Potential Use of Thalidomide in Glioblastoma Treatment: An Updated Brief Overview

Glioblastoma is the most common malignant primary brain tumor in adults. Thalidomide is a vascular endothelial growth factor inhibitor that demonstrates antiangiogenic activity, and may provide additive or synergistic anti-tumor effects when co-administered with other antiangiogenic medications. This study is a comprehensive review that highlights the potential benefits of using thalidomide, in combination with other medications, to treat glioblastoma and its associated inflammatory conditions. Additionally, the review examines the mechanism of action of thalidomide in different types of tumors, which may be beneficial in treating glioblastoma. To our knowledge, a similar study has not been conducted. We found that thalidomide, when used in combination with other medications, has been shown to produce better outcomes in several conditions or symptoms, such as myelodysplastic syndromes, multiple myeloma, Crohn’s disease, colorectal cancer, renal failure carcinoma, breast cancer, glioblastoma, and hepatocellular carcinoma. However, challenges may persist for newly diagnosed or previously treated patients, with moderate side effects being reported, particularly with the various mechanisms of action observed for thalidomide. Therefore, thalidomide, used alone, may not receive significant attention for use in treating glioblastoma in the future. Conducting further research by replicating current studies that show improved outcomes when thalidomide is combined with other medications, using larger sample sizes, different demographic groups and ethnicities, and implementing enhanced therapeutic protocol management, may benefit these patients. A meta-analysis of the combinations of thalidomide with other medications in treating glioblastoma is also needed to investigate its potential benefits further.


Introduction
Glioblastoma is adults' most common malignant primary brain tumor [1]. Maximal safe surgical resection followed by radiotherapy and/or chemotherapy is the glioblastoma and epigenetic profiles of primary and secondary GBM are not shown in histological examinations as different characteristics, even though they are believed to share different cell origins. Surprisingly, cells with mutated IDH1 and IDH2 tend to have better prognoses [28]. Methylation of MGMT prolongs survival compared to the unmethylated form of MGMT, which is involved in deoxyribonucleic acid (DNA) repair upon administrating alkylating treatment [29]. Myeloid-derived suppressor cells (MDSCs) play an essential role in decreasing the activity of the T-cell response in glioma, and it is an immature heterogeneous group of cells recognized for their myeloid lineage [30]. MDSCs weaken the first line of defense by inhibiting the natural killer cell activation receptor (NKG2D), and decreases interferongamma (IFNγ) production while transforming growth factor-beta (TGFβ) is present [31]. In addition to that, MDSCs also diminish adaptive immunity by disturbing the production of ARG1, inducible nitric oxide (NO) synthase 2 (iNOS2), TGFβ, depletion of cysteine, and the down-regulation of CD62L (L-selectin) [32,33]. Angiogenesis and vasculogenesis in the solid tumor are correlated with a poor prognosis, due to their promotion by MDSCs [34].

