ABSTRACTThe pharmacological property of indomethacin, an anti-inflammatory drug approved in 1965, has been lately highlighted for having multiple intermingling pathways, particularly during the COVID-19 epidemic. Originally used as a COX inhibitor and PG regulator. Indomethacin has been found to be effective in many other therapeutic areas. It is a promising anti-cancer and antiviral agent, a central nervous system (CNS) drug, and also useful in treating tuberculosis, tocolysis, aortic rupture, autophagy, and burning scrotum syndrome. Indomethacin’s multifaceted effects are likely the result of its synergistic and reparative modes of action, making it a versatile drug for various medical indications.Currently, much work is being done to develop various formulation approaches for indomethacin to enhance its efficacy while minimizing side effects. Wider exploration of indomethacin in these lesser-known capacities has profound implications in the management of several diseases. The purpose of this review is to catalog the less well-known medicinal roles of indomethacin and compile a succinct overview for researchers. This synthesis of literature could guide future research and contribute to discussions on additional ways indomethacin might be used, ultimately leading to improved medical approaches in various areas. The evolving role of indomethacin in medicine underscores its significance as a ”miracle drug” with many implications.Keywords: Indomethacin, Cancer, COVID-19.IntroductionNonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequently prescribed drugs worldwide. Indomethacin was approved in 1965 and licensed as an analgesic, anti-inflammatory, and antipyretic medication by the Food and Drug Administration (FDA) (Tonby et al., 2016). Indomethacin is produced from indole-acetic acid (1-(p-chlorobenzene)-5-methoxy-2-methylindole-3-acetic acid) and has been used to treat rheumatic diseases such as osteoarthritis, collagen issues, ankylosing spondylitis, and gout (Martín-Saborido et al., 2019; Tonby et al., 2016). Despite their powerful anti-inflammatory and analgesic properties, they also have some unexplored pharmacological properties, such as restorative and synergistic properties, increasing their efficacy, and making them a magical drug because of their stealthy property. Proteomics, genetic interactions, and expression profile investigations are examples of comprehensive pharmacological research that can aid in indomethacin treatment. It has also been associated with alterations in polymorphonuclear neutrophil (PMN) adherence, degranulation, phagocytosis, and reactive oxygen species (ROS) (Chen et al., 2019). Indomethacin is a photosensitive medicine that is water-insoluble and slightly soluble in alcohol and has a logarithmic acid dissociation constant (pKa) of 3-4.5 (Little, 2020). Aging has little effect on indomethacin absorption, which is an additional property of indomethacin (Tomida et al., 2019). Indomethacin may influence a variety of cellular processes by modifying the microenvironment within the membrane (Chamoun-Emanuelli et al., 2019). Indomethacin is used to treat arthritic pain, fever, a variety of headache syndromes, and dysmenorrhea. In addition, the patent ductus arteriosus is closed (Summ et al., 2021). In this review, we summarized the unexplored pharmacological information on indomethacin, such as its synergistic, restorative, and efficacy effects in different diseases, which proves that it is a miracle drug.Indomethacin as an anticancer drugApoptosis is a multigene-controlled mechanism of cell death. The regulation of cell apoptosis has recently emerged as a possible cancer therapeutic technique. The anti-apoptotic proteins from the Bcl-2 family are (Bcl-XL, Mcl-1, Bcl-2, Bcl-W, and A1) and pro-apoptotic proteins (BH1-3 and BH-3 proteins), which are crucial regulators of cell apoptosis. Apoptosis resistance is brought about by increased production of anti-apoptotic Bcl-2 proteins. Because of the strong affinity of indomethacin for the Bcl-2 protein, inhibiting its expression can assist traditional anticancer therapies in overcoming drug resistance and boosting their efficacy (Revuelta et al., 2020). It can be used as a targeted drug that can increase its binding affinity towards Bcl-2 proteins, thus increasing the ability of cancerous cells to resume apoptosis and synergistically work in the treatment of cancer (Figure 1). Breast cancer is the most prevalent cancer in women. Studies have shown that human mucin 1 (MUC1)-directed tumor vaccines are effective in the treatment of breast, colorectal, and pancreatic cancers (Xu et al., 2020). The activity of COX-2 is elevated in breast cancer and plays a role in several tumor-related processes. COX-2 inhibits the activation of immune cells such as cytotoxic natural killer cells, T-helper lymphocytes, CD8+ T lymphocytes, and dendritic cells by generating prostaglandin E2 (PGE2). Elevated COX-2 levels in breast cancer patients are associated with larger and more advanced tumors, which have been shown to negatively affect dendritic cell and T cell function. Recent research indicates that combining MUC1-specific peptide vaccination with COX-2 pathway inhibitors, such as indomethacin, can potentially enhance the effectiveness of treatment for invasive breast cancer. It demonstrates analgesic effects and protects against hepatotoxicity and nephrotoxicity. Additionally, it exhibited inhibitory activity against proliferative responses in estrogen-sensitive breast cancer cell lines (. et al., 2024). Preclinical studies using indomethacin have provided valuable insights into the potential benefits of combining COX pathway inhibition with immunotherapy for breast cancer treatment (Xu et al., 2020). Indomethacin increases apoptosis in colon cancer cells by lowering peroxisome proliferator-activated receptor (PPARδ) activation (Figure 2). Indomethacin therapy inhibits the proliferation of effector T cells and enhances their ability to destroy apoptotic tumor cells in their immediate surroundings. It is interesting to note that indomethacin in combination has been shown to boost arginase expression at the RNA and protein levels. The depletion of L-arginine in the tumor microenvironment is crucial for cancer cells to avoid the immune system, which is known to impair T-cell responses. (Xu et al., 2020). Viruses that lead to cancer can also be cured because indomethacin activates protein kinase R (PKR), causing rapid phosphorylation of eukaryotic translation initiation factor 2 (eIF) and blocking protein synthesis, thereby preventing viral reproduction and protecting host cells from viral harm (Chen et al., 2019). Non-COX-dependent mechanisms may interfere with the function of other cellular machinery, resulting in cell growth suppression, apoptosis, or necrosis. For example, tubulin and heat shock protein 27 (Hsp27) have been identified as anti-cancer molecular targets of COX inhibitors, which are well-known targets for anticancer therapeutic development. The use of COX inhibitors in cancer treatment has been restricted because of their association with cardiovascular side effects. Consequently, it is crucial to develop non-COX-active analogs derived from established COX inhibitors. This approach is essential for the discovery of new anticancer medications with both effectiveness and safety profiles (Rovers et al., 2021). These findings indicated that lysosomes could be potential targets for chemotherapy. Indomethacin elevated the levels of three autophagy substrates, p62, LC3-II, and NBR1. These results demonstrated that indomethacin suppresses the breakdown of autophagic components, thereby impeding autophagy (Marinella, 2020). Androgen receptors (AR) are important therapeutic targets in prostate cancer (PC). Most PC tumors ultimately turn into lethal condition castrated-resistant prostate cancer (CRPC) due to the overuse of ADT or AR-targeted therapy because AR is essential for the formation and increase of CRPC tumors. Novel indomethacin derivatives (CZ-212-3) that have been developed and used in CRPC conditions degrade AR-variant and AR proteins in CRPC cells, resulting in the suppression of CRPC, which shows the restorative property of indomethacin. It can be concluded that CZ-212-3 and its variants suppress CRPC tumor growth and can be used for therapy. Indomethacin significantly suppresses proliferation and promotes apoptosis in gastric cancer cells by inhibiting mitochondrial function (Curry et al., 2019).Indomethacin as an anti-viral drugIndomethacin has long been recognized to have antiviral characteristics (first shown in 2006) and is generally utilized as a strong anti-inflammatory drug by decreasing pro-inflammatory PG formation (Gliszczyńska & Nowaczyk, 2021). Indomethacin exhibits an effective antiviral effect against SARS-CoV-2 by directly targeting and downregulating the expression of transmembrane serine protease and angiotensin-converting enzyme 2 (ACE2). Studies have shown that ACE2 expression is particularly high