Possible mechanisms of action of clarithromycin and its clinical application as a repurposing drug for treating multiple myeloma

Clarithromycin (CAM), a semisynthetic macrolide antibiotic, is a widely used antibacterial drug. Recently, the efficacy of CAM as an add-on drug for treating multiple myeloma (MM) has been noted. Its effect on treating MM has been confirmed in combination chemotherapies that include CAM. However, a single treatment of CAM has no efficacy for treating MM. Many myeloma growth factors (MGFs) including interleukin (IL)-6 are known to be closely involved in the development of MM. CAM has been shown to suppress many MGFs, particularly IL-6. The possible mechanisms of action of CAM in treating MM have been suggested to include its immunomodulatory effect, autophagy inhibition, reversibility of drug resistance, steroid-sparing/enhancing effect and suppression of MGFs. In addition, MM is characterised by uncontrolled cell growth of monoclonal immunoglobulin (Ig)-producing neoplastic plasma cells. Large quantities of unfolded or misfolded Ig production may trigger considerable endoplasmic reticulum stress. Thus, MM is originally a fragile neoplasm particularly susceptible to autophagy-, proteasome- and histone deacetylase 6-inhibitors. Taken together, CAM plays an important role in MM treatments through its synergistic mechanisms. In addition, CAM with its pleiotropic effects on cytokines including IL-6 and indirect antiviral effects might be worth a try for treating COVID-19.


Antineoplastic effects of 14-membered macrolides including CAM
The first experimental report suggesting the presence of the antineoplastic effect of macrolide (erythromycin) dates back to 1995. Hamada et al [8] experimentally demonstrated that the erythromycin induced cytotoxic macrophages in tumour-bearing mice and that these cytotoxic macrophages might work as one of the effector cells together with NK cells against the tumours transplanted in the mice. Clinically, Mikasa et al [9] first reported that long-term treatment with CAM significantly increased the median survival time of patients with advanced nonsmall cell lung cancer (NSCLC). Sakamoto et al [10] demonstrated that NK activity was significantly increased in patients with unresectable NSCLC after 1-month treatment with CAM. Furthermore, using Lewis lung carcinoma (LLC)-inoculated mice, they demonstrated that the administration of CAM after anticancer chemotherapy strongly inhibited tumour growth and significantly increased the NK activity. Recently, antineoplastic or immunomodulatory effects of CAM have again attracted attention in clinical fields. Several investigators reported that the CAM monotherapy was effective in treating hematologic malignancies including extranodal marginal zone B-cell lymphoma [11], mucosaassociated lymphoid tissue lymphoma [12,13], follicular B-cell lymphoma [14,15] and Hodgkin's lymphoma [16].

IL-2
IL-2 plays an essential role in key functions of the immune system, which includes immuno-tolerance and immunity. It is mainly produced by activated CD4 + T-cells and activated CD8 + T-cells and to a lesser extent by activated dendritic cells, NK/NKT cells and macrophages. IL-2 mediates its effects by binding to IL-2-receptors, which are expressed by lymphocytes. IL-2 is known to strongly stimulate NK-cell and T-cell growth which consequently augments the cytolytic action, leading to enhanced cytotoxicity. Anti-tumour properties of IL-2 are mediated by the cellular immune system, but IL-2 itself has no direct effect on tumours [31]. CAM has been reported to suppress IL-2 production [30,[32][33][34] (Table 1). The suppression of IL-2 by CAM seems to be disadvantageous from the view point of cellular immunity. However, the effect of reduced IL-2 on MM cells has not yet been fully elucidated. Table 1. Cytokines and enzyme, which are positively or negatively influenced by CAM, with respect to MM cell proliferation.
Matsuoka et al [22] An inhibitory effect of CAM on costimulatory molecule expression and cytokine production by cultured synoviocytes was investigated. CAM suppressed the production of IL-1β in human synoviocytes obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients.
Miyanohara et al [24] CAM suppressed IL-1β gene expression in human nasal epithelial cell.
Fonseca-Aten et al [26] Children with a history of recurrent wheezing or asthma, who presented with an acute exacerbation of wheezing, were treated with CAM. Nasopharyngeal concentrations of IL-1β of the patients decreased after CAM treatment.
Perić et al [27] Nasal polyp patients (nonallergic and allergic) were treated with CAM for 8 weeks. Following CAM treatment, IL-1β in nasal fluid of allergic patients significantly decreased.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of chronic rhinosinusitis (CRS) was investigated. CAM suppressed the protein production of IL-1β in sinonasal mucosa of CRS without nasal polyp.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

IL-2
Morikawa et al [32] The immunomodulatory effects of three macrolides including CAM on human T-cell function were investigated. CAM suppressed IL-2 production induced by mitogen-stimulated T-cells.
Morikawa et al [33] The effects of macrolides including CAM on the generation of human Th1-and Th2-type cytokines were investigated. CAM showed a potent inhibitory activity on the release and gene expression of IL-2 induced by Con-A-stimulated human T-cells.
Sugiyama et al [34] Differing effects of macrolides including CAM on cytokine production by murine dendritic cells were investigated. CAM decreased IL-2 production by co-cultured dendritic cells and T-cells.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

IL-5
Kraft et al [38] The asthma patients treated with CAM demonstrated a reduction of IL-5 mRNA in bronchoalveolar lavage (BAL).
Morikawa et al [33] The effect of macrolides including CAM on the generation of human Th1-and Th2-type cytokines was investigated. CAM showed an inhibitory activity on the release and gene expression of IL-5 induced by Con-A-stimulated human T-cells.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of IL-5 in sinonasal mucosa of eosinophilic CRS with nasal polyp.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