Hypoxia, Endoplasmic Reticulum Stress, and Chemoresistance
GBM is characterized by hypoxic regions that are responsible for necrosis associated with GBM. The low-oxygen condition could activate the hypoxia-inducible factors (HIFs), critical transcriptional factors that play an essential role in a tumor's response against hypoxia [35,36]. HIFs are believed to be sensors and proteins that are released in hypoxic regions responsible for cell adaption to low levels of oxygen, by optimizing the cellular processes that could initiate transcription factors activation of specific genes responsible for angiogenesis, metabolic reprogramming, and chemo-radio resistance in GBM [37][38][39][40][41]. The PTEN mutation (20-40%) and P53 mutation have been found in GBM. A loss of their function leads to the overexpression of HIF-1α. Moreover, HIF-1α can increase VEGF levels as one of the angiogenesis proteins. The level of VEGF in the cyst fluid of glioblastoma patients was found to be 200-300 fold more than in the serum.
During hypoxia, HIF-1α induces anaerobic glycolysis in GBM, resulting in a reduction in mitochondrial respiration, an increase in lactate levels and tumor acidity, and an interruption in the pH ratio between the intracellular and extracellular matrix that could decrease the passive absorption for many drugs, therefore increasing the probability of drug resistance [42,43]. Several studies reported that as the tumor size increases, the pH decreases. An acidic pH also plays an essential role in regulating cell proliferation, angiogenesis, immunosuppression, invasion, and chemoresistance in solid tumors [44][45][46][47][48]. Vaupel et al. reported that the pH could vary among the localized region of the tumor [48]. Normal brain tissue has a pH of 7.1, and in brain tumors it can be 5.9 [49]. Interestingly, upon performing electrode measurements, Hjelmeland et al. [49] reported a significant reduction in pH at the edge of the tumor compared with that in normal tissues. Moreover, the pH at the center of the tumor was even lower. Such a shift in the pH in gliomas, and the low pH, may increase angiogenesis through the induction of VEGF [50,51]. Acidic stress in GSCs promotes the HIF-2α protein. In low pH, HIF-2α mRNA increased 7-fold in cultures used for GSCs; however, HIF-1α mRNA was repressed. These findings hypothesize that HIF-2α could be responsible for inducing VEGF in acidic conditions [49]. Notably, the stability of HIFs is known to be regulated at the protein level as well, since HIFs have been found to be stable at low pH [52].
Other involvements of HIF-2α in GBM have also been reported. Li et al. [53] highlighted a higher expression level of HIF-2α in GSCs than in non-GSC. However, minimal expression of HIF-2α was found in normal adult murine neural progenitors under both conditions of hypoxia, and normoxia. Interestingly, HIF-1α was not as dramatically upregulated as HIF-2α in response to hypoxia in GSCs. The HIF-2α mRNA half-life time was found to be shorter in GSCs in comparison with non-GSCS. This indicates an increased de novo synthesis of mRNA of HIF-2α rather than the stabilization of mRNA of HIF-2α [53]. VEGF promoter activity was severely affected by the knock-down of HIF-1α and HIF-2α in hypoxic conditions, and decreased VEGF mRNA levels and intracellular and secreted VEGF protein levels. In contrast to non-stem glioma cells, only HIF-1α was required to regulate VEGF [53]. In another study, where the mesenchymal shift is the process where cells lose their adhesion and become migratory and invasive, it was found that HIF-1α-ZEB1, not the HIF-2α signaling axis, promoted this feature in GBM [54]. Another protein was associated with HIF-2α is teneurin transmembrane protein 1 (TENM1), a family of transmembrane proteins located on chromosome X. In vertebrates, their expression occurs during the central nervous system development, and is also associated with cellular signaling, cell proliferation, and adhesion regulations [55,56]. A recent study also reported that HIF-2α silencing decreased the expression of TENM1 [57]. Cancer stem cells show increased expressions of CD133 and HIF-2α when they are exposed to hypoxia [53,[58][59][60].
As a result of hypoxia, endoplasmic reticulum (ER) stress occurs due to the accumulation of unfolded proteins and misfolded proteins because of the shortage of energy produced by the aerobic process; as the ERAD/ERAQ system does not work properly, it is becomes overburdened [61][62][63]. Consequently, it drives the cancer cell population in a similar fashion of selecting and allowing for the survival of the most resilient phenotype of glioma cells that can withstand hypoxic and endoplasmic reticulum stress, and are accustomed to resisting anti-tumor drugs [61,64]. The unregular and unorganized structure of solid tumor vasculature contributes to irregular drug delivery to the site of action, whereas the well-oxygenated regions are less susceptible to ER stress [65,66]. HIF-1α is associated with the multidrug resistance mutation 1 (MDR1) gene that is responsible for the expression of P-glycoprotein/ATP binding cassette transporter B1 [Pgp/ABCB1] [67]. Upon studying doxorubicin resistance, a direct link was found between HIF-1α and increased expression of P-glycoprotein (Pgp), which effluxes the medication outside the cell. [68]. It was observed that the efficacy of temozolomide was increased after the knockdown of HIF-1α, due to the down-regulation of DNA repair proteins [69,70].
It is suggested that THD binding to CRBN limits angiogenesis ability, and helps generate reactive oxygen species [78,83]. THD induces T helper 2 (Th2) production; on the contrary, it inhibits the Th1 release in peripheral mononuclear cells [71] (Figure 1).
TNFα down-regulation causes nuclear factor kappa B (NF-kB) inhibition that results in lower levels of interleukin-6 transcriptions. Oppositely, THD up-regulates the caspase-8 protein widely used in myeloma by inducing caspase-8 myeloma cell programmed cell death apoptosis [84]. However, THD's entire mechanism of action in reducing myeloma cells is still unclear. THD stimulates natural killer cells and T-lymphocytes, and prevents strong myeloma cells' adhesion to bone marrow [85], thus affecting the composition of the bone marrow microenvironment [86]. THD has an antiangiogenic effect by blocking growth factors such as VEGFs and FGFβ [74,[87][88][89][90]. Other studies have shown that THD inhibits HOXB7 [91] and Sp1 that have a binding site in the c-MYC promoter and suppress the TGFβ1-mediated non-SMAD ERK1/2 signaling pathways [92]. Several uses and mechanisms of actions in some clinical conditions exist in (Table 1)  .
It is suggested that THD binding to CRBN limits angiogenesis ability, and helps generate reactive oxygen species [78,83]. THD induces T helper 2 (Th2) production; on the contrary, it inhibits the Th1 release in peripheral mononuclear cells [71] (Figure 1).