in lung type II alveolar cells, enterocytes in the colon and ileum, upper esophageal stratified epithelial cells, and bladder urothelial cells. It has also demonstrated efficacy against canine coronavirus (CCoV) and SARS-CoV. Common laboratory findings include elevated levels of liver enzymes such as serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. Additionally, inflammatory markers such as serum ferritin and C-reactive protein (CRP) are often increased (Brizawasi et al., 2023, p. 19). According to the current study, indomethacin can activate a cellular antiviral defense mechanism by quickly and successfully activating PKR in an interferon- and dsRNA-independent manner, which is the sole obvious antiviral mechanism. Additional targets of indomethacin and its analogs have been identified in the analysis of indomethacin: aldose reductase (AKR1B1), aldo-keto reductase (AKR1C3, AKR1C4, AKR1C2), cannabinoid receptor 2 (CB2), and peroxisome proliferator-activated receptor gamma (PPARG) (Gliszczyńska & Nowaczyk, 2021). According to a recent study, indomethacin inhibits cytokine-mediated IL-6 production by lowering lipopolysaccharide (LPS). COVID-19 individuals have a ”cytokine storm,” with IL-6 levels in the body greatly elevated (Gliszczyńska & Nowaczyk, 2021). Symptom relief occurred faster in the indomethacin group than in the paracetamol group (Rajan et al., 2020). This study demonstrates that indomethacin, independent of its anti-inflammatory properties, has direct antiviral activity against SARS-CoV and CCoV by preventing viral RNA synthesis (>1,000-fold decrease in viral production in CCoV-infected dogs). Indomethacin prevents rotavirus infection in human intestinal Caco-2 cells by lowering viral protein production. The antiviral properties of indomethacin and its ability to reduce cytokine release syndrome make it helpful (Abou-Ghannam et al., 2012). In COVID-19 patients, indomethacin, in combination with usual therapy, appears to promote faster symptomatic relief and prevent pneumonia progression. This should be considered a replacement for paracetamol. Blocking proteases such as 3C-like cysteine proteases, Mpro, and Ppro play an important role in replication and transcription. Another therapeutic target is the RNA-dependent RNA polymerase (RdRp), a key enzyme in the replication process (Alkotaji & Al-Zidan, 2021). If the process which occurs normally after virus entrance and replication is disrupted or delayed by a cascade of pro-inflammatory cytokines is released in an uncontrolled manner culminating in a ”cytokine storm” (Alkotaji & Al-Zidan, 2021). A ”cytokine storm” involves several interleukins, the most prominent of which are IL-6, IL-17, and IL-1. Indomethacin suppresses this cytokine storm by inhibiting COVID-19, demonstrating its curative effects. SARS-Cov-2 appears to interact with IL-17. Indomethacin suppressed Cathepsin L activity, and no other NSAID had any effect on Cathepsin L activity. Cathepsin L activity reduces SARS-Cov-2 entrance by 76%. As a result, indomethacin might theoretically be a promising fusion inhibitor synergistically (Alkotaji & Al-Zidan, 2021). Indomethacin is one of the medications that has both antiviral and anti-inflammatory properties. Additionally, indomethacin has proven to be exceptionally effective in treating both dry cough caused by ACE2 inhibitors and the pro-inflammatory effects of extremely high bradykinin levels. In light of the diverse response efficacy of indomethacin, investigation of the mechanism and search for more relevant clinical trials for COVID-19 treatment are needed. La-related protein 1 (LARP1), RNA-binding proteins (RBPs), and poly adenylate binding protein 1 (PABPC1) are involved in the regulation of translation of certain downstream target mRNA transcripts (Chen et al., 2019). However, there was no indication of a direct impact of indomethacin on LARP1 or PABPC1, but it still showed a positive synergistic effect (Shekhar et al., 2022). This will help us refine our approach to identifying druggable targets in preclinical research with high confidence. When indomethacin was used in combination with the existing ICMR standard procedure (synergistic effect) for curing COVID-19-infected hospitalized patients, there was a significant decrease in the severity and duration of the disease, with no notable adverse drug responses (Chen et al., 2019). Further studies are needed to determine the potency of indomethacin alone in the management of COVID-19. The antiviral properties of