activity is not restricted to malaria and that it may also be of therapeutic interest for several other diseases, including cancer [4-12]. Should ARS-type drugs ever be used routinely in clinical oncology, then with high probability as part of combination therapy protocols rather than as monotherapy [13-15]. The principles, which are common sense for combination therapy protocols with established anticancer drugs, also have to be applied for combinations with ARS-type drugs, 3

i.e. (1) prevention of resistance development by combining drugs with different targets and modes of action; (2) dispersion of side effects to different tissues in the body to minimize the occurrence of severe or fatal toxicities; (3) each constituent of the combination protocol has to reveal anticancer activity, if applied as single drug etc. [16-18]. In the preclinical setting, synergy has to be proven by appropriate biostatistical methods [19, 20] in well suitable experimental biological models in vitro and in vivo [21-25]. Frequently, authors publish their data by claiming synergistic interactions of compound combinations without reliable proof, whether or not the observed effects are indeed synergistic in nature. This is a considerable limitation of many data available in the literature in pharmacology in general and also in the case of ARS-type drugs. A wealth of results has been published in the literature on additive or synergistic combinations of ARS-type drugs with other compounds to improve tumor growth inhibition in cell lines and in animal experiments (Figure 1). The intention of the present review was to give an overview of the published data and to outline perspectives for further research developing the most suitable combination therapy protocols for ARS-based cancer therapy. A central question in this context is about the molecular targets of ARS-type drugs in cancer cells. Despite a plethora of published data, this question is not ultimately resolved. Most likely, this class of compounds acts in a multi-specific manner exhibiting several modes of action at the same time. This is a typical feature of most - if not all – natural products [26]. The endoperoxide moiety of ARS-type drugs can be opened leading to the formation of radical oxygen species and carbon-centered radical molecules. This process is facilitated by free ferrous iron or iron-bound proteins (e.g. heme) in a Fenton-type reaction. These radical molecules may exert a broad range of detrimental effects in cancer cells. As previously outlined, the activity of antitumor drugs in general may be categorized as (1) mechanisms acting upstream of the actual drug target, (2) target site mechanisms, and (3) mechanisms acting downstream of the drug targets [27]. This model may also be applied for ARS-type drugs. As described in great detail in a recent review [28], molecular modes of action of ARStype drugs in cancer cells include: (1) Upstream mechanisms: antioxidant response mechanisms, receptor signaling pathways (e.g. EGFR, WNT/β-catenin, BCR/ABL) (2) Target site mechanisms: DNA damage and repair mechanisms, alkylation of target proteins (e.g. TCTP, LDH etc.), cell cycle arrest, neoangiogenesis, invasion and metastasis (3) Downstream mechanisms: apoptotic and non-apoptotic cell death (autophagy, ferroptosis).

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2 Combination with standard chemotherapy In the past few years, a plethora of papers appeared on the interaction of ARS-type drugs with clinically established anticancer agents, many of which are acting directly or indirectly on tumor DNA by damaging its integrity in one or another way (the DNA topoisomerase II inhibitor doxorubicin, the adduct-forming platin compounds, the alkylating agents temozolomide) or disturbing DNA-biosynthesis (gemcitabine, cytarabine) (Table 1). Since ARS-type drugs cause oxidative DNA lesions and double strand breaks [29-35], it is worth hypothesizing that their interaction with DNA-affecting anticancer drugs may provoke enhanced tumor cell killing. Indeed, additive to synergistic interactions of ARS, DHA, or ART have been observed in combination with standard anticancer drugs towards tumor cell lines of diverse origin, e.g. hematopoetic tumors (leukemia, multiple myeloma) as well as epithelial carcinoma (of breast, ovarian, lung, brain tumor, liver, prostate, or pancreas). Importantly, these effects were not only measured in vitro but also in vivo, which raises the potential clinical utility of this kind of drug combinations. In case that ARS-type drugs should ever reach clinical routine application for cancer therapy, it is important to investigate the molecular modes of action of these drug-drug interactions in preclinical models. As shown in Table 1, mechanisms contributing to additive or synergistic effects of ARS-type drugs in combination with established anticancer drugs include effects on DNA repair (RAD51) and signal transduction pathways (mTOR, NF-κB) as well as angiogenesis (VEGF, HIF-1α) and induction of cell death (apoptosis, autophagy). The enhancement of activity of ARS-type drugs was not only observed in combination with anticancer drugs that act on DNA, but also on other targets of tumor cells. As shown in Table 2, combinations with tyrosine kinase inhibitors (erlotinib), proteasome inhibitors (bortezomib), corticosteroides (dexamethasone), as well as immunomodulating and antiangiogenic drugs (thalidomide, lenalidomide) cereblon (CRBN) E3 ubiquitin ligase as the primary target, all led to additive or synergistic inhibition rates of tumor cells.

3 Activity against drug-resistant tumor cells The development of drug resistance and unwanted side effects represent main reasons for the failure of chemotherapy in clinical oncology. Severe and partly life-threatening side effects prevent the administration of drug concentrations high enough to kill all cell populations in a tumor. 5

A dreaded phenomenon in cancer chemotherapy is multidrug resistance (MDR). This type of drug resistance represents cross-resistance to a wide array of functionally and structurally distinct anticancer drugs, e.g. Vinca alkaloids,