Figure 1.
Thalidomide mechanism of action. Cells expressing the cereblon protein show higher expressions of p21 if thalidomide is administrated, leading to cell arrest. Thalidomide can also increase immuno-cytotoxicity through the regulation of different cytokines. Thalidomide has a direct effect on mitochondria through binding to c-JNK, followed by releasing oxygen species. The inhibition of NF-KB occurs as a means for thalidomide by interacting with TNFR-1, ICOM1, and other multistep signaling. Abbreviations, c-JNK-c-jun terminal kinase; TNFR1-tumor necrosis factor receptor 1; IFN-interferon, IL-interleukin; NF-kB-nuclear factor-kappa B; Smac-second mitochondria-derived activator of caspases; ICAM1-intercellular adhesion molecule-1.
TNFα down-regulation causes nuclear factor kappa B (NF-kB) inhibition that results in lower levels of interleukin-6 transcriptions. Oppositely, THD up-regulates the caspase-8 protein widely used in myeloma by inducing caspase-8 myeloma cell programmed cell death apoptosis [84]. However, THD's entire mechanism of action in reducing myeloma cells is still unclear. THD stimulates natural killer cells and T-lymphocytes, and prevents strong myeloma cells' adhesion to bone marrow [85], thus affecting the composition of the bone marrow microenvironment [86]. THD has an antiangiogenic effect by blocking growth factors such as VEGFs and FGFβ [74,[87][88][89][90]. Other studies have shown that THD inhibits HOXB7 [91] and Sp1 that have a binding site in the c-MYC promoter and suppress the TGFβ1-mediated non-SMAD ERK1/2 signaling pathways [92]. Several uses and mechanisms of actions in some clinical conditions exist in (Table 1)  .   THD has antipruritic action that can be due to its inhibition of TNFα, or due to its central depressive action [110] Oro mucosal disease -12 patients were given THD through a period of ulcerative oromucosal condition (recurrent aphthous stomatitis, HIV-related oral ulceration, and oral manifestation of Crohn's disease) THD had an excellent efficacy-to-safety ratio in managing oro-mucosal ulceration over a prolonged treatment period Modulation of inflammatory cascade and interaction with various cytokines, such as TNFα, IL-6, and IL-10 it can also co-stimulate primary human T lymphocytes, which increases their anticancer activity [97] Abbreviations: DLE-discoid lupus erythematosus; Treg-regulatory T; IBD-inflammatory bowel disease; HIMEC-human intestinal microvascular endothelial cells; HHT-hereditary hemorrhagic telangiectasia; LVADleft ventricular assist device; GIB-gastrointestinal bleeding; ENG-endoglin; ACVRL1-activin A eceptor like type 1; PNP-paraneoplastic pemphigus; has-hemangiosarcoma; IFNγ-interferon gamma; CD-cluster of differentiation; PPD-purified protein derivative; PDAI-pemphigus disease area index; ENL-erythema nodosum leprosum; POEMS-polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes; RASrecurrent aphthous stomatitis; DTH-delayed-type hypersensitivity; PBMCs-peripheral blood mononuclear cells; PV-pemphigus vulgaris; IL-interleukin; THD-thalidomide; TNF-tumor necrosis factor; BSA-body surface area.