indomethacin are shown in Figure 3.Indomethacin as a CNS drugIndomethacin is a strong cerebral vasoconstrictor that effectively reduces intracranial pressure (ICP), leading to improved brain perfusion and oxygenation. This property of indomethacin makes it potentially beneficial for aiding the recovery of individuals with traumatic brain injury (TBI) (Ivanyi & Zumla, 2013). It is predicted that 11% of those with severe brain injury will die. To reduce the risk of secondary brain damage and cerebral edema (Bykova et al., 2021; Jebamalar et al., 2016). Post-traumatic cerebral edema is prevalent and contributes to elevated ICP. One of the main initiating factors for the onset of brain edema is a disruption in capillary permeability of small solutes, which is controlled by the blood-brain barrier (BBB) when it is intact. Indomethacin has the potential to break the vicious cycle that occurs when ICP rises beyond 25 mmHg, putting an end to the waves and restoring brain perfusion and oxygenation. The methods by which indomethacin lowers ICP are unknown, but they show synergistic and restorative effects. In addition, COX inhibition and blocking of prostacyclin receptors directly constrict cerebral blood vessels and reduce brain swelling (Bykova et al., 2021). Indomethacin, on the other hand, may have a direct neuroprotective effect via a non-prostaglandin-mediated mechanism. Several trials have demonstrated that indomethacin can improve cerebral perfusion pressure (CPP) and lower ICP in patients with refractory ICP who have not responded to traditional therapy (Bykova et al., 2021). Indomethacin is commonly used to prevent patent ductus arteriosus in premature infants. It has also been shown to reduce the severity of intraventricular hemorrhage (IVH) in premature infants. According to experimental and clinical research, indomethacin treatment may reduce the incidence and severity of IVH through synergistic effects (Chennamaneni et al., 2016). According to a previous study, indomethacin may interfere with NO signalling pathways, which are known to cause headaches and migraines. Indomethacin has been proven to prevent NO-induced vasodilation in preclinical studies that cure headaches and migraines through its synergistic effects (Zhou et al., 2010). It was one of the first NSAIDs to be used in the management of headache diseases that were later referred to be ”indomethacin-responsive” headaches, such as migraines and headaches. Side effects have been reported in less than 1% of indomethacin users. High ICP causes cerebral vasoconstriction and decreased CBF, which raises concerns about possible cerebral ischemia, which can be treated synergistically with indomethacin. Indomethacin, by synergistic behavior, cured Alzheimer’s disease patients by reducing the production of the amyloid-42 ( amyloid-β peptide) amino acid, which is part of plaque formation and neurotoxic (Summ et al., 2021). The idea that indomethacin may act as an allosteric modulator of CB1R was motivated by the structural similarities between indomethacin and the type 1 cannabinoid receptor (CB1R). Endogenous cannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), their synthesis and degradation enzymes, and receptors such as CB1R and CB2R make up the endocannabinoid system. The promise of treating conditions including pain, depression, and neurodegenerative illnesses by focusing on these elements has been demonstrated. Given these linkages, indomethacin may be a useful tool for examining the relationship between the structure and function of COX enzymes and CB1R allosteric modulators. This study may contribute to the development of new medicinal substances that are selective for the endocannabinoid system subsystems. (Herminghaus et al., 2020).Indomethacin as an anti-inflammatory drugInjury, infection, damaged cells, pathogens, and irritants cause inflammation, which is a physiological response of body tissues to harmful stimuli. Through inflammatory processes, the immune system eliminates harmful stimuli and promotes tissue repair (Laprairie et al., 2019). Inflammatory cells would produce a slew of cytokines, PG, and reactive oxygen species (ROS), which hasten normal tissue impairment (Kiguchiya et al., 2018). The relative potencies of drugs in the prostaglandin synthesis are popular anti-inflammatory drugs in decreasing order indomethacin > naproxen > ibuprofen > phenylbutazone > aspirin based on COX selectivity. It has a COX inhibitory action that lowers PMN recruitment and inhibits the