taxanes, anthracenes,

epipodophyllotoxins and others. The underlying mechanisms of MDR phenomena are drug efflux pumps of the ATP-binding cassette (ABC) transporter family, which extrude drug molecules that passively diffused into cancer cells in an active ATP-consuming manner out of the cells. This drug efflux keeps intracellular drug concentrations at low sub-lethal levels, which ultimately favors survival of cancer cells and failure of chemotherapy with fatal consequences for cancer patients. Well-known ABC-transporters that confer MDR are Pglycoprotein (ABCB1, MDR1), MDR-related proteins (ABCCs, MRPs) and breast cancer resistance protein (ABCG2, BCRP) [36,37]. In addition to MDR, resistance mechanisms to single anticancer drugs without broad cross-resistance profiles also hamper the success of chemotherapy. Resistance to DNAdamaging agents (e.g. cisplatin), DNA-biosynthesis (methotrexate, hydroxyurea), tyrosine kinase inhibitors (e.g. imatinib) and other anticancer drugs have been described in the past years [38-41]. Therefore, there is an intensive ongoing search for novel compounds that are able to combat drug resistance. In principal there are three main categories to overcome drug resistance: (1) specific inhibitors of resistance mechanisms, e.g. efflux inhibitors of ABCtransporters, (2) cytotoxic compounds that are not recognized by resistance mechanisms and which thereby bypass drug resistance, and (3) compounds that exert hypersensitivity (collateral sensitivity) in drug-resistant cells [42,43]. The therapeutic potential of ARS-type drugs in oncology is not only visible by the fact that they show profound activity in vitro and in vivo against cancer cells [44], but also that they do not exert cross-resistance or even do reveal collateral sensitivity (hypersensitivity) against ABC-transporter-mediated MDR and also resistance to methotrexate, hydroxyurea, cisplatin or imatinib (Table 3, Figure 2). Interestingly, some ARS-derivatives were also able to inhibit P-gp at the blood brain barrier making them interesting candidates to improve glioblastoma therapy [45].

4 Combination with radiotherapy or photodynamic therapy Since their discovery in 1895, X-rays are used for diagnosis and treatment in medicine. Nowadays, radiotherapy represents one of the main pillars of cancer therapy [46-48]. Radiotherapy kills tumors by damaging their DNA. As more and more sophisticated 6

technologies are available, the normal tissue surrounding the tumor is less affected. Furthermore, normal cells repair damaged DNA better than tumor cells. Nevertheless, the development of resistance represents also a major impediment of radiotherapy, and radiosensitizers are investigated to overcome radioresistance [49,50]. Some authors attempted to sensitize tumor cells to radiotherapy by the application of ART or DHA and found that resensitization effects were associated with ROS or nitric oxide generation, cell cycle arrest and induction of apoptosis [51-55] (Table 4). In vivo experiments are missing as of yet and it remains to be seen, whether or not the side effects of ARS-based radiochemotherapy are tolerable. Another therapeutic mode to combat tumors is photodynamic therapy. A systemically given photosensitizing agent is preferentially accumulated in tumor cells. Then, light is applied, which causes oxidative reactions with the photosensitizer inducing tumor cell killing [56]. Although there is not much experience thus far with the use of ARS-type drugs for photodynamic therapy, first favorable results were reported [57]. DHA enhanced PDTinduced growth inhibition and apoptosis in human esophageal cancer cell lines. DHA inhibited NF-κB activated by photodynamic therapy, which in turn led to down-regulation of the NF-κB target, Bcl-2.

5 Combination with drugs for indications other than cancer The rationale for investigating, whether drugs established for other diseases might cooperate together with ARS-type drugs is that patients occasionally do not only suffer from cancer, but also from other diseases at the same time [58,59]. Comorbidity in cancer patients occasionally can lead to unwanted drug interactions because of multiple medications. On the other side, multiple medications might also enhance growth inhibition of tumors. For instance, the angiotensin I converting enzyme (ACE) inhibitors, which are widely used for the treatment of cardiovascular diseases, also exert synergistic activity towards tumors in combination with anticancer drugs [60,61]. Recently, drug repurposing came into the center of interest. ARS-type drugs as antimalarial drugs are an example, since they also show activity against tumors. It is not beyond the expectations that their combination with drugs with indications other than cancer may also improve anticancer treatment efficacy. Indeed, there are some publications substantiating this speculation (Table 5). The ACE inhibitor captopril enhanced the effects of ART by inhibition of angiogenesis [62]. Elderly patients can suffer from congestive heart failure or arterial hypertension Therefore, captopril-ART combinations are reasonable. 7

Remarkably, the use of ACE inhibitors was correlated with a lower incidence of skin cancer [63]. Especially in tropical countries, it is not a rare condition that patients suffer from both malaria and cancer at the same time. Comparable to ARS-type drugs, the antimalarial drug chloroquine also showed anticancer effects [64]. The combination of chloroquine and ARS exerted synergistic interaction by ROS generation and induction of autophagy in lung cancer cells [65]. Another example is the antifunal agent miconazole, which is used for the treatment of antifungal infections in cancer patients [66]. An interaction of ARS and miconazol in killing tumor cells has been reported [67]. The published data on the interaction of ARS-type drugs with drugs approved for other diseases than cancer were found by chance and were not a result of systematic screening for synergistic interactions against tumors. More detailed analyses might unravel novel interesting drug combinations justifying the repurposing of established drugs for cancer therapy. Analyses for novel drug combinations should, however, also consider that drug interactions could cause unwanted toxicities, as will be discussed in the chapter on hepatotoxicity below.

6 Combination with novel synthetic compounds The synthesis of novel cytotoxic compounds represents an important part of the anticancer drug development process in academia and industry. Therefore, there is a constant interest to test experimental combinations of new compounds with clincially established or investigational new drugs. In this contest, the combination of ARS-type drugs with several novel synthetic compounds has been reported (Table 6). ROS or carbon-centered radical molecules generated by drugs or irradiation can be detoxified by reduced glutathione (GSH). Cellular GSH contents as well as enzymes related to GSH metabolism are long known as determinants of cancer cells to chemo- and radiotherapy [68,69]. This has also been shown for ART. The GSH biosynthesis inhibitor, Lbuthionine sulfoximine and the glutathione S-transferase (GST) inhibitor, ethacrynic acid sensitize tumor cells to ART [70]. Another enzyme related to the cellular detoxification capacity is glucose-6-phosphate dehydrogenase (G6PD). This is the key enzyme of the pentose-phosphate pathway, which catalyzes the conversion of glucose-6-phosphate to D8