IL-6
Sakamoto et al [41] Patients with NSCLC were treated with CAM. The level of serum IL-6 was significantly depressed after CAM treatment.
Matsuoka et al [22] CAM significantly suppressed the production of IL-6 in IL-1β-added cultured synoviocytes obtained from RA and OA patients.
Khan et al [42] The effect of IL-6 production by LPS-and Pansorbin-stimulated human monocytes was investigated. CAM significantly decreased an IL-6 production.
Sassa et al [43] The therapeutic effect of CAM on subcutaneously inoculated mammary adenocarcinoma cells in rats was investigated. The expression of IL-6 gene of spleen cells of tumour-bearing rats significantly decreased after CAM treatment.
Majima et al [44] Mice were inoculated subcutaneously with Lewis lung carcinoma (LLC) cells. The treatment of CAM (for 7 days) started 1 week after tumour inoculation. Immediately after finishing the treatment, the mice were sacrificed, and the expression of IL-6 mRNA in spleen cells was evaluated. The expression of IL-6 mRNA decreased after CAM treatment.
Morikawa et al [33] The effect of macrolides including CAM on the generation of human Th1-and Th2-type cytokines was investigated. CAM showed an inhibitory activity on the release and gene expression of IL-6 induced by Con-A stimulated human T-cells.
Sugiyama et al [34] The differing effects of macrolides including CAM on cytokine production by murine dendritic cells were studied. CAM decreased IL-6 production in co-cultured dendritic cells and T-cells.
Morris et al [45] Phase II study to treat patients with relapsed and refractory MM. With respect to the effect of added CAM on MM, the possibility that CAM acts through suppression of production of various cytokines including IL-6 was suggested.
Kanoh and Rubin [2] The mechanisms of action and clinical application of macrolides as immunomodulatory medications (review of literature).
Cai et al [28] CAM inhibited spontaneous and LPS-stimulated IL-6 production by alveolar macrophages in BOOP.
Van Nuffel et al [3] Repurposing drugs in oncology (ReDO)-CAM as an anticancer agent (review of literature).
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of IL-6 in sinonasal mucosa of CRS.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

IL-8
Matsuoka et al [22] CAM suppressed the production of IL-8 by IL-1β-added human synoviocytes obtained from RA or OA patients.
Suzuki et al [50] The effect of CAM on IL-8 secretion from cultured human nasal epithelial cells was studied.
The LPS-stimulated IL-8 secretion was significantly inhibited by CAM.
Kikuchi et al [49] The effects of CAM on IL-8 production using stimulated human peripheral monocytes and human monocytic leukaemia line, THP-1, were investigated. CAM suppressed the production of IL-8 induced by stimulated monocytes and THP-1 cells.
Kanoh and Rubin [2] The mechanisms of action and clinical application of macrolides as immunomodulatory medications (review of literature).
Cai et al [28] CAM induced a dose-dependent attenuation of LPS-stimulated IL-8 production by alveolar macrophages in BOOP.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).  [54] Patients with unresectable NSCLC were treated with CAM for 3 months. The expression of IL-10 mRNA was measured during CAM treatment. The expression of IL-10 mRNA significantly decreased during CAM treatment.
Majima et al [44] Patients with advanced NSCLC were treated with CAM. The expression of IL-10 mRNA in PBMNCs was evaluated during CAM treatment. In an additional experimental model, the mice were inoculated subcutaneously with LLC cells and treated with CAM for 1 week. Thereafter, the mice were sacrificed, and the expression of IL-10 mRNA in spleen cells was evaluated. The expression of IL-10 mRNA in PBMNCs and the spleen cells decreased after CAM treatments.
Majima et al [55] Patients with advanced NSCLC were treated with CAM for 3 months. The expression of IL-10 mRNA in the patients' PBMNCs decreased during CAM treatment.
Fonseca-Aten et al [26] Children with an acute exacerbation of recurrent wheezing were enrolled in double-blind, randomised trial of CAM. The nasopharyngeal concentrations of IL-10 were significantly decreased in children treated with CAM.
Cai et al [28] CAM inhibited the LPS-stimulated IL-10 production by alveolar macrophages in BOOP.

IL-12*
Kraft et al [38] The asthma patients treated with CAM demonstrated a reduction of IL-12 mRNA in BAL.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of IL-12 in sinonasal mucosa of CRS.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

IL-17
Fouka et al [58] Patients with stable non-cystic fibrosis bronchiectasis were treated with CAM. IL-17 concentrations in exhaled breath condensate (EBC) and peripheral blood Th17 cells were evaluated. Post-treatment absolute count of CD4 + IL-17 + cells in PB and IL-17 levels in EBC decreased significantly.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

TNF-α
Sakamoto et al [41] Patients with NSCLC were treated with CAM. The level of serum TNF-α significantly decreased after CAM treatment.
Morikawa et al [23] CAM suppressed the synthesis of TNF-α by LPS-stimulated healthy human monocytes.
Khan et al [42] The effect of IL-6 production by LPS-and Pansorbin-stimulated human monocytes was investigated. CAM significantly decreased TNF-α production.
Sassa et al [61] Rat mammary adenocarcinoma cells were treated with CAM, and total RNAs were extracted from the tumour. CAM inhibited the expression of TNF-α gene.
Kraft et al [38] Asthma patients treated with CAM demonstrated a reduction of TNF-α mRNA in BAL.
Morikawa et al [33] The effect of macrolides including CAM on the generation of Th1-and Th2-type cytokines by mitogen-stimulated human T-cells was investigated. CAM showed a potent inhibitory activity on the release and gene expression of TNF-α.
Fonseca-Aten et al [26] Children with an acute exacerbation of recurrent wheezing were treated with CAM. The nasopharyngeal concentrations of TNF-α significantly decreased after CAM treatment.
Perić et al [27] Patients with nasal polyp (nonallergic and allergic) received CAM treatment for 8 weeks. Following the treatment, TNF-α in nasal fluid of allergic patients significantly decreased.
Cai et al [28] CAM inhibited the LPS-stimulated TNF-α production by alveolar macrophages in BOOP.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of TNF-α in sinonasal mucosa of CRS without nasal polyp and eosinophilic CRS with nasal polyp.

