**2.8 Multiple-drug combination therapy**

A combination therapy with different drugs targeting on multiple molecules that contribute to malignancy is rational and enhances antitumor effects, reduces side effects, and avoids resistance. This section provides an overview of the treatment of recurrent GBM with multiple existing drugs (**Figure 5**).

## *2.8.1 CLOVA cocktail*

**2.7 Anti-inflammatory drugs**

*2.7.1 Acetylsalicylic acid (ASA)*

of ASA in malignant glioma.

*2.7.2 Sulfasalazine (SAS)*

**Figure 4.**

**144**

ASA, a nonsteroidal anti-inflammatory drug, is used worldwide. Previous studies have shown the molecular signaling changes by aspirin (**Figure 4**). ASA exerts an anticancer via the inhibition of prostaglandin, including prostaglandin E2 (PGE2), synthesis through the acetylation, and inhibition of cyclooxygenase [77, 78]. ASA treatment suppresses the invasion of glioma cells via the activation of the expression of connexin 43 (Cx43), which is a major gap junction protein in astrocytes. Cx43 is normally suppressed by PGE2. Thus, ASA-treated glioma cells would overexpress Cx43 and the invasion would be inhibited [79]. Other studies have revealed that ASA suppresses the Wnt/β-catenin/T-cell factor (TCF) signaling pathway, which plays a key role in glioma progression [79]. Wnt/β-catenin/TCF pathway suppression would suppress glioma via the regulation of downstream genes, *c-myc* and *cyclin D1*. ASA inhibits the sonic hedgehog (SHH)/gliomaassociated oncogene homolog 1 (GLI1) pathway and adjusts the epithelial-tomesenchymal transition [80]. The SHH/GLI1 pathway is also associated with recovery from the damage by TMZ [80]. Based on these studies, a retrospective cohort study was performed to investigate the therapeutic effect of ASA in patients with malignant glioma. The results revealed that the use of ASA is associated with a higher OS and PFS in patients with WHO grade III glioma; however, there was no difference in OS and PFS in patients with WHO grade IV glioma [81]. In the future, prospective multicenter randomized studies are warranted to determine the effect

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

SAS, which is approved for the treatment of rheumatoid arthritis and inflammatory bowel diseases, may be a therapeutic drug for malignant glioma [82]. SAS exerts anti-inflammatory effects by blocking the activation of NF-κB and the Xc

antiporter system, which usually causes the uptake of cystine, release of glutamate, and increase in the levels of reactive oxygen species (ROS) [83]. NF-κB is activated in GBM tissues and promotes cell proliferation and survival. SAS blocks

*Antitumor mechanisms of acetylsalicylic acid. ASA indicates multimodal effects for glioma cells. ASA suppresses the invasion of glioma cells by activating the expression of connexin 43. ASA also suppresses* c-myc *and* cyclin D1 *through Wnt/β-catenin/TCF pathway and interfered the recovery of DNA damages and adjusted the epithelial-to-mesenchymal transition through SHH/GLI1 pathway. Abbreviation: PGE2, prostaglandin E2.*

The CLOVA cocktail, composed of cimetidine, lithium, olanzapine, and valproate, targets dysregulated GSK3β in GBM [87–89]. The therapeutic effects of GSK3β inhibition are the suppression of tumor cell survival and proliferation, synergy with TMZ and irradiation, attenuation of invasion, and induction of GSC differentiation via various pathways [90]. Olanzapine stimulates AMPK catabolic action, followed by the induction of p53-dependent autophagy. VPA, as an HDACi, enhances the effect of radiation. A phase I/II clinical study to investigate the efficacy and safety of the CLOVA cocktail in patients with TMZ-resistant recurrent GBM revealed that this regimen is well tolerated and results in a higher OS than the control group treated with TMZ alone [87].

### *2.8.2 CUSP9\* treatment*

The rational of the coordinated undermining of the survival paths active in GBM by nine repurposed drugs [aprepitant, artesunate, auranofin, captopril, celecoxib, disulfiram (DSF), itraconazole, ritonavir, and sertraline], termed CUSP9\*, was developed to prevent therapeutic resistance in tumor cells. CUSP9\* targets the diverse complementary redundant pathways to render tumor cells susceptible to the cytotoxic effects of TMZ [91] by the simultaneous administration of nine drugs with low-dose daily TMZ. Each drug exerts different inhibitory effects on the 17 molecules and pathways shown in **Figure 5**. Auranofin and DSF increase the level of intracellular reactive oxygen species [96]. Recently, the experimental CUSP9\* strategy with TMZ was shown to suppress the stemness of GSCs and tumorigenesis via the blockade of the Wnt/β-catenin pathway [92].