glucono-1,5-lactone-6-phosphate by reduction of NADP+ to NADPH. NADPH is a cofactor of glutathione reductase, which converts oxidized glutathione (GSSG) to its reduced form (GSH). G6PD fosters cancer cell proliferation and pharmacological inhibition of G6PD is considered as novel therapeutic approach against cancer [71-73]. Increased pentose phosphate pathway and G6PD activities accompanied by increased amounts of GSH are determinants of drug resistance [74]. On the other hand, peripheral mononuclear blood cells from patients suffering from inherited G6PD-defficiency reveal increased oxidative stress, DNA damage and susceptibility to undergo apoptosis upon treatment with cytostatic drugs or radiation [7577]. Therefore, it can be hypothesized that pharmacological inhibition of G6PD may also sensitize tumor cells to ARS-type drugs. Indeed, the G6PD inhibitors physcion and S3 interacted with DHA in a synergistic manner to inhibit leukemia cell growth [78]. Another attractive treatment target to inhibit tumor growth is glutamine utilization. Glutaminolysis represents the first and rate-limiting step of glutamine utilization and is catalyzed by glutaminase (GLS). Glutaminase-1 (GLS1) was upregulated in tumor cells [79], and inhibition of GLS1 resensitized tumor cells to standard anticancer agents [80,81]. The GLS1 inhibitor 968 revealed synergistic interaction with DHA by excessive ROS generation [82]. Aberrant histone deacetylase (HDAC) activity has been implicated in cancer and HDACs are interesting targets for treatment [83,84]. Inhibition of HDACs increased the cytotoxicity of diverse established anticancer drugs, including oxaliplatin, docetaxel, 5fluorouracil and others [85-87]. Comparable results have been published for the combination of HDAC inhibitors and DHA in vitro and in vivo [88]. In a comparable manner as discussed for drug repurposing in the chapter above, unwanted interactions also are considered for combinations of ARS-type drugs with novel synthetic compounds. The nitrone compound, α-phenyl-tert-butylnitrone, and related nitrone substances revealed anticancer activity in preclinical models [89]. Their combination with DHA led, however, to attenuation of DHA cytotoxicity [90], indicating that this drug combination should be avoided.

7 Combination with natural products or natural product derivatives Natural products and derivatives thereof always played an important role for the treatment of diseases. This is especially true for cancer therapy. Drugs such as Vinca alkaloids (vincristine, vinlastine), taxanes (paclitaxel, docetaxel), camptothecins (topotecan, irinotecan), and 9

epipodophyllotoxins (etoposide, teniposide) are just a few prominent examples. A previous survey of the National Cancer Institute in USA revealed that the vast majority of all clinically established anticancer drugs are of natural origin [91,92]. Hence, it does not come as a surprise that natural products exert synergistic interactions with ARS-type drugs to inhibit tumor cells (Table 7). This has been reported for natural compounds used as constituents of dietary supplements such as vitamins, curcumin (a diarylheptanoid from Curcuma long L., Zingiberaceae), allicin (an organosulfor compound from Allium sativum L., Alliaceae), and butyrate (a bacterial product of anaerobic fermentation of milk products). Furthermore, chemical constituents of medicinal plants also enhanced growth inhibitory properties of ARStype drugs, e.g. dictamnine (a furoquinoline alkaloid from the roots of Dictamnus albus L., Rutaceae), salicylic acid (a phenolic acid that acts as plant hormone), resveratrol (a stilbenoid that is produced by many food plants as phytoalexin in response to stress and injury), triptolide (a diterpene epoxide from Triperygium wilfordii Hook.f., Celastraceae), and halofuginone (a quinazolinone alkaloid from Dichroa febrifuga Lour., Hydrangaceae). Some of these plants are used in traditional Chinese medicine (TCM). Inorganic compounds such as arsenic trioide, which also used in TCM, also enhance growth inhibition in combination with ARS-type drugs. ARS did not only exert additive or synergistic interactions with isolated compounds of other plants, but also with other phytochemicals (flavonoids, scopoletin, 1,8-cineole etc.) present in Artemisia annua [93,94]. This indicates that plant extracts from this plant may act as genuine combination therapies to combat cancer growth.

8 Combination with therapeutic antibodies or recombinant proteins The rapid development of gene- and biotechnological technologies during the past years facilitated the production of monoclonal antibodies and recombinant proteins for therapeutic purposes. Nowadays, antibodies belong the standard armory to treat cancer, and frequently they are combined with standard chemotherapy. Importantly, antibodies and clinically established chemotherapeutics are known to exert synergistic interactions leading to improved tumor eradication [95-97]. It can be envisaged that novel treatment regimens combining chemotherapy, adoptive antibody therapy and specific immunotherapy will be developed in an endeavor to develop novel strategies of precision medicine [98,99]. The chimeric anti-CD20 monoclonal antibody, rituximab, was the first FDA-approved therapeutic antibody for B-non-Hodgkin's lymphoma (B-NHL) therapy. The combination regimen R-CHOP (rituximab, cyclophosphamide, vincristine, prednisone) led to significant 10

prolonged survival times of B-NHL patients [100].

Rituximab inhibited several signal

transduction pathways that confer resistance to conventional chemotherapy, e.g. the p38 MAPK, NF-κB, (ERK 1/2) and AKT anti-apoptotic survival pathways, which mediated apoptosis resistance by regulating the expression of members of the Bcl-2 family. This antibody also inhibited the transcription factor YY1, which mediated apoptosis resistance via Fas (Apo-1) and TRAIL-R1 (DR5, Apo-2) regulation [101,102]. Rituximab increased the cytotoxicity towards ART in Ramos NHL cells [103]. ART induced both receptor-driven extrinsic and mitochondrial intrinsic apoptosis by induction of Fas expression, ROS generation and loss of mitochondrial membrane potential. Rituximab augmented these cellular effects. The expression of the transcription factors YY1 and Sp1 was reduced by the combination of rituximab and ART. While ART increased the expression of antioxidant proteins (catalase, glutathione S-transferase-π), rituximab counteracted this effect and, thereby, diminished the antioxidant capacity of cells. The effects of rituximab on antioxidant stress response represent a novel mode of action of rituximab to overcome drug resistance. Defective apoptosis contributes to the development of tumors to chemo- and radiotherapy [104]. In addition to Fas (Apo1) and Fas ligand (FasL, Apo1L), other members of this receptor family also induce apoptosis. Tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL, Apo2L) bound to TRAIL receptors 1 and 2 (TRAIL-R1, TRAILR2), also known as death receptors 4 and 5 (DR4 and DR5). TRAIL-targeted therapy attracted much attention, but the clinical results were disappointing, since most human tumors were resistant to TRAIL-induced apoptosis [105,106]. Therefore, the quest for novel TRAIL-based therapies is ongoing. Among other compounds, phytochemicals from medicinal plants produced synergistic effects on TRAIL-induced apoptosis in tumor cells [107] Recently, several investigations described the combination of TRAIL with DHA or ART (Table 8). The authors observed enhanced induction of apoptosis due to ROS generation, induction of DR5 expression, as well as downregulation of other apoptosis-regulating proteins (XIAP, Bcl-xL, NF-κB, and Akt) [108-110].