Thalidomide Efficacy
It has been over sixty years since THD has been on the market, and it is still frequently used in a wide range of therapeutic applications. The clinical trials and pharmacovigilance research have shown that THD is an effective medication in the treatment of idiopathic pulmonary fibrosis (IPF), severe lung injuries caused by swine flu subtype H1N, and lung injuries caused by the toxic fast-acting herbicide called paraquat, with a well-defined mode of action [118]. To determine THD efficacy, we performed a systematic search on various databases, and reported the results of clinical trials that used THD for GBM treatment (Figure 2). It has been shown that THD may be a promising therapeutic option for treating GBM through the following three trials conducted on patients with recurrent GBM who received conventional chemotherapy followed by THD [120][121][122]. During the trials, the daily dose of THD was between 100-1200 mg, with good tolerance in general, although there were uncommon responses. The first study included patients with recurrent GBM (n = 18), 77.8%. The median overall survival (OS) for 17 patients was 36 weeks (12-40) [123]. The second trial included patients with recurrent GBM (n = 42), 38 of whom were eligible for assessment; only 42% had stable disease, and 5% had a partial response. The OS was 31 weeks, and 35% of the patients achieved one-year survival [124]. The third trial included patients with recurrent GBM (n = 39), of whom 36 were eligible for assessment; only 33% had stable disease, 6% had a partial response, the OS was 28 weeks, and eight patients achieved one-year survival [125].
The use of THD concurrently with irinotecan has limited efficacy for the treatment of newly diagnosed or recurrent GBM; this was proven through two studies. In the first study (n = 26), 24 were eligible for assessment; only 79% had stable disease and 7% had a It has been shown that THD may be a promising therapeutic option for treating GBM through the following three trials conducted on patients with recurrent GBM who received conventional chemotherapy followed by THD [120][121][122]. During the trials, the daily dose of THD was between 100-1200 mg, with good tolerance in general, although there were uncommon responses. The first study included patients with recurrent GBM (n = 18), 77.8%. The median overall survival (OS) for 17 patients was 36 weeks (12-40) [123]. The second trial included patients with recurrent GBM (n = 42), 38 of whom were eligible for assessment; only 42% had stable disease, and 5% had a partial response. The OS was 31 weeks, and 35% of the patients achieved one-year survival [124]. The third trial included patients with recurrent GBM (n = 39), of whom 36 were eligible for assessment; only 33% had stable disease, 6% had a partial response, the OS was 28 weeks, and eight patients achieved one-year survival [125].
The use of THD concurrently with irinotecan has limited efficacy for the treatment of newly diagnosed or recurrent GBM; this was proven through two studies. In the first study (n = 26), 24 were eligible for assessment; only 79% had stable disease and 7% had a partial response. The six-month progression-free survival (PFS) of the recurrent group was 19%, and the six-months PFS of the newly diagnosed group was 40% [126]. In the second study, patients with recurrent GBM only (n = 32) were eligible for assessment. Only 19 patients had stable disease, one had a partial response, one had a complete response, the one-year OS was 34%, and the six-month PFS was 25% [127]. It is worth mentioning that THD was reported in eight studies to be an ineffective drug for the treatment of GBM [87,[128][129][130][131][132][133].
A systematic review and meta-analysis compared only two cohorts of THD (n = 81) to 7 cohorts of bevacizumab (n = 351). The ORR favored bevacizumab over THD (RR 6.8, 95%CI 2.64-17.6; p < 0.001); however, both drugs showed comparable results in the progression-free survival and 1-year median overall survival rates (RR 1.68, 95%CI: 0.84-3.34, p = 0.07; and RR 0.89, 95%CI: 0.59-1.37; p = 0.31, respectively) [16]. In addition to using THD in the direct treatment of GBM, Hassler et al. [134] suggested using THD as a palliative treatment in patients with advanced secondary GBM. The patients (n = 23) were advised to administer 100 mg of THD at bedtime. Symptomatic improvement was most prominent in restoring a normal sleep pattern, and 47.8% of the patients survived longer than one year [134]. The palliative effects of THD can be a topic for further research. A list of the combined possible uses of THD with other drugs is shown in Table 2 [74,[85][86][87][88][89][90][91][92].

Combination Mechanism of Action Uses
Ref.

Capecitabine and THD
THD exerts its immunomodulatory effect, and capecitabine is a prodrug transformed to 5-fluorouracil in tissue via numerous enzymatic routes, and has the same effectiveness in colorectal cancer as 5-fluorouracil -Treatment of refractory metastatic colorectal cancer (n = 34) revealed a low level of toxicity, such as thromboembolism, constipation, and hand-foot syndrome; however, 38% of the patients had stable disease, and the median progression-free survival rate was 2.6 months, while median overall survival was 7.1 months -There were no radiographic responses observed with therapy -No treatment response in cases treated previously from metastatic colorectal cancer [145] Interferon-α and THD Interferon and THD are angiogenesis inhibitors that slow the progression of renal carcinoma -The response of renal carcinoma is moderate to interferon and THD -In a study (n = 30) involving patients with renal cell carcinomas, the median follow-up time was 49.6 weeks, while the study's median time of participation was 11.1 weeks -There were 29 patients with grade 2 toxicity -At 12 weeks, there was no complete response; there were 2 partial response patients, 8 with stable disease, and 11 patients showed disease progression -The median survival was 68 weeks The plasma concentration of THD has no connection to the dose -There was no link between medication level and treatment response or toxic effects [138] THD and FCT Pathophysiology of acute leukemia depends on angiogenesis, contributes to the survival of leukemia cells, and resistance to chemotherapy-induced apoptosis The combination between THD and other chemotherapeutic drugs such as FCT may decrease the angiogenic process due to the ability of THD to block VEGF, thus inhibiting the formation of new vessels -FCT is an effective therapy in recurrent leukemia patients (n = 42). Adding THD to this combination did not significantly increase efficacy or modify angiogenic markers such as VEGF or MVD -The total response rate in this trial with THD added to the FCT regimen is similar to the regimen seen in phase I when FCT was used alone (24% vs. 26%) -Adding THD results in thrombotic events and dermatological problems, which were not found in phase I using only FCT [90] Carboplatin and THD Carboplatin is an antineoplastic agent that enhances the cell cycle, stimulates apoptosis, and inhibits cancer cell proliferation. Combining both drugs has an antiangiogenic effect through inhibiting cytokines VEGF, TNF, and Factor VIII expression in neoplastic cells -THD and carboplatin combination effectively inhibit tumor proliferation and 4T1 murine breast cancer metastasis in the animal model -The treated group had a significantly increased animal survival rate compared with the control group (p = 0.0005) -Compared with the control group, there was a (62%) significant reduction in tumor growth after treatment (p < 0.05) -The number of lung metastases in the treated group was fewer than in the control group (p < 0.001) -There was a significant reduction in mitosis and an increase in apoptosis in the treated group compared with the control group (p < 0.05 and p < 0.001, respectively).
-Immunohistochemical analysis of tumor vascularization revealed a higher reduction in the number of blood vessels in the intervention group than in the control (p < 0.001) [74]  TMZ, THD, and celecoxib TMZ is cytotoxic chemotherapy for the treatment of malignant gliomas. Malignant glioma cells' secrets of angiogenic factors such as acidic FGF, bFGF, angiogenin, VEGF, platelets-derived growth factors, and IL 8 induce angiogenic process, increasing tumor proliferation. Combining TMZ with two angiogenic inhibitors such as THD and celecoxib inhibits these growth factors, inhibiting angiogenesis The combination of TMZ, THD, and celecoxib is moderately well-tolerated but unlikely to significantly increase the survival of patients newly diagnosed with GBM (n = 50) [87] Bortezomib, THDdexamethasone Bortezomib causes inhibition of the action of proteasome, which results in cell apoptosis. Moreover, it inhibits the activation of NF-κB and regulatory protein toxic levels accumulation, but the mechanism leading to apoptosis of cells remains unclear. THD prevents angiogenesis. Finally, dexamethasone suppresses tumor growth and enhances the apoptosis of myeloma cells, but it does not release cytochrome C from mitochondria -Bortezomib, THD, and dexamethasone combination were successful therapies for multiple myeloma patients (n = 38) -THD and dexamethasone increased induction remission in 66% of patients, but when adding bortezomib to the combination, remission occurred very fast in 87% of patients, including 16% with complete remission -Most side effects of the combination were mild [88] Thoracic radiation, paclitaxel, carboplatin, and THD

