Expanding Horizons: Opportunities for Diclofenac Beyond Traditional Use—A Review

Abstract

This study systematically reviews the non-traditional pharmacological effects of diclofenac, a well-known nonsteroidal anti-inflammatory drug, to explore its potential for drug repositioning beyond its established analgesic and anti-inflammatory applications. A comprehensive literature search was conducted using the PubMed, Scopus and Web of Science databases, covering studies from 1981 to 2025. It was revealed that over 94% of records in Scopus and Web of Science are duplicated in PubMed, so the latter was used for the search in our study. After duplicate removal and independent screening, 89 from 1123 retrieved studies were selected for the search. The analysis revealed a broad spectrum of diclofenac’s non-traditional pharmacological activities, including neuroprotective, antiamyloid, anticancer, antiviral, immunomodulatory, antibacterial, antifungal, anticonvulsant, radioprotective, and antioxidant properties, primarily identified through preclinical In vitro and In vivo studies. These effects are mediated through diverse molecular pathways beyond cyclooxygenase inhibition, such as modulation of neurotransmitter release, apoptosis, and cellular proliferation. Diclofenac showed potential for repositioning in oncology, neurodegenerative disorders, infectious diseases, and immune-mediated conditions. Its hepatotoxicity and cardiovascular risks necessitate strategies like advanced drug formulations, dose optimization, and personalized medicine to enhance safety. Large-scale randomized clinical trials are essential to validate these findings and ensure safe therapeutic expansion.

1. Introduction

The modern pharmaceutical science and industry constantly search for new, more effective, and safer therapeutic solutions to combat various human diseases. However, the traditional de novo drug development pathway, which begins with target identification and synthesis of new chemical compounds, is facing increasing and often insurmountable challenges. This process is highly time-consuming, usually taking 10–15 years from an idea to enter the market. This requires tremendous financial investments, reaching billions of dollars per successful drug, and is characterized by an extremely high failure rate. Only a small part of molecules from preclinical and early clinical trials eventually receives regulatory approval [1,2].

Drug repositioning (also known as repurposing or “drug repurposing”) is one of the most attractive and strategically essential paradigms in modern pharmacology. This approach consists of identifying, substantiating, and developing new therapeutic indications for existing medicinal products previously approved for use for other indications [3]. The key advantage of drug repurposing is the ability to use a large array of existing data on the molecules investigated. This approach significantly reduces the time and cost of developing new drugs, reducing the risk of unforeseen side effects and clinical trial failures. It is estimated that repositioning can be 40–90% cheaper than creating a fundamentally new drug from scratch [4].

Particular attention merits the repositioning of generic drugs, which have been widely used in clinical practice for decades. These drugs possess a well-studied primary mechanism of action and an established safety profile. [5]. Diclofenac, a nonsteroidal anti-inflammatory drug from the phenylacetic acid derivatives group, is a prominent representative of such drugs. Introduced into clinical practice [6] in the 1970s, diclofenac has become one of the most common drugs for treating pain and inflammation of various genesis, particularly in rheumatic diseases, arthritis, postoperative pain, and injuries. Its classical mechanism of action is well studied. It involves non-selective inhibition of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. This inhibition reduces the synthesis of prostaglandins, which are key mediators of inflammation, pain, and fever. The effectiveness of diclofenac in this role is undeniable. The use of diclofenac, like other NSAIDs, carries a risk of side effects, primarily affecting the gastrointestinal tract and cardiovascular system. These side effects are mainly due to COX-1 inhibition. This inhibition impacts the homeostatic functions of prostaglandins [7].

However, over the past two decades, an increasing body of evidence has accumulated suggesting that the pharmacological action of diclofenac is not limited solely to its effect on COX. Preclinical In vitro and In vivo studies, some clinical observations, and computer modeling indicate that diclofenac has several additional pharmacological properties. Recent studies reported on the potential anticancer activity of diclofenac, which is mediated through its effects on angiogenesis, apoptosis, and cellular proliferation [8]. Its effect on bacterial and viral strains is being studied [9]. Its impact on the processes involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease, is also being studied [10]. These potential new effects are likely mediated through mechanisms independent or only partially dependent on COX inhibition, including effects on other signaling pathways. Such multifaceted pharmacological action opens new prospects for expanding the therapeutic horizons of Diclofenac through its repositioning beyond its traditional applications.

The aim of this study was to analyze and systematize scientific information on the non-traditional pharmacological effects of diclofenac, beyond its well-known anti-inflammatory and analgesic properties. The study focused on its potential repositioning for treating conditions not covered by its established clinical indications.