9 Combination with RNA interference Gene therapy is a collective term for the delivery of nucleic acids to treat diseases. There are numerous experimental approaches from DNA incorporated into engineered vectors to naked DNA

vaccines,

antisense

oligodeoxynucleotides, 11

ribozymes,

RNA

interference,

nanotechnological delivery, and CRISPR. The targeted manipulation of gene expression may be used as strategy to improve response of tumor cells to standard chemotherapy [111]. In principle, this strategy is may be transferrable to any new drug, as long as the determinants of cellular responsiveness are known. Some authors reported on the combination of ARS-type drugs with short interference RNA or short hairpin RNA (Table 9). Vascular cell adhesion molecule-1 (VCAM1) is expressed in brain tumors and has a role for cell surface adhesion. A combination of ARM and VCAM1 shRNA promoted apoptosis in glioblastoma cells [112]. Moloney murine leukemia virus insertion site 1 (BMI1) regulated tumor cell proliferation in nasopharyngeal carcinoma. ARS inhibited growth of nasopharyngeal carcinoma cells, induced G1 cell cycle arrest and inhibited BMI1. These effects were augmented by combinational treatment of ARS plus BMI1 siRNA [113]. Rac1 is important in activation of NF-κB-mediated transcriptional processes. Down-regulation of Rac1 expression by siRNA enhanced DHA-induced growth inhibition, cell cycle arrest, apoptosis, and migration in tumor cells [114]. The PARK7 (DJ-1) protein was overexpressed in DHA-resistant cells as identified by a proteomic approach. Compared to DHA-sensitive cells, this protein was translocated to the mitochondria and oxidized [115]. Although the exact function is still elusive, PARK7 might reveal cytoprotective effects. The causative role of PARK7 for DHA resistance was demonstrated by siRNA, which resensitized cells to DHA [115]. The stress-regulated protein p8 was upregulated upon DHA treatment, and this effect was reversed by siRNA leading to increased cell death [116]. Whether therapeutic approaches based on the combination of ARS-type drugs plus siRNA for specific target genes can be translated from the bench to the bedside will largely depend on the bioavailability of siRNA drugs and whether concentrations high enough to exert therapeutic effects in vivo can be reached. Nanotechnology might provide solutions to this problem in the future [117].

10 Hepatoxicity and drug interactions ARS is a natural product and is generally considered as safe and well tolerated. However, there is no rational reason to assume that natural products per se act in a gentle manner. Secondary metabolites from plants serve as chemical weapons to deter microbial attacks by viruses, bacteria and protozoans as well as to provide protection from being eaten by herbivores (insects and mammals including human beings). Therefore, some plant-based drugs or preparations might be toxic and exert side effects. 12

Recently, we published two cases, where the combination of ART with other medications led to unwanted adverse events. A glioblastoma multiforme patient treated with a combination of temozolomide, ART and Chinese herbs (Coptis chinensis, Siegesbeckia orientalis,

Artemisia

scoparia,

Dictamnus

dasycarpus)

suffered

from

reversible

hepatotoxicity [118]. While all drugs alone may bear a minor risk for hepatotoxicity, this specific combination increased liver enzyme activities in this patient. In the present case, there was no obvious reason to expect such toxicity at first sight. Artesunate is an approved antimalarial drug, whose good tolerability is well known [119]. In a large meta-analysis with 8000 patients, hepatotoxicity occurred in 0.9% of the patients [120]. The Chinese herbs used (Coptis chinensis, Siegesbeckia orientalis, Artemisia scoparia, Dictamnus dasycarpus) are also generally considered as safe and clinical toxicity is very rare. Temozolomide is an approved anti-cancer drug routinely used for glioblastoma therapy, although case reports of hepatotoxicity have been reported in the post-marketing phase [121]. The fact that hepatotoxicity occurred in this patient indicates that this specific combination was not well tolerated. A genetic predisposition of the patient (e.g. by single nucleotide polymorphisms in CYP genes) might be considered as explanation. Another glioblastoma patient was treated by an alternative practitioner with a combination of dichloroacetate and artesunate. The patient showed clinical and laboratory signs of severe liver damage and bone marrow toxicity (leukopenia, thrombocytopenia) and died a few days later [122]. The compassionate use of dichloroacetate/ART combination therapy outside of clinical trials cannot be recommended. These drastic examples illustrate that even if ART alone is considered to be well tolerated, non-approved combinations with medications from complementary and alternative medicine should be avoided. In general, the question arises, whether or not ARS-type drugs have to be considered as toxic. A recent report on human leukemia cells xenografted to nude mice described that single-dose ART (120 mg/kg) produced human equivalent exposures, but multiple doses daily administration required for in vivo efficacy were not tolerated [123]. Liver metabolism plays an important role for drug tolerability. Many compounds taken up with food or other sources cannot be directly excreted from the body, because they are too lipophilic. The liver is the main organ bearing the capability to convert lipophilic compounds into hydrophilic ones that can be excreted by urine and faeces (biotransformation). This process is divided into three phases. In phase I, non-polar compounds are oxidized to increase their polarity and, hence, water solubility. In phase 2, molecules are conjugated to other water soluble molecules so that they can be transported outward (phase 3). The most important 13