Inhibition of the angiogenesis process
Combining THD with chemoradiotherapy increased levels of toxicity (grade 3) such as fatigue, dizziness, thromboembolism, tremors, constipation, dyspnea, hypoxia, hypokalemia, rash, edema, sensory neuropathy, and depressed consciousness -Aspirin failed to decrease thromboembolism -THD did not increase survival in people with NSCLC (n = 546), so it was not recommended to add THD to these patients [141] THD, vincristine, liposomal doxorubicin, and dexamethasone (T-VAD Doxil) VAD causes a reduction in tumor cells, especially liposomal doxorubicin, which increases the exposure of tumors cells to doxorubicin. THD works by its anti-myeloma mechanism, and hence, when combined with dexamethasone, it shows synergistic action The combination was effective in treating myeloma patients (n = 39). Almost 74% of patients responded to treatment; of these, 10% had a complete response, 64% a partial response, 8% a minor response, and 18% did not respond to the treatment -Some patients showed grade 3 or 4 toxicity symptoms such as rash, thrombocytopenia, peripheral neuropathy, constipation, neutropenia, deep venous thrombosis, and 2 patients died due to infections [85] THD, IL-2 The effective drug in RCC treatment is the combination immunotherapy drug such as THD with IL-2, which has immunomodulating and antineoplastic activity. Even though the entire mechanism is unknown, the THD/IL-2 combination was found to maintain RCC stability in some patients -THD and IL-2 were effective in treating metastatic RCC according to phase 1 and phase 2 trials (n = 50), with the benefit ranging from 5-71 percent, with disease stability -The combination was relatively safe, and the only side effects were flu-like symptoms, hypotension, and hypothyroidism, all of which were linked to IL-2 treatment. Constipation, neuropathy, and rash are among the THD toxicity symptoms, and two patients had deep venous thromboses [89] Retinoic acid and THD Retinoic acid induces the activity of resistance genes (HOXB7, bFGF, VEGF, and IL-8) that participate in GBM cell proliferation, hypoxia, and angiogenesis. THD inhibits HOXB7 and bFGF. THD causes suppression of HOXB7 and bFGF. Therefore, the combination shows synergistic action, as THD inhibits the induction of IL-8, IGFBP-3, HILPDA, and ANGPTL4 in GBM tumors, which are elevated by retinoic acid and linked to hypoxia and angiogenesis The addition of THD to retinoic acid inhibited the development of human U251 GBM xenografts, and suppressed the resistance genes induced by retinoic acid; thus, this combination prevented hypoxia and angiogenesis linked to these genes [91]

Combination Mechanism of Action Uses
Ref.