Table 1. Cytokines and enzyme, which are positively or negatively influenced by CAM, with respect to MM cell proliferation. (Continued)
IFN-γ* Morikawa et al [33] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. The effect of macrolides including CAM on the generation of Th1-and Th2-type cytokines by mitogen-stimulated human T-cells was investigated. CAM showed a potent inhibitory activity on the release and gene expression of IFN-γ.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of IFN-γ in sinonasal mucosa of CRS.

References Supplementary explanations PDGF
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of PDGF in sinonasal mucosa of CRS.

VEGF
Hu et al [67] CAM decreased the protein expression of VEGF in the nasal mucosa of CRS with nasal polyp.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of VEGF in sinonasal mucosa of CRS.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).

TGF-β
Sassa et al [43] The therapeutic effect of CAM on subcutaneously inoculated mammary adenocarcinoma cells in rats was investigated. The expression of TGF-β gene of spleen cells of tumour-bearing rats significantly decreased after CAM treatment.
Sassa et al [61] Rat mammary adenocarcinoma cells were treated with CAM, and total RNAs were extracted from the tumour. CAM inhibited the expression of TGF-β gene.

FGF2 (basic FGF)
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of FGF2 in sinonasal mucosa of CRS.

G-CSF
Matsuoka et al [22] CAM suppressed the production of G-CSF by IL-1β-added human synoviocytes obtained from RA and OA patients.

GM-CSF
Matsuoka et al [22] CAM suppressed the production of GM-CSF by IL-1β-added human synoviocytes obtained from RA and OA patients.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of GM-CSF in sinonasal mucosa of CRS patients.

MMP-9
Sassa et al [61] Rat mammary adenocarcinoma cells were treated with CAM, and total RNAs were extracted from tumour cells at 6, 12, 24, 48, and 72 hours. CAM inhibited the expression of MMP-9 gene in rat mammary adenocarcinoma cells.
Zeng et al [29] Anti-inflammatory effect of CAM on ethmoid mucosal tissue of CRS was investigated. CAM suppressed the protein production of MMP-9 in sinonasal mucosa of CRS patients.
Zimmermann et al [30] The immunomodulatory effects of macrolides (review of literature).
Takahashi et al [71] CAM suppressed induction of MMP-9 in mouse monocytes and improved pathological changes in the lung and heart of mice infected with influenza A virus.

IL-4
Hamada et al [36] LLC-bearing mice received anticancer chemotherapy after tumour inoculation, and CAM treatments (for 7 days) started immediately after the chemotherapy or 1 week later after the chemotherapy (i.e., delayed initiation of CAM treatment). The mice were sacrificed after finishing CAM treatments. The tumour growth and the number of IL-4-producing T-cells in the spleen were evaluated. The delayed initiation of CAM treatment was effective for reducing tumour growth and resulted in increased IL-4-producing T-cells.
Zeng et al [29] Experiment using ethmoid mucosal tissue from CRS showed that CAM enhanced the protein production of IL-10 in sinonasal mucosa of CRS.

IL-12*
Teramoto et al [53] Patients with inoperable NSCLC were treated with CAM. The level of IL-12 mRNA was elevated after CAM treatment.
Majima et al [54] Patients with unresectable NSCLC were treated with CAM for 3 months. The expression of IL-12 mRNA was measured during CAM treatment. The expression of IL-12 mRNA significantly increased during the treatment.
Majima et al [44] Patients with advanced NSCLC were treated with CAM. The expression of IL-12 mRNA in PBMNCs obtained from the patients was measured during CAM treatment. In an additional experimental model, mice were inoculated subcutaneously with LLC cells and treated with CAM for 1 week. Thereafter, the mice were sacrificed, and the expression of IL-12 mRNA in spleen cells was evaluated. The expression of IL-12 mRNA in PBMNCs and mouse spleen cells increased after CAM treatments.
Majima et al [55] Patients with NSCLC were treated with CAM for 3 months. The expression of IL-12 mRNA in PBMNCs increased during CAM treatment.

IFN-γ*
Majima et al [54] Patients with unresectable NSCLC were treated with CAM. The expression of IFN-γ mRNA in PBMNCs was evaluated during CAM treatment. The expression of IFN-γ mRNA significantly increased during the treatment.
Majima et al [44] Patients with advanced NSCLC were treated with CAM. The expression of IFN-γ mRNA in PBMNCs was evaluated during the treatment. In an additional experimental model, mice were inoculated subcutaneously with LLC cells and treated with CAM for 1 week. Thereafter, the mice were sacrificed, and the expression of IFN-γ mRNA in spleen cells was evaluated. The expression of IFN-γ mRNA in PBMNCs and mouse spleen cells increased after CAM treatments.
Majima et al [55] The patients with advanced NSCLC were treated with CAM for 3 months. The expression of IFN-γ mRNA in PBMNCs increased during CAM treatment.
Hamada et al [36] LLC-bearing mice received anticancer chemotherapy after tumour inoculation, and CAM treatments (for 7 days) started immediately after the chemotherapy or 1 week later after the chemotherapy (i.e., delayed initiation of CAM treatment). The mice were sacrificed after finishing CAM treatments. The tumour growth and the number of IFN-γ-producing T-cells in the spleen were evaluated. The delayed initiation of CAM treatment was effective in reducing IFN-γ-producing T-cells.
*The influence of CAM on IL-10, IL-12 and IFN-γ showed conflicting results.