## *2.8.3 FTT cocktail*

A unique therapeutic approach to reprogram and reverse cancer cells to normal somatic cells has attracted attention. The combination of fasudil, tranilast, and TMZ was identified to reprogram GBM cells into neuronal like cells [93]. GBM cells treated with the FTT cocktail show normal neuronal morphology, gene expression, and electrophysiological properties and lower malignancy than untreated cells. This might be caused by the synergistic effect of the three drugs [93]. In addition, the FTT cocktail suppresses tumor growth and prolongs survival in a GBM xenograft

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

model more than TMZ alone. Fasudil inhibits the ROCK2/moesin/β-catenin pathway in TMZ-resistant glioma cell lines and downregulates the ATP-binding cassette super-family G member 2 transporter to increase sensitivity to TMZ [94]. The inhibition of ROCK with mTOR inhibition exerts neuronal reprogramming more effectively in vitro and in vivo than the inhibition of ROCK alone [95], which suggests the possibility of more drug combinations. Tranilast alone inhibits glioma progression via TGF-β restriction [96]. Although the mechanism underlying the tumor-suppressive function of the FTT cocktail is not fully elucidated, this cocktail

*Drug Repositioning for the Treatment of Glioma: Current State and Future Perspective*

DSF, the FDA-approved drug for the treatment of alcohol abuse, may be a therapeutic drug for GBM. DSF is an irreversible inhibitor of aldehyde dehydrogenase [97], which is a functional marker of cancer stem cells [98]. An in vitro study revealed that DSF is an inhibitor of MGMT and enhances the efficacy of alkylatorinduced tumor death [99]. Another study revealed that DSF suppresses the growth and self-renewal of GSCs via the inhibition of polo-like kinase-1, which controls cell progression and cytokinesis [97]. The activity of DSF is potentiated by copper and induces GSC death [100]. However, an open-label, single-arm phase II study of TMZ plus DSF for patients with recurrent TMZ-resistant GBM showed that the objective response rate is 0% and DSF combination therapy would have only

Statins, a therapeutic drug for dyslipidemia, inhibit 3-hydroxy-3-methylglutaryl-

[102, 103]. In vitro, simvastatin induces the apoptosis of C6 glioma cells by phosphorylation of activating transcription factor-2 and c-Jun [102]. Lovastatin suppresses the proliferation and migration of glioma cell lines via the suppression of the activation of ERK [103]. A retrospective cohort study suggested that the long-term prediagnostic statin intake increases the OS in patients with GBM [104]. Another retrospective cohort study suggested that statin intake is associated with fewer seizures in patients with GBM [105]. However, other cohort studies have not indicated a survival relationship between malignant glioma and statin intake. Finally, a meta-analysis of these retrospective cohort studies revealed that statins do not increase the PFS and OS in patients with GBM [106]. In the future, prospective multicenter randomized studies

Kenpaullone, a potent and nonselective inhibitor of GSK3 [107], is a serine/ threonine kinase that regulates numerous signaling pathways involved in cell

coenzyme A reductase. Some statins have a therapeutic effect on glioma cells

are warranted to determine the effect of statins in malignant glioma.

cycle control, proliferation, differentiation, and apoptosis [108, 109]. Kenpaullone treatment inhibits glioma cell proliferation, suppresses antiapoptotic mechanisms in the mitochondria, inhibits pro-survival factors, and

might improve the current therapy for malignant glioma.

*DOI: http://dx.doi.org/10.5772/intechopen.92803*

limited therapeutic effects for patients with GBM [101].