THD and cisplatin
Cisplatin is a toxic agent against murine erythroleukemic cells, and is known for its antimicrobial, immunosuppressive, and mutagenic effects, while THD exerts its effect through its anti-inflammatory and immunomodulatory properties THD failed to suppress tumor and metastasis when given alone to DBA2/J mice, while cisplatin administration alone or with THD inhibited the multiplication of tumor cells [150] Bevacizumab, THD, docetaxel, and prednisone THD suppresses FGF activity, the proliferation of endothelial cells, circulating endothelial cells, and the appearance of TNF. Bevacizumab keeps the balance of VEGF. Docetaxel has anti-tumor action The combination helped reduce tumor volume of castration-resistant prostate cancer (n = 60), as the antiangiogenic agents THD and bevacizumab reduced the prostatic specific antigen and achieved an unprecedented response [148] Semaxanib and THD THD down-regulates TNF α, IL-6, VEGF, and FGF. Semaxanib binds to VEGF-2 and causes its inhibition. It also inhibits the growth of A375 melanoma, C6 glioma, Calu-6 lung, and A431 epidermoid xenografts in athymic mice -The combination of semaxanib and THD is proved to be a successful therapy in malignant melanoma (n = 12) -Headache, asthenia, constipation, edema in the lower limbs, neuropathy, hyperglycemia, hypercholesterolemia, and thrombosis were among the toxicity symptoms encountered by some individuals, whereas headache and thromboembolic symptoms were linked to semaxanib [147] THD and infliximab Infliximab acts as a monoclonal antibody, helps balance TNFα, and down-regulates granulocyte-macrophage colony-stimulating factor expression, which was beneficial in treating TH1 disorders. THD suppresses the formation of TNFα by a mechanism that is different from infliximab The combination is valuable in the management of refractory entero-Bechet's disease (n = 1) [144] THD and antibiotics (rifampicin and isoniazid) The anti-inflammatory THD agent enters the CSF and decreases TNFα in CSF and blood. Moreover, it decreases leukocytosis, and its penetration of CSF does not interfere with the action of antimycobacterial drugs The combination of THD and (rifampicin and isoniazid) improved the survival rate of infected rabbits with mycobacterium tuberculosis [152] Pyrotinib and THD Pyrotinib has been licensed as an effective and irreversible inhibitor of the epidermal growth factor receptor (EGFR)/HER2 identified in NSCLC. THD is effective in solid tumors due to its antiangiogenic and immunomodulatory effects Since THD lowers diarrhea caused by pyrotinib, a combination of pyrotinib with THD is beneficial in managing NSCLC (n = 39) [135] THD and TP chemotherapy THD and TP chemotherapy reduces VEGF and NRP-1, which are involved in esophageal cancer tumor angiogenesis Effective in treating esophageal cancer and relieving associated nausea and vomiting (n = 133) [155] Glucocorticoids and THD THD's immunomodulatory and anti-inflammatory properties allow it to boost T cells, suppress cell growth, and reduce lung fibrosis and damage. THD also calms anxiety and reduces oxygen consumption due to its sedative effect. Glucocorticoids reduce cytokinesis levels Glucocorticoids and THD in combination effectively relieve symptoms associated with pulmonary effusion without side effects (n = 1) [137] THD, IFNα-1b, and IL 2 The combination of IL 2, IFNα-1b, and THD lowers minimal residual disease and reverses AML1-ETO fusion gene positivity in individuals with AML IFNα-1b, IL-2, and THD are effective in the treatment of AML patients (n = 20), and the effective rate reached 72.2% [146] Table 2. Cont.

Combination Mechanism of Action Uses
Ref.

THD and lenalidomide
THD and lenalidomide have direct anti-tumor effects and affect the bone marrow microenvironment: THD has a cytotoxic effect in tumor cells, and lenalidomide inhibits the proliferation of multiple myeloma cells. They induce apoptosis by the down-regulation of NF-kB signaling and enhancing the intrinsic apoptosis pathway. Lenalidomide inhibits the expression of two anti-apoptotic proteins, cIAP-2 and FLICE inhibitory protein. Both proteins block apoptosis induced by TNF-related apoptosis A broad spectrum of activities to treat multiple myelomas and other hematologic and solid malignancies [153] THD, gemcitabine, and carboplatin THD has an anti-tumor impact on multiple myeloma by inhibiting angiogenesis, mediated by VEGF and bFGF. When coupled with chemotherapeutic drugs, it has a synergistic effect. In mice injected with NSCLC cell lines, THD slows tumor growth -Overall survival did not improve when THD was combined with chemotherapy -In patients with NSCLC (n = 722), THD was not related to a survival benefit, although survival was lower in the group with non-squamous histology -The most severe side effects of THD were thrombotic events [142] THD and irinotecan THD has immunomodulatory and antiangiogenic activities against malignant gliomas, while irinotecan is a topoisomerase-II suppressor The combination of THD with irinotecan did not achieve enough effectiveness to warrant further investigation against anaplastic glioma (n = 39) although some patients achieved prolonged progression-free survival rate/overall survival rate [140],