IL-4
IL-4 plays an essential role in promoting Th2 cell differentiation. It is primarily produced by Th2 cells, NKT cells, mast cells and eosinophils. It is also known that IL-4 induces B-cell class switching to IgE. The relationship between IL-4 and MM cell growth was demonstrated by Herrmann et al [35]. They showed that recombinant human IL-4 blocked endogenous IL-6 synthesis in a dose-dependent fashion and caused a significant reduction of plasma cell growth that could be reversed by exogenous recombinant human IL-6. Hamada et al [36] reported that CAM induced IL-4-producing T-cells in the spleen of tumour-bearing mice. Considering the suppressive effect of IL-4 on MM cell proliferation [35], it is probable that the activation of IL-4 by CAM might lead to MM cell reduction.

IL-5
IL-5 is produced by Th2 cells and mast cells and is known to stimulate B-cell growth and increase immunoglobulin secretion, primarily IgA. This cytokine is known to synergise with IL-6 to support myeloma cell proliferation [4,37]. The suppression of IL-5 by CAM [29,30,33,38] ( Table 1) might result in the reduction of MM cell.

IL-8 (CXCL8)
IL-8, a member of CXC chemokine family, was originally described as a neutrophil chemoattractant. IL-8 is known to promote cancer cell growth, survival, angiogenesis and metastasis in various human cancers. Herrero et al [46] demonstrated that IL-8 did not affect the growth of MM cells but protected them from death induced by serum starvation. Kline et al [47] reported that MM cells expressed IL-8 receptors (i.e., CXCR1 and CXCR2), BMSCs produced IL-8 in active MM and IL-8 production by BMSCs paralleled the MM disease activity. Pellegrino et al [48] demonstrated that IL-8 (CXCL8) and stromal cell-derived factor 1α (SDF-1α, a member of CXC chemokine which is also known as CXCL12) stimulate the proliferation and chemotaxis of human myeloma cells. Kikuchi et al [49] reported that CAM suppressed lipopolysaccharide (LPS)-induced IL-8 production by human monocytes through activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) transcription factors. Thus, the suppression of IL-8 by CAM [2,22,25,28,30,49,50] (Table 1) might be related to MM cell reduction.

IL-10
IL-10 is a cytokine with multiple and pleiotropic effects in immunoregulation and inflammation. IL-10 belongs to MGFs and represents a potent IL-6-unrelated MM cell proliferation factor but not a differentiation factor [51]. Gu et al [52] reported that IL-10 induced MM cell proliferation through an OSM autocrine loop. However, the studies on the influence of CAM on IL-10 showed conflicting results. Some investigators reported that CAM suppressed IL-10 production, secretion or regulation [26,28,44,54,55] (Table 1), whereas others [23,29] ( Table 1) showed the enhancement of IL-10 by CAM. These conflicting results might be attributed to different experimental conditions. Thus, it is very difficult to draw a definite conclusion on the effect of CAM on IL-10, pending further studies.

IL-12
IL-12 is a cytokine that exerts potent antitumor activity through a combination of immunomodulatory and antiangiogenic mechanism. According to Airoldi et al [56], IL-12 mediates the development and maintenance of Th1 cells and induces IFN-γ production by Th1 and NK cells. They demonstrated that IL-12Rβ2 was expressed in primary MM cells but downregulated as compared with normal polyclonal plasmablastic cells and plasma cells. They showed that IL-12 reduced the proangiogenic activity of primary MM cells in vitro and significantly decreased the tumourgenicity of the NCI-H929 MM cells transplanted in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. However, the study of CAM effect on IL-12 seems to show conflicting results. Some investigators reported that CAM enhanced IL-12 production or gene expression [44,[53][54][55], whereas others reported the suppressive effect of CAM on IL-12 [29,30,38] ( Table 1). The contradictory results might be caused by the difference in the experimental settings. Further detailed studies are needed to define the actual effect of CAM on IL-12.

IL-17
IL-17, a member of MGFs, is a proinflammatory cytokine produced by Th-17 cells. It is closely related to TGF-β, nerve growth factor, bone morphogenic protein and platelet-derived growth factor (PDGF) with similar structural motifs. IL-17 induces the expression of a number of chemokines and cytokines including IL-6, TGF-β, GCSF, GM-CSF, MMP and intercellular adhesion molecule-1 in a variety of cell types, including BMSCs [57]. Prabhala et al [57] demonstrated that Th17 cells and serum IL-17 increased in MM, and IL-17 promoted MM cell growth both in vitro and vivo through IL-17 receptors and inhibited Th1 responses [57]. The suppressive effect of CAM on IL-17 [30,58] (Table 1) might be manifested as a reduction of MM cells.