**2.9 Other drugs**

*2.9.1 Disulfiram*

*2.9.2 Statins*

**2.10 Pre-drugs**

**147**

*2.10.1 Kenpaullone*

#### **Figure 5.**

*Multiple molecular-targeted therapies by multiple-drug treatment with temozolomide. Multiple existing drug combination, CLOVA cocktail, CUSP9\* treatment, and FTT cocktail, targets multiple signaling pathways which attribute GBM malignant phenotype. Abbreviations: 5-LO, 5-lipoxygenase; ABCG2, ATP-binding cassette super-family G member 2; ACE, angiotensin-converting enzyme; ALDH, aldehyde dehydrogenase; AMPK, adenosine monophosphate; CA, carbonic anhydrase; CDK, cyclin-dependent kinase; COX, cyclooxygenase; FAK, focal adhesion kinase; GSK3β, glycogen synthase kinase-3β; HDAC, histone deacetylase; HH, hedgehog; JNK, c-Jun N-terminal kinase; MGMT, O<sup>6</sup> -methylguanine-DNA methyltransferase; MMP, matrix metalloproteinase; MT, membrane type; mTOR, mammalian target of rapamycin; NK-1, neurokinin-1; NF-κB, nuclear factor-kappaB; P-gp, P-glycoprotein; ROCK, rhoassociated protein kinase; ROS, reactive oxygen species; TCTP, translationally controlled tumor protein; TF, tissue factor; TGF-β, transforming growth factor-β; TMZ, temozolomide; VEGF, vascular endothelial growth factor.*

*Drug Repositioning for the Treatment of Glioma: Current State and Future Perspective DOI: http://dx.doi.org/10.5772/intechopen.92803*

model more than TMZ alone. Fasudil inhibits the ROCK2/moesin/β-catenin pathway in TMZ-resistant glioma cell lines and downregulates the ATP-binding cassette super-family G member 2 transporter to increase sensitivity to TMZ [94]. The inhibition of ROCK with mTOR inhibition exerts neuronal reprogramming more effectively in vitro and in vivo than the inhibition of ROCK alone [95], which suggests the possibility of more drug combinations. Tranilast alone inhibits glioma progression via TGF-β restriction [96]. Although the mechanism underlying the tumor-suppressive function of the FTT cocktail is not fully elucidated, this cocktail might improve the current therapy for malignant glioma.

## **2.9 Other drugs**

## *2.9.1 Disulfiram*

DSF, the FDA-approved drug for the treatment of alcohol abuse, may be a therapeutic drug for GBM. DSF is an irreversible inhibitor of aldehyde dehydrogenase [97], which is a functional marker of cancer stem cells [98]. An in vitro study revealed that DSF is an inhibitor of MGMT and enhances the efficacy of alkylatorinduced tumor death [99]. Another study revealed that DSF suppresses the growth and self-renewal of GSCs via the inhibition of polo-like kinase-1, which controls cell progression and cytokinesis [97]. The activity of DSF is potentiated by copper and induces GSC death [100]. However, an open-label, single-arm phase II study of TMZ plus DSF for patients with recurrent TMZ-resistant GBM showed that the objective response rate is 0% and DSF combination therapy would have only limited therapeutic effects for patients with GBM [101].

### *2.9.2 Statins*

Statins, a therapeutic drug for dyslipidemia, inhibit 3-hydroxy-3-methylglutarylcoenzyme A reductase. Some statins have a therapeutic effect on glioma cells [102, 103]. In vitro, simvastatin induces the apoptosis of C6 glioma cells by phosphorylation of activating transcription factor-2 and c-Jun [102]. Lovastatin suppresses the proliferation and migration of glioma cell lines via the suppression of the activation of ERK [103]. A retrospective cohort study suggested that the long-term prediagnostic statin intake increases the OS in patients with GBM [104]. Another retrospective cohort study suggested that statin intake is associated with fewer seizures in patients with GBM [105]. However, other cohort studies have not indicated a survival relationship between malignant glioma and statin intake. Finally, a meta-analysis of these retrospective cohort studies revealed that statins do not increase the PFS and OS in patients with GBM [106]. In the future, prospective multicenter randomized studies are warranted to determine the effect of statins in malignant glioma.

#### **2.10 Pre-drugs**

**Figure 5.**

*factor.*

**146**

*Multiple molecular-targeted therapies by multiple-drug treatment with temozolomide. Multiple existing drug combination, CLOVA cocktail, CUSP9\* treatment, and FTT cocktail, targets multiple signaling pathways which attribute GBM malignant phenotype. Abbreviations: 5-LO, 5-lipoxygenase; ABCG2, ATP-binding cassette super-family G member 2; ACE, angiotensin-converting enzyme; ALDH, aldehyde dehydrogenase; AMPK, adenosine monophosphate; CA, carbonic anhydrase; CDK, cyclin-dependent kinase; COX, cyclooxygenase; FAK, focal adhesion kinase; GSK3β, glycogen synthase kinase-3β; HDAC, histone*