THD and ciclosporin
THD works similarly to ciclosporin in that it attaches to intracellular proteins, such as calmodulin and cyclophilin, which play a key role in immune response modulation; however, this has yet to be determined. THD interferes with neutrophil and lymphocyte function, causing suppression of the immune system THD is an effective and potent immunosuppressive drug in preventing early refusal of cardiac allograft transplantation in rats when paired with low dosages of ciclosporin [139] THD and intermediatedose dexamethasone THD is beneficial in multiple myelomas because it decreases drug resistance in multiple myeloma plasma cells when combined with dexamethasone, so the study used THD and dexamethasone together to treat primary amyloidosis based on these findings Second-line therapy for amyloidosis (n = 31) [149] 5-ALA-PDT and THD 5-ALA-PDT is dependent on prodrug 5-ALA administration. It increases protoporphyrin IX in tumor cell production, and acts as a photosensitizer. After radiation therapy, protoporphyrin is excited, leading to ROS formation that causes a cytotoxic effect on tumor cells. Photodynamic therapy enhances VEGF expression and destroys vessels' walls, leading to a reduction in tumor proliferation. THD inhibits VEGF and TNFα -This combination was more effective than monotherapy of each agent on the 2H11 endothelial cell line and 4T1 breast carcinomas -In 2H11 cells, THD alone did not affect VEGF expression, but THD lowered VEGF expression once 5-ALA-PDT was administered [154] Isotretinoin and THD Due to P1 or P2 promoter activity, isotretinoin and THD suppress c-MYC expression. Isotretinoin modifies TGFβ expression and also decrease TGFβ signaling degradation of pSMAD1 on the c-MYC promoter. AP-2 and c-MYC interact at the protein level, and AP-2 over-expression reduces c-MYC mRNA expression -In hepatocellular carcinoma, c-MYC mRNA expression was significantly decreased by 80% (p < 0.05) in cells treated with THD and isotretinoin -Isotretinoin effects reduce HepG2 cell viability [92]

Combination Mechanism of Action Uses
Ref.

THD and hydroxyurea
The immunomodulating and antiangiogenic effects of THD induce c-globin gene expression, and improve erythroid cell proliferation by amplifying ROS-p38 mitogen-activated protein kinase signaling and histone H4 acetylation during erythropoiesis Hydroxyurea increases hemoglobin by reducing inflammation, hypercoagulability, and induction of HbF expression, which could suppress ineffective erythropoiesis by reducing the build-up and precipitation of precipitation a-globin chains -For patients with a high risk of organ rejection (n = 25), very high-risk transplant-related mortality and morbidity, or patients who are not willing to undergo a bone marrow transplant -May be used for transplantation in patients with ineligible thalassemia whose iron overload indicates a high risk of treatment-related harm, or who are awaiting donor identification [151] Abbreviations

Thalidomide Safety
The proven teratogenicity of THD made it strictly prohibited from being used during pregnancy. Before THD may be taken, all patients have to be enrolled in the System for THD Education and Prescribing Safety (STEPS) program. Prior to commencing treatment with THD, women of reproductive potentiality (less than two years postmenopausal) are required to show a negative pregnancy test. In addition, they have to use two different effective ways of birth control and be examined for pregnancy every four weeks; meanwhile, men who are going to take THD have to quit any sexual activities, or use condoms made of latex [121]. THD is known to cause many side effects, including numbness, nervousness, confusion, paresthesia, aural buzzing, nausea, up to 20%, increased appetite, 30% of burning sensation and deep vein thrombosis, 5% of bradycardia and neutropenia, 25% of skin rash, hangover feeling, decreased libido, edema, and hypothyroidism up to 25% [121]. THD's common side effect was initially marketed as a sedative drug. The intensity of sedation tends to lessen with prolonged usage at a steady dose, which could be decreased and avoided by taking medicine at night three hours before bed. Constipation is a typical side effect that can vary depending on the dosage. Patients should be encouraged to eat food that contains a high amount of fiber, and to take laxatives as needed. In the case of dry skin side effects, it is pretty common to see it with pruritus, with the severity based on the drug's dosage. Nevertheless, this effect may be reduced by using lubricants that are free of alcohol [83]

Thalidomide Toxicity
THD is known for its considerable risk to the unborn and developing child. As a result, THD and its derivatives are highly controlled, and necessitate the use of contraception when used as a therapy. Patients should cease using THD if they have serious side effects, especially neuropathic symptoms [156]. THD toxicity in the treatment protocol for patients with GBM was studied, and a trial was conducted on 39 patients; 26 received the full dosage of the THD regime. The results showed that the majority of patients tolerated THD well. However, four incidences were graded as 4 for cortical toxicity, and were all seizures. All of the participants in this experiment had a previous seizure history, and it was verified that tumor development increased during the activity of new seizures. Additionally, one incident involved constipation grade 1, and there were six incidences of grade 2 constipation episodes. Moreover, twelve patients had somnolence grade 1, three had grade 2, and six had grade 3. These were the most prevalent toxicities linked to THD [157]. A further phase II trial was conducted on 17 GBM patients to assess THD toxicity, and it was found that a few cases were graded. Two patients (17%) showed leukopenia progression grade 3-4 with neutropenia. Furthermore, other serious adverse events comprised (4 patients, 24%) those with fatigue grade 3. Moreover, another patient suffered from neurotoxicity grade 3. In this intervention sample, the medication-related toxicity had no clinical consequences, and no participant was discontinued from the experiment due to THD toxicity ( Figure 3) [125]. related toxicity had no clinical consequences, and no participant was discontinued from the experiment due to THD toxicity ( Figure 3) [125].