TNF-α
TNF-α, a member of TNF superfamily, is a major proinflammatory cytokine involved in the innate immune response. TNF-α is known to be produced mainly by activated macrophages. According to Jourdan et al [59], TNF-α is a survival and proliferation factor for human MM cells, though less potent than IL-6. Its survival activity is not affected by anti-IL-6 or anti-gp130 monoclonal antibodies, and it induces MM cells into the cell cycle to promote long-term MM cell growth. Hideshima et al [60] investigated the role of TNF-α using human IM-9 MM cells and demonstrated that TNF-α secreted from MM cells induced only a modest increase of cell proliferation, as well as MAPK and NF-κB activation. They also showed that TNF-α upregulated MAPK and NF-κB and markedly increased IL-6 secretion from BMSCs. Furthermore, they demonstrated that TNF-α induced the adhesion molecules on both MM cells (VLA-4, LFA-1 and MUC-1) and BMSCs (ICAM-1 and VCAM-1), which resulted in increased adhesion between MM cells and BMSCs. An increase in adhesion activity, in turn, induces IL-6 secretion from BMSCs, leading to MM cell proliferation. Therefore, the important role of TNF-α in MM is to augment paracrine IL-6-mediated MM cell growth. Thus, the suppression of TNF-α by CAM [23, 26-29, 33, 38, 41, 42, 61] (Table 1) might cause MM cell reduction.

IFN-γ
IFN-γ is crucial for immunity against intracellular pathogens and tumour control. Cells from the innate immune system (e.g., NK cells, NKT cells, macrophages and myelomonocytic cells) and those from the adaptive immune system (e.g., Th1 cells, cytotoxic T lymphocytes and B cells) are known to produce IFN-γ [62,63]. Antitumour potential of INF-γ is well known. Portier et al [64] experimentally demonstrated that IFN-γ inhibited IL-6-dependent MM cell growth and downregulated IL-6R. According to Klein and Bataille [4], IFN-γ downregulates the expression of IL-6R on MM cells and completely inhibits the IL-6-mediated MM cell proliferation. In this respect, some investigators reported that CAM suppressed IFN-γ production or gene expression [29,33]

VEGF/PDGF family
The VEGF/PDGF family belongs to MGFs. VEGF is a multifunctional cytokine that plays an important role in triggering tumour cell migration and angiogenesis. It also possesses a modest proliferative effect on myeloma cells. The production of VEGF in the bone marrow environment is upregulated by myeloma cell adhesion to BMSCs and by IL-6. Moreover, VEGF reciprocally enhances the secretion of IL-6 from BMSCs, suggesting the existence of paracrine interaction between MM cells and BMSCs [65].PDGF plays a significant role in blood vessel formation, mitogenesis and chemotaxis. PDGF is produced mainly by megakaryocytes and alpha granules of platelets and is released by platelets on activation. It is also produced by other cells such as smooth muscle cells, activated macrophages and endothelial cells. It was shown that PDGF receptor (PDGFR)-α and β were frequently expressed on MM cells of newly diagnosed MM patients, and high PDGFR-β expression at diagnosis was associated with advanced stage of the disease [66]. PDGF-β/PDGFR-β kinase axis plays an important role in angiogenesis and proliferation in MM. The suppressive effect of CAM on VEGF/PDGF [29,30,67] (Table 1) might thus be led to MM cell reduction.

TGF-β
TGF-β, a member of MGFs, is a multifunctional cytokine belonging to the TGF superfamily and has pleiotropic biological effect in haematopoiesis and tumourigenesis. It is produced by a large variety of cells including epithelial cells, fibroblasts, eosinophils, macrophages and Treg cell [62]. Urashima et al [68] reported that MM cells secreted more TGF-β1 than splenic B-cells or CD40L pretreated B-cells, and BMSCs of MM produced more TGF-β1 than normal BMSCs [53]. According to Dong and Blobe [69], TGF-β is secreted at higher levels from both MM cells and BMSCs in MM and the increased production of TGF-β correlates well with increased IL-6 and VEGF secretion by BMSCs. Furthermore, neutralising antibody to TGF-β can block increased IL-6 and VEGF secretion, supporting TGF-β as the major inducer for the secretion of IL-6 and VEGF from BMSCs. The suppression of TGF-β by CAM [25,43,61] (Table 1) might cause MM cell reduction.

FGF2 (Basic FGF)
FGF2 (also called basic FGF) is a growth factor with angiogenetic properties and represents one of the MGFs [6]. FGF2 is known to be a significant mediator supporting MM cell expansion and survival. According to Mahtouk et al [6], BMSCs are the main source of FGF2, and they demonstrated that FGF2 gene was expressed in MM cells, but its expression was lower than that of BMSCs. The suppression of FGF2 by CAM [29] (Table 1) was noted.

G-CSF
G-CSF, a member of MGFs, is a potent growth factor for MM cells as well as a hematopoietic growth factor with structural homology to IL-6. G-CSF receptor shares some homology with gp130. Both G-CSF and IL-6 induce the activation of nuclear factor for IL-6 (NF-IL-6), which is a transcription factor involved in the synthesis of IL-6 [18]. The suppressive effect of CAM on G-CSF [22] (Table 1) was documented.

GM-CSF
GM-CSF, a member of MGFs, is a hematopoietic growth factor which has a broad impact on haematopoiesis; it stimulates the development and differentiation of committed stem cells to neutrophils, eosinophils and monocytes. According to Zhang et al [17], GM-CSF is a strong stimulator of in vitro myeloma cell proliferation by potentiating the response of myeloma cells to IL-6. The suppression of GM-CSF by CAM [22,29] (Table 1) might indirectly lead to MM cell reduction.