synthesis of lipoxins and resolvins, which delay inflammatory resolution (Lucas, 2016).Role of indomethacin in pregnancyIndomethacin has diverse effects on pregnancy-related disorders. Indomethacin suppresses PG synthesis in embryonic organs by passing through the placental barrier. It plays an important role in preterm labor (PTL) (. et al., 2023; Kim et al., 2021). Women with labor pain had higher plasma and amniotic fluid PG levels. Hypothyroidism has been linked to adverse maternal outcomes including gestational diabetes, preeclampsia, preterm labor, postpartum hemorrhage, and placental abruption. Additionally, it is associated with various fetal complications such as impaired neurodevelopment (Prabhat et al., 2023). Neural tube defects are a prevalent form of congenital malformations that can be effectively prevented through the periconceptional use of folic acid supplementation (Jain & Prabhat, 2023; Prabhat et al., 2023). The maternal serum levels of prostaglandin metabolites are reduced when tocolysis is treated with indomethacin, showing synergistic effects. The effect of indomethacin on uterine contractility is not solely attributed to the inhibition of prostaglandin synthesis. Additional pathways influence uterine contractility (Nalamachu & Wortmann, 2014). NF-κB and its target genes, including TNF-α and IL-6, have been identified as contributors to insulin resistance (. & Bhattacharjee, 2022). Nuclear factor kappa B (NF-kB) is involved in another mechanism of indomethacin-induced tocolysis. Interleukin IL-8 and COX-2 is regulated by this protein. NF-kB activity increases during labor pain and has an anti-progesterone effect. Furthermore, IL-8 genes and COX-2 are also upregulated before labor pain. The activity of NF-kB has been demonstrated to be decreased by the treatment of indomethacin. As a result, using indomethacin to treat PTL would diminish PG synthesis and show a synergistic effect, while also potentially reducing the anti-progesterone action of NF-kB on other pro-labor genes such IL-1b and IL-8 by showing restorative effects (Nalamachu & Wortmann, 2014).Role of indomethacin in aortic ruptureAortic ruptures occur when the layers of the aortic wall burst in any condition. Because of this, a lethal condition arises where the aortic intima tears (known as hematomas) and blood flows forcing the layers of the aortic wall to separate, weakening the aortic wall. It becomes more lethal if untreated because monocytes/macrophages infiltrate after aortic rupture and are marked by the segregation of aortic wall layers (Ravichandran et al., 2021). Elevated TSH levels, indicating subclinical hypothyroidism, were identified as significant predictors of postoperative mortality in patients with aortic dissection undergoing aortic surgery. There is no evidence suggesting that indomethacin affects thyroid function (Banerjee et al., 2024). Low-density lipoprotein cholesterol (LDL-C) levels, along with the ratio of LDL-C to high-density lipoprotein cholesterol (HDL-C), are crucial parameters for comprehensive evaluation and management of cardiovascular disease risk (. et al., 2022; Lnu et al., 2024). The use of indomethacin shows restorative properties through its synergistic behavior of lowering the risk of aortic rupture by reducing the infiltration of monocytes and macrophages in the aortic wall and curing hematomas. An earlier study revealed that indomethacin downregulates platelet endothelial cell adhesion molecule 1 (PECAM-1), resulting in decreased trans-endothelial migratory activity, which reduces inflammation by its hidden restorative properties (Ravichandran et al., 2021). Prostaglandin E2 PGE2 and prostaglandin F2 (PGF2) synthesis are known to be inhibited by indomethacin and antagonize the PGF2 receptor and the PGE2 receptor subtype 4 (EP4) cured the aortic rupture (Ravichandran et al., 2021).Role of indomethacin in burning scrotum syndromeBurning scrotum syndrome (BSS) is a mysterious illness with no known cause or difficult treatment. Dysesthesia and erythema in the front area of the scrotum are the symptoms of BSS. There are several possible causes of BSS, including rebound vasodilation following corticosteroid use, localized erythromelalgia, localized neurogenic inflammation, and rosacea-like vasodilation. The authors suggested that the vasoconstrictive qualities of indomethacin were responsible for the restorative effects of indomethacin because it influences mesenteric blood flow. It has been hypothesized that indomethacin may have