Thalidomide Drug Interactions
THD is mainly eliminated from the body by a hydrolysis reaction that occurs across the distribution area of the drug, and there is some influence on hepatic elimination and excretion via the kidney. The metabolization of THD in our bodies is minimally done by CYP450, and 2C19 is engaged in the synthesis process of 5-OH metabolites [160]. In liver microsomes, THD was found to suppress CYP2C19 function while increasing the activity of CYP3A, which means the elevation of CYP3A is due to a heterotopic interactivity rise in CYP3A5. In animal trials, THD revealed a possibility of human CYP3A up-regulation with a greater midazolam clearance with midazolam usage as a pharmacological probe. Based on a recent study, THD acts as a ligand for the pregnane X receptor (PXR) and the constitutive androstane receptor CAR, increasing the activity of the CYP450 enzyme. Since in vitro studies indicated that THD needs CYP450 metabolic stimulation, and that metabolites are detectable in urine, weak metabolizers of CYP2C19 (about 15% of Asians and 25% of Caucasians) may need greater THD dosages. Extensive metabolizers may be at a high risk of side effects. As a result, the CYP2C19 genotype and the CYP2C19 inhibitors or inducers could influence exposure to the active metabolite of THD [161]. Furthermore, adverse effects of THD on the central nervous system CNS, including dizziness, sleepiness, and focusing problems, may be exacerbated by alcohol intake while using THD. Some patients may also have difficulty concentrating and making decisions. While using THD, patients must abstain from or minimize their alcohol use. They should not exceed the prescribed dosage, and refrain from engaging in tasks requiring wakefulness, such as driving or working with dangerous equipment [162]. Figure 4 represents some of adverse effects of THD drug interactions.

Thalidomide Drug Interactions
THD is mainly eliminated from the body by a hydrolysis reaction that occurs across the distribution area of the drug, and there is some influence on hepatic elimination and excretion via the kidney. The metabolization of THD in our bodies is minimally done by CYP450, and 2C19 is engaged in the synthesis process of 5-OH metabolites [160]. In liver microsomes, THD was found to suppress CYP2C19 function while increasing the activity of CYP3A, which means the elevation of CYP3A is due to a heterotopic interactivity rise in CYP3A5. In animal trials, THD revealed a possibility of human CYP3A up-regulation with a greater midazolam clearance with midazolam usage as a pharmacological probe. Based on a recent study, THD acts as a ligand for the pregnane X receptor (PXR) and the constitutive androstane receptor CAR, increasing the activity of the CYP450 enzyme. Since in vitro studies indicated that THD needs CYP450 metabolic stimulation, and that metabolites are detectable in urine, weak metabolizers of CYP2C19 (about 15% of Asians and 25% of Caucasians) may need greater THD dosages. Extensive metabolizers may be at a high risk of side effects. As a result, the CYP2C19 genotype and the CYP2C19 inhibitors or inducers could influence exposure to the active metabolite of THD [161]. Furthermore, adverse effects of THD on the central nervous system CNS, including dizziness, sleepiness, and focusing problems, may be exacerbated by alcohol intake while using THD. Some patients may also have difficulty concentrating and making decisions. While using THD, patients must abstain from or minimize their alcohol use. They should not exceed the prescribed dosage, and refrain from engaging in tasks requiring wakefulness, such as driving or working with dangerous equipment [162].

Conclusions
GBM is still one of the most life-threatening types of primary malignant brain tumors, with a poor prognosis and a low 5-year survival rate. Investigating the different mechanisms of action of THD could lead to new potential uses for the drug, especially when combined with other medications. Future research on treatment options should also consider existing drugs that have different mechanisms for fighting tumors. Additionally, more efforts are needed to develop accurate preclinical models to evaluate treatments' effectiveness and improve methods for the early diagnosis of GBM.

Conclusions
GBM is still one of the most life-threatening types of primary malignant brain tumors, with a poor prognosis and a low 5-year survival rate. Investigating the different mechanisms of action of THD could lead to new potential uses for the drug, especially when combined with other medications. Future research on treatment options should also consider existing drugs that have different mechanisms for fighting tumors. Additionally, more efforts are needed to develop accurate preclinical models to evaluate treatments' effectiveness and improve methods for the early diagnosis of GBM.