Matrix metalloproteinases (MMPs)
MMPs play an important role in cell growth, invasion, angiogenesis, metastasis and bone degradation, which are all important events in the pathogenesis of cancer. Van Valckenborgh et al [70] showed the multifunctional role of several MMPs in the development of MM, especially MMP-9, which is closely related to angiogenesis. The suppressive effect of CAM on MMP-9 [29,30,61,71] (Table 1) might be related to MM cell reduction through the downregulation of angiogenesis.
Taken together, CAM can suppress MMP-9 and the following MGFs: IL-1, IL-2, IL-5, IL-6, IL-8, IL-17, TNF-α, PDGF, VEGF, TGF-β, FGF2, G-CSF and GM-CSF (Table 1). Of these MGFs, IL-6 seems to be a crucial factor for MM cell proliferation. Thus, it is probable that the suppression of these MGFs by CAM will cause MM cell reduction. On the contrary, CAM enhanced IL-4 activity [36] (Table 1). However, IL-10, IL-12 and IFN-γ, which are positively or negatively involved in MM cell proliferation, were reported to show the conflicting patterns of response to CAM (Table 1)

CAM-monotherapy for MM
The first report on the use of CAM in MM treatment dates back to 1997. Durie et al [72] pioneered a unique trial of CAM monotherapy to treat active MM. In their trial, 500 mg of CAM was administered to the patients twice a day approximately for 1 year as the longest followup period. They reported that greater than 50% response rate was obtained. However, several responding patients in their study were on concurrent antimyeloma agents that include steroids. Moreau et al [73] reported that no response was achieved in 35 MM patients receiving CAM, at 500 mg, twice a day for 4-20 weeks (median 8 weeks) without chemotherapy or steroids; MM progressed in 80% of cases, requiring other therapeutic approaches. Musto et al [74] reported that among 38 evaluable patients with relapsed or refractory MM (RRMM), who were treated with CAM, at 500 mg, twice a day for 12 weeks, an objective response or minor response (MR) was obtained only in one and two patients, respectively. On the other hand, Stewart et al [75] reported no significant response among 20 evaluable MM patients at various phases of the disease treated with CAM alone. CAM monotherapy seems to have no effect on treating MM.

Low-dose thalidomide and low-dose Dex combined with CAM (Biaxin ® ) (BLTd) for MM treatment
On contrary to the low efficacy of CAM-monotherapy, CAM has added value when combined with thalidomide or its analogues and Dex. Coleman et al [76] pioneered low-dose thalidomide and low-dose Dex combined with CAM (BLTd) to treat previously untreated or treated MM as well as Waldenström's macroglobulinemia and succeeded in achieving a high overall response rate (ORR) of 93%. Niesvizky et al [77] carried out a prospective, randomised trial which compared the safety and efficacy of standard pulsed Dex to those of low-dose thalidomide and low-dose Dex (LTd). A total of 23 advanced MM patients were randomised to either pulsed D or LTd. The patients given pulsed Dex displayed almost similar drops in paraprotein at 8 weeks of treatment as the LTd patients (median: 45% versus 52%). Nine patients (D=3, LTd=6) showed a <50% response (median 26%, SD 15.9) to induction therapy after 8 weeks. Then, Niesvizky et al [77] added CAM to the pulsed Dex or LTd treatments, and they showed that the addition of CAM produced further tumour mass reduction at 16 and 20 weeks of treatment. Overall response rate (>50% response in paraprotein) for pulsed Dex and LTd patients was 80%. They suggested that the addition of CAM contributes to tumour mass reduction by potentiating Dex's effect. Morris et al [45] also reported ORR of 96% in RRMM patients when they received BLTd.

Lenalidomide (Len: Revlimid ® ) and Dex/low-dose Dex combined with CAM (BiRD/BiRd) or pomalidomide (Pom) and low-dose Dex combined with CAM (Clapd) for MM treatments
Len is one of the immunomodulatory drugs (IMiDs) with a structural and functional analogue of thalidomide [78,79]. In 2008, Niesvizky et al [80] first reported the BiRD regimen for the treatment of naive symptomatic MM. The addition of CAM allowed a significant reduction of Dex, and an objective response rate of 90.3% was achieved. A combined stringent and conventional CR rate of 38.9% was achieved, and 73.6% of the patients achieved at least a 90% decrease in M-protein levels. Gay et al [81] performed a case-matched study and compared the efficacy and toxicity of BiRd versus Len combined with low-dose Dex (Rd) for newly diagnosed MM. BiRd regimen, compared to Rd regimen, showed increased CR rates (45.8% versus 13.8%), very good partial response (VGPR) rates (73.6% versus 33.3%) and a longer progression-free survival (PFS) (median: 48.3 months versus 27.5 months). The superiority of BiRd regimen was thus suggested. Rossi et al [82] reported that 72 previously untreated MM patients who were treated with BiRd achieved ORR of 93% (VGPR or better of 68%), and median progression-free survival (PFS) was 49 months. They demonstrated that BiRd was highly effective.
Pomalidomide (Pom) is a distinct immunomodulatory agent similar to Len. Mark et al [83] reported that Clapd proved to be effective for treating relapsed or refractory MM (RRMM). In their study, 117 patients with heavily pretreated RRMM including prior Len-treated RRMM were analysed. ORR (≧PR) and clinical benefit response rates (≧MR) were 60% and 67%, respectively. They concluded that Clapd is a highly effective and tolerable regimen for heavily treated RRMM.