phase 1 enzymes in the liver are the cytochrome P450 monooxygenases (CYPs), which belong to a large gene family of 57 members in the human genome. CYPs metabolize exogenous xenobiotics, chemicals and therapeutic drugs as well as endogenous substrates. Many xenobiotics and drugs can increase CYP activity by enzyme induction or decrease their activity by enzyme inhibition. Altered CYP activities, may however, influence the biotransformation of other drugs. If different drugs are co-administered, drug-drug interactions may occur leading either to decreased drug activity or increased drug toxicity [124-127]. CYP isoenzymes frequently involved in drug metabolism are CYP3A4/5, 2D6, 2C8/9, 1A2, 2C19, 2E1, 2B6, and 2A6. ARS and its derivatives are also metabolized in the liver. They are mainly converted to the bioactive metabolite DHA (artenimol) after either parenteral or gastrointestinal administration [128]. The main isoenzymes participating in metabolization of ARS-type drugs are CYP2A6, 2B6, and 3A4 (Table 10). Potential drug-drug interactions were generated by the induction or inhibition of CYP 1A2, 2B6, 2C19, and 3A4 (Table 10, Figure 3). CYP induction or inhibition by ARS-type drugs are not only of theoretical interest. There is a number of publications reporting on the interaction of ARS and its derivatives with drugs used to treat diverse diseases. Examples are shown in Table 11 and include drugs to treat infectious diseases (e.g. the antifungal imidazole ketonazole and the macrolidic antibiotic troleandomycin), omeprazole, which is used for treating gastroesophageal reflux and peptic ulcer, and the anticonvulsants S-mephenytoin and carbamazepine [129-134]. Furthermore, some authors published drug-drug interactions with natural products, e.g. paraxanthine as metabolite of caffeine and theobromide and 8-methoxypsoralen [131,135,136]. This compound is used for the therapy of psoriasis, eczema, vitilago and cutaneous lymphoma together with UVA light (PUVA therapy = psoralen plus UVA). These examples illustrate that negative drug-drug interactions have to be taken into account, if ARS-type drugs are used in combination with other drugs.

11 Conclusions and perspectives There is no doubt that there are numerous clues that ARS-type drugs can be combined with other drugs to reach improved tumor cell killing in vivo and in vitro. In general, it can be stated that the underlying mechanisms for these additive or synergistic effects are the same that are also responsible for the cytotoxicity of ARS and its derivatives if applied as single drugs, i.e. DNA damage, angiogenesis inhibition, inhibition of specific signal transduction 14

pathways, inhibition of apoptosis and autophagy etc. [44]. The spectrum of drugs that can be combined with ARS-type drugs is remarkable broad and comprises not only synthetic or natural chemical entities (Figure 4), but also radiotherapy and macromolecules such as antibodies, recombinant proteins, or therapeutic nucleic acids. While the exact mechanisms for synergistic interactions between ARS-type drugs and other drugs are largely unknown as yet, the cellular targets of the artemisinin’s partner drugs are known in many cases (Figure 5). This information may provide a basis to start more detailed investigations, how the modes of action of ARS-type drugs and the partner drugs in combination therapy may cooperate to generate synergistic tumor cell killing. This opens avenues for more systematic investigations to unravel the most effective and save combination treatments. It is important not only to search for drug combinations with optimal tumor inhibitory activities, but also to combinations with no or negligible effects on normal tissues and organs. It has been excluded that some combinations may also lead to antagonistic effects. The fact that 8-methoxypsoralen inhibited biotransformation of ARS indicates that PUVA therapy is not suited to be combined with ARS-type drugs [131]. A clinical trial in advanced non-small cell lung cancer reported on efficacy and toxicity of the standard combination vinorelbine and cisplatin with or without intravenous ART injections (120 mg) for 8 days [14]. Each treatment group consisted of 60 patients. Statically significant improvements in short-term survival were measured. The disease control rate of the trial group (88.2%) was significantly higher than that of the control group (72.7%) and the time to progression of the ART-treated patients (24 weeks) was significantly longer than that of the control group (20 weeks). ART was well tolerated and increased toxicity was not observed. Nevertheless, other ART-based combinations may be less well tolerated, as discussed above in two case reports with severe hepatotoxicity [118,122]. Controlled, doubleblind, randomized clinical trials are required to test efficacy and tolerabolity of novel combinations between ARS-type drugs and other treatment modalities for cancer therapy.

Conflict of interest: There is no conflict of interest.

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22

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26

Table 1: Enhanced activity of DNA-targeting anticancer drugs by ARS-type compounds. Combination drug

ARStype drug

Cell line

Doxorubicin

DHA

MCF-7 breast Ca

Doxorubicin

DHA

Doxorubicin

ART

Cisplatin

DHA

Cisplatin

DHA

SKOV3 and SKOV3/DDP ovarian Ca

Cisplatin

ART

ovarian Ca

Carboplatin Oxaliplatin

Effect

Synergistic interaction; induction of apoptosis various (HeLa, OVCAR-3, Enhanced doxorubicin activity in vitro and in MCF-7, PC-3 and A549) vivo. Induction of apoptosis and pyknosis; 10 multiple myeloma cell lines Synergistic interaction

Wu et al., 2013 [137] Tai et al., 2016 [138] Papanikolaou et al., 2014 [139] A549 and A549/DDP lung Ca Synergistic interaction in vitro and in vivo. Zhang et al., Inhibition of angiogenesis by downregulation 2013 [140] of HIF-1α and VEGF Enhanced cisplatin activity in cisplatinresistant cells by mTOR inhibition and apoptosis induction

Downregulation of RAD51 by ART sensitizes to cisplatin DHA A2780 and OVCAR-3 ovarian Enhanced carboplatin activity in vitro and in Ca vivo ArteMCF-7 breast Ca; HCT116 Enhanced oxaliplatin activity misone and SW480 colon Ca; PANC1 and MIAPaCa pancreas Ca

Temozolomide DHA

C6 rat glioma

Temozolomide ART Temozolomide DHA

U87MG and A172 glioblastoma 10 glioma cell lines

Temozolomide ART

Glioblastoma

Gemcitabine

HepG2 and Hep3B liver Ca

Gemcitabine

ART, DHA DHA

Gemcitabine

DHA

Reference

Feng et al., 2014 [141] Wang et al., 2015 [35] Chen et al., 2009 [142] Gravett et al., 2011 [143]

Enhanced temozolomide activity by generation of reactive oxygen species Enhanced temozolomide activity

Huang et al., 2008 [144] Karpel-Massler et al., 2014 [145] Enhanced temozolomide activity by Zhang et al., induction of autophagy 2015 [146] Enhanced temozolomide activity in vitro and Berte et al., 2016 in vivo by inhibition of homologous [147] recombination and senescence

Enhanced gemcitabine activity in vitro and in vivo independent of p53 mutational status BxPC-3 and Panc-1 pancreas Enhanced gemcitabine activity in vitro and in Ca vivo. Abolishing the NF-κB inducing activity of gemcitabine

Hou et al., 2008 [148] Wang et al., 2010a [149]