similar effects on testicular arteries, and it has also been shown to be effective in treating BSS through its unexplored stealthy restorative and synergistic effects (Vallecillo-Hernández et al., 2018).Indomethacin as an anti-tuberculosis drugHost-directed immune modifying treatment has been proposed as an alternative to tuberculosis (TB) medicine. In vitro findings have shown that the COX-1/2 inhibitor (indomethacin) modulates T-regulatory and Th1-effector cells in Mycobacterium TB (Mtb) infection through its synergistic properties (Object, n.d.). As a result, targeting FOXP3+ T regulatory cells (Tregs) with indomethacin might be useful as a host-directed supplementary therapy that increases its treatment efficacy by several folds (Nunes-Alves et al., 2014). Mtb antigen-triggered Tregs, where antigen-induced cytokine responses (particularly T cell proliferation and TNF-α+ cells) are all inhibited by indomethacin, show restorative effects. They showed for the first time that indomethacin inhibits Mtb antigen-induced Tregs (CD8+ and CD4+ cytokine responses), TNF-cytokine production, and T-cell proliferation, confirming that indomethacin is a better drug to cure this disease in the future (Miyara & Sakaguchi, 2007). However, further clinical research is needed to explore the potential of indomethacin as an alternative host-directed therapy for various TB infections and disease contexts. Additionally, investigating the effects of indomethacin on tuberculosis infection in human models is crucial for gaining a deeper understanding of its efficacy (Sloan, 2021).Role of indomethacin in autophagyIndomethacin therapy lowered the expression of lysosomal-associated membrane protein 2 (LAMP-2), which is necessary for the fusion of lysosomes and autophagosomes. The effects of indomethacin on autophagy have been demonstrated in two studies: one shows that indomethacin initially promotes autophagy but thereafter inhibits autophagic flux, while the other suggests that indomethacin causes cell death and uncontrolled autophagy activation. Indomethacin also impairs autophagy-dependent intestinal epithelial cell (IEC) activities that are required for symbiotic host-microbiota connections (Mozolewski et al., 2017).Indomethacin as a gastrointestinal drugIndomethacin decreases COX-1-dependent mucosal-protective gastric PG production, which causes significant stomach damage and increases the likelihood of gastrointestinal ulcers and bleeding, all of which are substantial side effects. COX-2 selective inhibitors, on the other hand, have fewer gastrointestinal side effects but increase the risk of thrombosis and cardiovascular disease (Laprairie et al., 2019). A new type of hybrid was formed by covalently linking indomethacin to phospholipids as a unique strategy to overcome indomethacin toxicity. They synthesized a phosphatidylcholine bioconjugate with indomethacin (DP-155). The DP-155 molecule has also been shown to function in brain tissue by reducing neurodegeneration caused by beta-amyloid (A) peptide buildup in cells and inflammation. The ester compound indomethacin was combined with menthol to reduce indomethacin toxicity (Summ & Evers, 2013). Indomethacin has strong binding affinities and penetrates phospholipids (Tan et al., 2018). Owing to its lipophilic nature and weak acidic properties, indomethacin can readily cross the outer mitochondrial membrane. Once inside, it transports protons back to the mitochondrial matrix from the intermembrane space (Wang et al., 2022). Through this approach, we can reduce the side effects of indomethacin, making it a miracle/novel drug that performs multiple functions in different types of ailments. In recent investigations, hydrogen sulfide (H2S) has emerged as a pivotal signaling molecule characterized by its antioxidative properties, notably contributing to defence mechanisms and reparative processes within the gastrointestinal (GI) mucosa. Conversely, indomethacin, a non-steroidal anti-inflammatory drug (NSAID), is a therapeutic agent renowned for its efficacy in mitigating inflammation associated with conditions such as gout or osteoarthritis. Nevertheless, its clinical applicability remains circumscribed owing to its propensity to impede gastric mucosal prostaglandin (PG) biosynthesis, thereby precipitating or exacerbating ulcerogenic manifestations. Notably, the advent of research indicates the potential of H2S to ameliorate the adverse gastrointestinal effects typically associated with certain NSAIDs (Głowacka et al., 2023).