Steroid-enhancing/sparing effect of CAM
The role of CAM in BiRd, BLTd and Clapd regimen has been attributed to its steroid-enhancing/sparing effect [3,76,77,80,81,82,84]. It is well known that CAM is a potent CYP3A4 inhibitor [3,84]. CAM added in BiRd, BLTD and Clapd regimen slows the hepatic clearance of Dex, leading to the augmentation of Dex responsiveness to MM cells [3]. Fost et al [85] investigated the inhibitory effect of CAM on the elimination of methyl-prednisolone (methyl-PSL) and prednisolone (PSL) in six patients with asthma. In their study, the patients received a single oral dose of methyl-PSL (40 mg/1.73 m 2 ) or PSL (40 mg/1.73 m 2 ) before and post administration of 7 days of CAM (500 mg twice a day). Pre-and post-CAM pharmacokinetic profiles of the two steroids showed a 65% reduction in clearance of methyl-PSL but no significant reduction in clearance of PSL. The interactions of CAM with methyl-PSL and PSL were found to be different. To the best of the authors' knowledge, studies directly evaluating the effect of CAM on Dex metabolism have not been reported.

Reversibility of drug resistance by CAM
Juge-Morineau et al [86] demonstrated that the gp130 family cytokines, such as IL-6, LIF and OSM, inhibit the antiproliferative effect of Dex on human MM cells. Yang and Lin [87] reviewed the mechanisms of resistance against Dex in MM. According to them, IL-6 induces drug resistance against Dex by stimulating JAK/STAT to BCL-XL/MCL1 transduction cascade and PI3K/AKT transduction cascade, consequently leading to the upregulation of antiapoptotic signalling [88,89,90]. It is noteworthy that IGF-1, a member of MGFs, is also involved in the development of drug resistance against current standard-of-care agents for MM [91]. MM cells express IGF-I receptor, and its stimulation activates two distinct signal transduction cascades (PI3K/Akt and MEK/ERK cascades), leading to the proliferation of MM cells as well as protection against apoptosis [91,92]. As far as we know, no studies demonstrating the effect of CAM on IGF-1 have been done. Since CAM can potently suppress IL-6, the reversibility of drug resistance including steroid resistance by CAM in MM treatments seems more important than the CYP3A4-related steroid-sparing/enhancing effect thus far reported.

Specific character of MM as a monoclonal immunoglobulin (Ig)-producing neoplasm
Since MM is characterised by the uncontrolled cell growth of monoclonal immunoglobulin-producing neoplastic plasma cells, the large quantities of unfolded or misfolded Ig production itself trigger endoplasmic reticulum stress (ER stress) which is followed by the unfolded protein response (UPR) [93,94,95,96]. According to Obeng et al [95], MM cells are inherently sensitive to proteasome inhibitors because of their large volume of Ig production which requires the expression of physiologic UPR genes. If the ER stress is severe or prolonged, UPR activation leads to cell-cyclic arrest and the induction of apoptosis of MM cells. Thus, it is probable that MM is a specific neoplasm susceptible to proteasome-, autophagy-and histone deacetylase 6 (HDAC6)-inhibitors. In this study, the bone marrow of MM patients obtained before chemotherapy showed that some of the myeloma cells were already under ER stress at the electron microscopic level (Figure 1).

Role of CAM as a potent autophagy inhibitor
It is well known that CAM is a potent and continuous inhibitor of autophagy. Nakamura et al [97] analysed the direct effect of CAM on MM cells in vitro. They demonstrated that CAM attenuated autophagy by blocking the late phase of the autophagic process, probably after the fusion of autophagosomes with lysosomes at clinically relevant concentrations (6-50 μg/mL). In other words, CAM halts the autophagy process and induces the inhibition of MM cell growth. Thus, CAM can serve as a potential adjuvant for MM treatment modalities, where autophagy is used by the tumour as an escape mechanism from apoptosis.

Synergistic effect of CAM with proteasome inhibitor or histone deacetylase 6 (HDAC6) inhibitor
Bortezomib (Bor, Velcade ® ), the first-in-class proteasome inhibitor of the 26S proteasome, was initially approved for the treatment of patients with RRMM as a single agent [98]. Bor is now widely used in combination regimens: Bor, thalidomide and low-dose Dex (VTd) [99], Bor and low-dose Dex (Vd) [100], Bor, Len and low-dose Dex (VRd) [101], Bor, cyclophosphamide and low dose Dex (VCd) [102], Bor, melphalan and prednisolone (VMP) [103], daratumumab, Bor and low-dose Dex (DVd) [104] or pomalidomide, Bor and low-dose Dex (PVd) [105] in RRMM and newly diagnosed MM. The effect of Bor was reported due to its blocking of the ubiquitin-proteasome system, leading to the accumulation of unfolded or misfolded protein in the ER in myeloma cells. This results in ER stress followed by a coordinated cellular response known as UPR [93,94,95]. UPR is known to induce activation of the chaperone protein GRP-78 (Bip: binding immunoglobulin protein) to maintain ER integrity, and it also upregulates the transcription factor CHOP (i.e., C/EBP homologous protein) to mediate cell death when ER stress is beyond the tolerance of the cell adaptation [95]. The combined inhibition of the ubiquitin (Ub)-proteasome system by Bor and autophagylysosome system by CAM synergistically activates UPR, resulting in MM cell apoptosis. Moriya et al [93] experimentally demonstrated that combined treatment of CAM and Bor resulted in increased cytotoxicity as compared to the use of Bor alone. The effectiveness of this combined treatment with CAM, Len and Bor was recently demonstrated at the clinical level [106]. The apoptosis-inducing effect was further enhanced when vorinostat (HDAC6 inhibitor), which is known to inhibit aggresome formation, was added to Bor and CAM [94] (Figure 2). However, no clinical studies have been done using this combination.
Carfilzomib (Kyprolis ® ) is a selective and irreversible epoxyketone proteasome inhibitor of the 20S proteasome. Recently, carfilzomib, Len and low-dose Dex (KRd) [107] combined with CAM (KRd-CAM) were also found to be effective for treating refractory MM (unpublished data).