Gemcitabine

BxPC-3 and Panc-1 pancreas Enhanced gemcitabine activity in vitro and in Ca vivo. Inhibition of NF-κB ArteMCF-7 breast Ca; HCT116 Enhanced gemcitabine activity misone and SW480 colon Ca; PANC1 and MIAPaCa pancreas Ca

Wang et al., 2010b [150] Gravett et al., 2011 [143]

Gemcitabine

DHA

A549 lung Ca

Synergistic interaction. Activation of Bak, Caspases 3,8 and 9, loss of mitochondrial membrane potential, inhibition of Fas

Zhao et al., 2014 [151]

Cytarabine

ART, DHA

10 acute myeloic leukemia cell lines

Synergistic interaction

Drenberg et al., 2016 [123]

27

Table 2: Enhanced activity of anticancer drugs with diverse modes of actions by ARS-type compounds. Combination drug

ARStype drug

Erlotinib

ART

Thalidomide

Cell line

11 glioblastoma cell lines ArteMCF-7 breast Ca; misone HCT116 and SW480 colon Ca; PANC-1 and MIAPaCa pancreas Ca

Effect

Reference

Additive and synergistic interactions Enhanced thalidomide activity

Efferth et al., 2004 [152] Gravett et al., 2011 [143]

Lenalidomide

ART

MCF-7 breast Ca, Enhaced lenalidomide Liu et al., 2011 [153] HCT116 colon Ca, activity. Simultaneous arrest A549 lung Ca at all phases of the cell cycle by reduction of cell cycle transition proteins

Bortezomib

ART

10 multiple No cross-resistance of myeloma cell lines bortezomib-resistant cells; non-caspase-mediated cell death; synergy if combined with bortezomib

Papanikolaou et al., 2014 [139]

Dexamethasone ART

10 multiple Synergistic interaction myeloma cell lines Molt-4 leukemia Enhanced growth inhibition

Papanikolaou et al., 2014 [139] Gerhardt et al., 2015 [154]

Dexamethasone DHA

28

Table 3: Activity of ARS-type drugs towards drug-resistant tumor cell lines.

Resistance type

ARS-type drug

P-gp-mediated MDR

ART

P-gp-mediated MDR

CEM/ADR5000 and CEM/VCR100 acute lymphoblastic leukemia ARS, ART, K562/adr chronic DHA myeloic leukemia

P-gp-mediated MDR

ART

P-gp-mediated MDR P-gp-mediated MDR

ART

P-gp-mediated MDR P-gp-mediated MDR

ART

P-gp-mediated MDR P-gp-mediated MDR P-gp-mediated MDR P-gp-mediated MDR P-gp-mediated MDR MRP1-mediated MDR MRP1-mediated MDR

Cell line

Effect

Reference

no cross-resistance

Efferth et al., 2001 [155]

no cross-resistance by loss of mitochondrial membrane potential

Reungpatthanaphong and Mankhetkorn, 2002 [156]

CEM/ADR5000 acute lymphoblastic leukemia CEM/VBL0.05 leukemia cells HT29 colon Ca

No cross-resistance by induction of Efferth et al., 2007 ROS-induced apoptosis [157]

pig brain endothelial cells MCF-7/adr breast cancer

Inhibition of daunorubicin efflux

Li et al., 2008 [158]

Increase of doxorubicin resistance by SERCA inhibition and Pglycoprotein induction

Riganti et al., 2008, 2009 [159,160]

Enhanced calcein-AM uptake by inhibition of blood brain barrier Inhibition of P-gp efflux; collateral sensitivity

Soomro et al., 2011 [45] Zhong et al., 2016 [161]

Artesunic CEM/ADR5000 acute acid dimers lymphoblastic leukemia ARTK562/ADR chronic podophyllo- myeloic leukemia toxin hybrid

no cross-resistance or collateral sensitivity

Reiter et al., 2012 [162]

no cross-resistance by disrupting microtubule network

Zhang et al., 2016 [163]

Artesunic CEM/ADR5000 acute acid dimers lymphoblastic leukemia ARS CEM/ADR5000 acute hybrids lymphoblastic leukemia ARS CEM/ADR5000 acute hybrids lymphoblastic leukemia ART CEM/E1000 acute lymphoblastic leukemia ARS, ART, GLC4/adr lung Ca DHA

no cross-resistance

Horwedel et al., 2010 [164]

no or low degree of crossresistance

Reiter et al., 2014 [165]

no or low degree of crossresistance

Reiter et al., 2015 [166]

Enhancement of daunorubicin uptake

Efferth et al., 2002 [167]

no cross-resistance by loss of mitochondrial membrane potential

Reungpatthanaphong and Mankhetkorn, 2002 [156]

Downregulation of BCRP expression in vitro and in vivo Reversal of drug resistance

Ma et al., 2011 [168]

no cross-resistance

Efferth et al., 2001 [155]

no cross-resistance

Efferth et al., 2001 [155]

ARS

ARS derivatives

BCRP-mediated MDR BCRP-mediated MDR Methotrexate resistance

ART

A549 lung Ca

ART

Hydroxyurea resistance

ART

Eca109/ABCG2 esophageal Ca CEM/MTX1500LV acute lymphoblastic leukemia CEM/HUR90 acute lymphoblastic leukemia

ART

29

Liu et al., 2013 [169]

Cisplatin resistance

ART, DHA

Imatinib resistance DHA

UKF-NB-3rCDDP1000 neuroblastoma

sensitivity of ART or DHA was increased by L-buthionine-S,Rsulfoximine

Michaelis et al., 2010 [170]

chronic myeloic leukemia

Inhibition of BCR/ABL expression and tyrosine kinase function in imatinib-resistant cells

Lee et al., 2013 [171]

30

Table 4: Enhanced activity of radiotherapy or photodynamic therapy by ARS-type compounds. ARS-type Cell line drug Radiotherapy: DHA U373MG glioblastoma ART A549 lung Ca

Effect

Reference

Radiosensitization by ROS generation and GST inhibition Kim et al., 2006 [51] Radiosensitization by nitric oxide-mediated induction of G2M cell cycle arrest.