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

*methyltransferase; MMP, matrix metalloproteinase; MT, membrane type; mTOR, mammalian target of rapamycin; NK-1, neurokinin-1; NF-κB, nuclear factor-kappaB; P-gp, P-glycoprotein; ROCK, rhoassociated protein kinase; ROS, reactive oxygen species; TCTP, translationally controlled tumor protein; TF, tissue factor; TGF-β, transforming growth factor-β; TMZ, temozolomide; VEGF, vascular endothelial growth*

*-methylguanine-DNA*

*deacetylase; HH, hedgehog; JNK, c-Jun N-terminal kinase; MGMT, O<sup>6</sup>*

#### *2.10.1 Kenpaullone*

Kenpaullone, a potent and nonselective inhibitor of GSK3 [107], is a serine/ threonine kinase that regulates numerous signaling pathways involved in cell cycle control, proliferation, differentiation, and apoptosis [108, 109]. Kenpaullone treatment inhibits glioma cell proliferation, suppresses antiapoptotic mechanisms in the mitochondria, inhibits pro-survival factors, and

attenuates the stemness and viability of GSCs via the downregulated activity of GSK3β [88, 110, 111]. The combination of low-dose kenpaullone with TMZ enhances cytotoxicity against glioma via the induction of c-Myc-mediated apoptosis [110]. These results suggest that kenpaullone is a potential compound for the treatment of glioma.

However, a malignant glioma is a complicated aggregation, once called "glioblastoma multiforme" [121]. If one candidate agent exerts therapeutic effects for some glioma cells, other resistant glioma cells would multiply. To overcome this problem, several previous studies have performed multiple-drug combination therapy. This therapy would focus on multiple therapeutic targets at once with minimal side effects [85]; however, currently, there are no combination treatments that can replace the current treatments. Second, despite its clinical aggressiveness, 60–70% of the tumor cells in malignant glioma are in the nonproliferating phase [122]. This indicates that not only heterogeneous cells but also the cell cycle must be considered because resting cells indicate resistance to chemoradiotherapy [122]. Based on this, some studies have focused on candidate agents that can change the phase of

*Drug Repositioning for the Treatment of Glioma: Current State and Future Perspective*

The strategy to discover the most effective drug is the key to accomplish a successful drug repositioning. One of the main methods is an in vitro or in vivo drug screening system in which target cells are treated by various existing drugs and the alteration to the malignant phenotype, such as by cytotoxicity, is analyzed. Drugs that exert cytotoxicity in GBM cells, especially GSCs, at low concentrations would be good candidates. Since the previous reports mention that GSCs were the cause of recurrence of GBM [100], GSCs can be good target. Lower drug concentration can minimize side effects. However, to achieve this strategy, appropriate experimental resources, including candidate agents, drug screening systems, and established cell lines are required. Epidemiological discovery is another option, such as the measurement of the incidence of a certain disease in the population to which specific drugs are administered. Serendipity is an important factor in this strategy. For instance, a prospective cohort study revealed a lower cancer incidence in people with schizophrenia [123]. This led us to the idea that antipsychotic drugs possess therapeutic effects against cancers including glioma [57, 62]. However, the most efficient method might be mutual molecular and structure analyses between target cells and drugs using artificial intelligence (AI). Different biochemical and mathematical techniques have been designed and optimized to accurately infer links between target cells and drugs. Drug-target interaction prediction is an important part of most rational drug repositioning pipelines. The major target molecules for malignant glioma are Akt, ERK, and STAT3, which sustain malignant phenotype

The supply of research resources is also important. Pharmaceutical companies hold the materials for drug repositioning such as drug libraries and useful knowledge for bringing new drugs to market. Thus, a collaboration between researchers who establish efficient screening systems and pharmaceutical companies that own various drugs, including those that failed in clinical trials, can lead to a successful

Although drug repositioning may be useful in the future, there are hurdles to the transition of this research into clinical practice owing to financial problems. Drug repositioning involves reinvestment in inexpensive drugs with expired patents; therefore, the benefits to pharmaceutical companies are small, which results in a reluctance to cooperate to broaden the indications of their drugs. This is especially true for rare diseases, such as glioma. Currently, the only way for researchers to raise public and private funds is by themselves, and they must conduct physicianled clinical trials without the support of pharmaceutical companies. An effective system in which the government supports drug repositioning is required to

the cell cycle [18, 45].

*DOI: http://dx.doi.org/10.5772/intechopen.92803*

**4. Perspective**

[62, 103, 113].

drug repositioning.

**149**