Safety of long-term administration of CAM
Antibacterial treatment with CAM is usually recommended for a duration of 1-2 weeks. Safety of the long-term administration of CAM has been proved [2,108]. However, there remains a concern regarding the development of resistance of Streptococcus pneumoniae against CAM due to the long-term administration of CAM. Recently, concerns have been raised on the long-term effect regarding the risk of cardiovascular events and mortality in both patients with stable coronary heart disease and patients without heart disease, following the short-term use of CAM (e.g., daily for 2 weeks) [3].

Adverse effects of CAM
The frequent and common adverse effects of CAM are diarrhoea, nausea, abnormal taste, dyspepsia, abdominal pain/discomfort, headache, insomnia, tooth discolouration, smell loss and taste loss. These adverse reactions are usually mild in intensity and resolve after the discontinuation of treatment. All other adverse reactions are uncommon or rare [3]. However, in post-marketing surveillance, allergic reactions ranging from urticaria and mild skin eruptions to rare cases of anaphylaxis, Stevens-Johnson syndrome, toxic epidermal necrosis, liver dysfunction, somnolence and confusion have occurred [https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/050662s044s050,50698s026s03 0,050775s015s019lbl.pdf]. In particular, CAM is also known to prolong QT interval. Rarely, it can be fatal, leading to fatal ventricular arrhythmias, including ventricular tachycardia and torsades de pointes [109]. Thus, CAM therapy should not be given to the patients with a history of QT prolongation and arrhythmia. Since CAM inhibits CYP3A4, the coadministration of CAM with some drugs metabolised by CYP3A4 should be carefully done. In general, antibiotics are known to be ineffective against virus infection. Shinahara et al [110] treated 195 influenza-A-infected children with oseltamivir (OSV) and zanamivir (ZNV) with or without CAM. They demonstrated that the combination of CAM plus OSV or ZNV boosted and restored the production of mucosal secretory IgA (S-IgA) and systemic IgG. Furthermore, they reported that CAM supplementation reduced the reinfection rate in the subsequent year in the patients treated with OSV and ZNV. They suggested the possibility that CAM enhanced influenza virus-specific S-IgA production through the induction of IgA class switching recombination.

Indirect effects of CAM against virus infections
It is interesting to note that hydroxychloroquine treatment combined with azithromycin, 15-membered macrolide, reinforced viral load reduction/disappearance in COVID-19 patients [111]. Just recently, Millán-Oñate et al [112] first reported that a 34-year-old Columbian man with COVID-19 pneumonia was successfully recovered after receiving chloroquine and CAM. Emerging data suggest that many patients with COVID-19 may die due to an excessive response of their immune system, characterised by the abnormal release of circulating cytokines including IL-1β, IL-6, IL-12, IL-18, TNF-α, TGF-β, GM-CSF, IFN-γ and various chemokines. This phenomenon is referred to as cytokine release syndrome (CRS), and a crucial role seems to be played by IL-6. CRS plays a major role in the deterioration of COVID-19 patients with pneumonia to acute respiratory distress syndrome, cumulating in systemic inflammation and multiorgan failure [113]. In addition, clarithromycin is also known to be effective for treating organising pneumonia [114]. Cai et al [28] demonstrated that spontaneous and lipopolysaccharide-stimulated alveolar macrophages from patients with BOOP upregulated the production of TNF-α, soluble TNF receptor 1/2, IL-1β, IL-6, IL-8, IL-10 and chemokine ligand 18. They suggested that CAM significantly attenuated or inhibited the production of these cytokines. Considering the pleiotropic immunomodulative effects of CAM, the suppressive effects of CAM on cytokines related to CRS, particularly on IL-6, should be noted. The administration of CAM as a single agent or combined with other drugs might be worth a try for treating COVID-19. One trial of CAM against COVID-19 has already started in Greece in May, 2020 (https://www.clinicaltrialsregister.eu/ ctr-search/trial/2020-001882-36/GR).

Conclusion
We reviewed the current preclinical, clinical and experimental evidence for supporting the efficacy of CAM for treating MM, especially when it is combined with IMiDs, steroids or proteasome inhibitors. IMiDs, steroids, proteasome inhibitors and CAM might synergistically play an important role in inducing apoptosis of MM cells. In addition, the reversibility of drug resistance by CAM to chemotherapeutic drugs including steroids seems to be more important than steroid-sparing/enhancing effect thus far reported in MM treatments. Since multiple myeloma is characterised by the uncontrolled cell growth of monoclonal Ig-producing neoplastic plasma cells, the large quantities of unfolded or misfolded immunoglobulin production itself may trigger ER stress. Thus, MM is a specific neoplasm particularly susceptible to proteasome, autophagy and HDAC6 inhibitors. The development of CAM analogues specific for more potent immunomodulatory effects may provide a novel approach to MM treatment in the future.
In addition, considering the pleiotropic effects of CAM on cytokines including IL-6 and its indirect antiviral effects, CAM might be worth a try for treating COVID-19.