Zhao et al., 2011 [52]

ART

LN229 and U87MG Radiosensitization by induction of DNA damage, G2M cell Reichert et al., 2012 glioblastoma cycle arrest and apoptosis. [53]

ART

HeLa cervical Ca

Radiosensitization in vitro and in vivo by induction of G2M Luo et al., 2013; 2014 cell cycle arrest and apoptosis. Involvement of multiple [54,172] pathways (RNA transport, the spliceosome, RNA degradation and p53 signaling).

DHA

GLC-82 lung Ca

Radiosensitization by induction of G0G1 cell cycle arrest and apoptosis.

Zuo et al., 2014 [55]

Enhanced growth inhibition and apoptosis.

Li et al., 2014 [173]

Photodynamic therapy: DHA Eca109 and Ec9706 esophageal Ca

31

Table 5: Enhanced cytotoxic activity of ARS-type compounds by drugs established for other indications than cancer.

Combination ARSdrug type drug

Cell line

Effect

Reference

Captopril

ART

HUVEC endothelial cells, Synergistic interaction by inhibition chorioallantoic of angiogenesis membrane of quail embryos

Krusche et al., 2013 [62]

Chloroquine

ARS

A549 lung Ca

Ganguli et al., 2014 [65]

Miconazole

ARS

5637 bladder Ca, 4T1 breast Ca

Synergistic interaction by ROS generation and induction of autophagy Necrosis was more prominent than apoptosis

32

Shahbazfar et al., 2014 [67]

Table 6: Interaction of anticancer activity of ARS-type compounds by novel synthetic compounds.

Combination compound

ARStype drug

Cell line

Effect

Reference

L-Buthionine sulfoximine Ethacrynic acid

ART

MSV-HL13 cells

Sensitization to ART

ART

MSV-HL13 cells

Sensitization to ART

6PGD inhibitors DHA Physcion and S3

Leukemia cells

Synergistic interaction by activation of AMP-activated protein kinase

Efferth and Volm, 2005 [70] Efferth and Volm, 2005 [70] Elf et al., 2016 [78]

Glutaminase-1 inhibitor 968 Histone deacetylase inhibitors N-tert-butylalphaphenylnitrone

Liver Ca cell lines Synergistic interaction by excessive ROS generation HepG2 liver Ca Enhanced DHA activity in vitro and in vivo

Wang et al., 2016 [82] Zhang et al., 2012 [88]

MOLT-4 acute lymphoblastic leukemia

Chan et al., 2013 [90]

DHA DHA

DHA

Attenuation of DHA cytotoxicity

33

Table 7: Enhanced anticancer activity of ARS-type compounds by natural products or natural product derivatives.

Combination compound

ARS- Cell line type drug

Effect

Reference

1 alpha,25dihydoxyvitamin D3 [1,25OH2D3] All-trans retinoic acid Sodium butyrate Dictamnine

ARS

HL-60 leukemia

Enhanced cell differentiation

Kim et al., 2003 [174]

ARS

HL-60 leukemia

Enhanced cell differentiation

Kim et al., 2003 [174] Singh and Lai, 2005 [175] An et al., 2013 [176] Liu and Cui, 2013 [177]

DHA Molt-4 leukemia

Enhanced growth inhibition

DHA A549 lung Ca

Enhanced induction of S phase arrest and apoptosis

Triptolide

ART

Pancreatic Ca

Inhibition of growth, induction of apoptosis, induction of HSP20 and HSP27

Allicin

ART

Osteosarcoma

Enhanced growth inhibition in vitro and in vivo

Arsenic trioxide ART

K562 and U937 leukemia

Enhanced growth inhibition. Induction of apoptosis and downregulation of Fos

Butyric acid

ARS

Resveratrol

ARS

5637 bladder Ca, Necrosis was more prominent than apoptosis Shahbazfar et al., 4T1 breast Ca 2014 [67] HeLa cervical Ca Enhanced growth inhibition, cell migration, apoptosis, Li et al., 2014 [179] and HepG2 liver necrosis and ROS generation Ca

Sodium salicylate Curcumin

DHA leukemia cells

Vitamin C , vitamin D3 Halofuginone

DHA Molt-4 leukemia

Enhanced growth inhibition

ARS

Growth inhibition in vivo by increasing p21CIP, p27KIP and CDK2 to induce G1 cell cycle arrest

ARS

Drosophila melanogaster larvae

MCF-7 breast Ca, HCT116 colon Ca

Jiang et al., 2013 [178] fehlt Li et al., 2014 [179]

Additive interaction

Wickerath and Singh, 2014 [180] Inhibition of brain tumor, prolongation of life span and Das et al., 2014 restoration of locomotor activity [181]

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Gerhardt et al., 2015 [154] Chen et al., 2016 [182]

Table 8: Enhanced cytotoxic activity of therapeutic antibodies or recombinant proteins by ARS-type drugs.

Combination ARSdrug type drug

Cell line

Effect

Reference

Rituximab

ART

B-cell lymphoma

Sieber et al., 2009 [103]

TRAIL

DHA

Prostate Ca

TRAIL

ART

HeLa cervical Ca

Enhanced inhibition of growth and ROS generation and induction of apoptosis. Downregulation of YY1, Sp1, and Fas Enhanced cell killing. Induction of DR5 Enhanced apoptosis induction. Downregulation of XIAP, Bcl-XL, NFκB, Akt

TRAIL

DHA

BxPC-3 and PANC-1 pancreas Ca

He et al., 2010 [108] Thanaketpaisarn et al., 2011 [109]

Enhanced induction of apoptosis and Kong et al., 2012 ROS generation. DR5 induction [110]

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Table 9: Enhanced cytotoxic activity of ARS-type drugs by RNA interference.

Combination drug

ARS- Cell line type drug

Effect

Reference

BMI siRNA

ARS

CNE-1 and CNE-2 Enhanced inhibition of growth and G1 Wu et al., 2011 nasopharyngeal Ca cell cycle arrest [113]

VCAM1 shRNA

ARM

U97MG glioblastoma

Enhanced inhibition of cell viability, migration, invasiveness, apoptosis, and MMP-2/-9 expression

Wang et al., 2013 [112]

RAC1 siRNA

DHA

HCT116 and RKO colon Ca

Enhanced inhibition of growth and migration and induction of cell cycle arrest and apoptosis

Han et al., 2013 [114]

PARK7 siRNA

DHA

HeLa cervical Ca

Zhu et al., 2014 [115]

p8 siRNA

DHA

HeLa cervical Ca; HCT116 colon Ca; HepG2 liver Ca; SKOV3 ovarian Ca

Enhanced inhibition of growth. Enhanced induction of ROS Enhanced generation of ROS and apoptosis Reversion of DHA-induced p8 expression led to increased cell death

36

Chen et al., 2015 [116]

Table 10: Role of CYP enzymes for ARS-type drugs. Combination drug

ARS-type drug

Investigation Model

Effect

Reference

CYP2B6, 3A4, and 3A5 CYP2B6 and 3A4 CYP2B6 and 3A4 CYP2B6 and 2A6 CYP2B6

ARE

Liver microsomes

Biotransformation

ARS

B-cell microsomes

Biotransformation

CYP2C19

ARS

CYP2B6 and CYP3A4 CYP2B6 and CYP2C19 CYP2B6

ARS

Grace et al., 1998;1999 [129,183] Svensson et al., 1999 [130] Giao and de Vries, 2001 [184] Svensson et al., 2003 [185] Asimus et al., 2009 [186] Giao and de Vries, 2001 [184] Burk et al., 2005 [187]

ARS

Biotransformation

ARS

Liver microsomes

Biotransformation

ARS

Liver microsomes

Biotransformation Enzyme induction

human hepatocytes

Enzyme induction

ARS, ARE, Healthy volunteers Enzyme induction ARM ARS Human hepatocytes Enzyme induction

CYP3A4 and CYP2B6

ARS

CYP2B6 and CYP3A4 CYP1A2

ARS + piperaquine ARS, DHA Liver microsomes

Enzyme inhibition

CYP2B6

ARS, DHA

Enzyme inhibition

CYP2B6 CYP1A2, 2B6, 2C19 and 3A4 CYP2B6

Liver microsomes, Enzyme induction Human hepatocytes, recombinant enzyme Healthy volunteers Enzyme induction

Liver microsomes, human hepatocytes, recombinant enzyme ARS, ARM Liver microsomes, recombinant enzyme ARS, ARM, Liver microsomes, ART, DHA recombinant enzymes ARM Molecular docking

Elsherbiny et al., 2008 [133] Rhodes et al., 2011 [188] Xing et al., 2012 [189]

Zang et al., 2014 [190] Bapiro et al., 2001 [191] Xing et al., 2012 [189]

Enzyme inhibition

Ericsson et al., 2012 [193]

Enzyme inhibition

Ericsson et al., 2014 [193]

Enzyme inhibition

Kobayashi et al., 2014 [194]

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Table 11: Interaction of ARS-type drugs with other drugs via hepatic CYP enzymes.

Combination drug

ARS- InvestigationModel Effect type drug

Ketoconazole

ARE

Liver microsomes

Ketoconazole

ARS

Liver microsomes

Troleanodomycin 8-Methoxypsoralen

ARE

Liver microsomes

ARS

Liver microsomes

Omeprazole

ARS

Healthy volunteers

Inhibition of biotransformation of Mihara et al., omeprazole 1999 [131]

Paraxanthine

ARS

Healthy volunteers

CYP1A2-mediated inhibition of biotransformation of paraxanthine to caffeine

Bapiro et al., 2005 [135]

Paraxanthine

ARS, Healthy volunteers ARE, DHA

CYP1A2-mediated inhibition of biotransformation of paraxanthine to caffeine

Asimus et al., 2007 [136]

CYP2B6-mediated biotransformation of Smephenytoin CYP2B6- and CYP2C19mediated biotransformation of Smephenytoin Inhibition of biotransformation of carbamazepine

Simonsson et al., 2003 [132]

S-mephenytoin ARS

Liver microsomes

S-mephenytoin ARS

Healthy volunteers

Carbamazepine ARS, Rabbits ARE, ARM

Reference

Inhibition of biotransformation of Grace et al., ARE 1998, 1999 [129,183] Inhibition of biotransformation of Svensson et al., ARS 1999 [130] Inhibition of biotransformation of ARE Inhibition of biotransformation of ARS

38

Grace et al., 1998 [129] Svensson et al., 1999 [130]

Elsherbiny et al., 2008 [133] Sukhija et al., 2006 [134]

Legends to figures:

Figure 1: Synopsis of mechanisms involved in combination treatments between ARS-based drugs and other treatment modalities for cancer therapy. Figure 2: Combating resistance to clinically established anticancer drugs by ARS and its derivatives. Figure 3: Role of CYP enzymes for artemisinin and its derivatives.

Figure 4: Cellular targets of ARS-type drugs and partner compounds in combination therapy approaches. Figure 5: Chemical structures of ARS-type drugs and partner compounds in combination therapy approaches.

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• Artemisinin • Artesunate • Dihydroartemisinin

Standard anticancer drugs Radiotherapy Photodynamic therapy Drugs for other indications Novel synthetic compounds Natural products Therapeutic antibodies and recombinant proteins • RNA interference • • • • • • •

Combination therapy of cancer • • • • •

Inhibition of cell growth and cell viability Induction of cell cycle arrest Induction of apoptosis and autophagy Inhibition of invasion, migration and metastasis Inhibition of angiogenesis

Hepatotoxicity and drug-drug interactions

40

• Artemisinin • Artesunate • Dihydroartemisinin

P-gp-mediated MDR MRP-mediatedMDR BCRP-mediated MDR Cisplatin resistance Methotrexate resistance • Hydroxyurea resistance • Imatinib resistance • • • • •

• Cross-resistance • Bypassing • Functional inhibition of resistance proteins • Collateral senstivity

41

Biotransformation CYP2A6, 2B6, 3A4

Artemisinin Artesunate Dihydroartemisinin

Induction

Inhibition

CYP2B6, 2C19, 3A4

CYP1A2, 2B6, 2C19, 3A4

42

43

Butyric acid

Chloroquine

Allicin

Halofuginone

Omeprazole

n-Tert-butyl α phenylnitrone

Resveratrol

Vitamin D3

Vitamin C

Sodium salicylate

Miconazole

Paraxanthine

Ketoconazole

S-Mephenytoin

Sodium butyrate

Curcumin

8-Methoxypsoralen

Carbamazepine

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45