2. Materials and Methods

2.1. Search Strategies

The information resources of the PubMed (a widely recognized resource for biomedical and pharmacological research), Scopus and Web of Science database were used to search for information. A scoping probe in Scopus (94.9%) and Web of Science (94%) showed that >94% of records retrieved there were duplicates of PubMed citations, with no additional primary studies meeting the a priori inclusion criteria. Therefore, the study included data search, analysis, and systematization, and generalization was used from the PubMed database [11]. The study covered the period from 1981 to 2025, when various studies of different non-traditional pharmacological properties of diclofenac started (Figure 1). This broad time frame ensured a comprehensive collection of the relevant literature. The search strategy was likely structured to combine these terms using Boolean operators (AND, OR) to capture studies addressing diclofenac’s diverse pharmacological applications. It includes an analysis of 89 literature sources (Figure 1, for Section 3.1, Section 3.2, Section 3.3, Section 3.4, Section 3.5, Section 3.6, Section 3.7, Section 3.8, Section 3.9 and Section 3.10 below in Section 3, Results and Discussion). The search combined free-text keywords relating to “diclofenac” and “drug repurposing” OR (“pleiotropic”) AND specific effect-related terms. These included “neuroprotective,” “amyloid”, “antiamyloid”, “Alzheimer disease”, “Parkinson’s disease”, “cancer”, “anticancer,” “antitumor”, “antiviral,” “immunomodulatory,” “antibacterial,” “antimicrobial”, “antifungal,” “anticonvulsant,” “antiseizure”, “epilepsy”, “antiepileptic”, “radiation”, “radioprotective,” “prooxidant”, and “antioxidant”.

2.2. Filters Applied

Both preclinical (In vitro and In vivo) and clinical studies were included to provide a holistic view of diclofenac’s non-traditional effects. No specific filters for study type (e.g., randomized controlled trials, observational studies) were noted, indicating a broad approach to capturing relevant literature. We focused our search on studies reported in English.

2.3. Screening Process

The screening process involved the following stages: initial screening, full-text assessment, and resolution of disagreements. The initial screening included reviewing titles and abstracts to identify studies relevant to diclofenac’s non-traditional pharmacological effects. Then evaluating the full texts of potentially relevant studies to confirm eligibility based on inclusion and exclusion criteria was carried out. Duplicate removal was performed and verified manually. Two reviewers (M.D. and M.S.) independently screened records against pre-defined eligibility criteria. Any discrepancies between reviewers were resolved through discussion.

2.4. Inclusion and Exclusion Criteria

Studies were included if they

• were original research articles, reviews, or meta-analyses reporting on diclofenac’s non-traditional pharmacological effects (any formulation or derivative of diclofenac) beyond COX inhibition (e.g., neuroprotection, anticancer activity);
• reported data from In vitro assays, animal experiments, or clinical studies in contexts beyond its traditional anti-inflammatory and analgesic uses;
• provided sufficient methodological detail to allow extraction of dose, model, and outcome.

Exclusion criteria were non-original publications (descriptive, theoretical papers, letters, comments), studies focusing solely on analgesic/anti-inflammatory endpoints, and articles not in English or without access to the full text.

2.5. Data Extraction

Data extraction was conducted to synthesize information from the 89 studies included. Two reviewers extracted data independently to minimize bias, adopting the dual-extraction practice. Detailed tables summarize study methods, doses, models, effects, and references for each non-traditional property. The extracted data included

• Study characteristics: study design and experimental models (e.g., In vitro, In vivo, clinical studies);
• Intervention details: doses of diclofenac used in the studies;
• Outcomes: observed pharmacological effects (e.g., neuroprotective, anticancer) and underlying mechanisms;
• Safety data: reported adverse effects or toxicity concerns;
• References: bibliographic details for each study.

3. Results and Discussion

The analysis of information sources on diverse studies of diclofenac’s pharmacological activities identified 10 main non-traditional directions, primarily investigated at the preclinical stage. These include neuroprotective, anti-amyloidogenic, anticancer, antiviral, immunomodulatory, antibacterial, antifungal, anticonvulsant, radioprotective, and antioxidant properties (Figure 2).

3.1. Neuroprotective Properties

It is known that the mechanism of neuroprotective action is identical to anti-inflammatory and analgesic effects. It involves the inhibition of the cyclooxygenase enzyme, which reduces the synthesis of prostaglandins. Reducing the latter’s level can help reduce neuroinflammation, which plays an essential role in the development of neurodegenerative processes [12]. Diclofenac may affect other molecular pathways related to neuroprotection, such as modulation of neurotransmitter release and stabilization of cell membranes [13]. Several scientific studies provided evidence of diclofenac’s potential benefits in treating neurodegenerative diseases. In particular, a study [14] investigated the effect of diclofenac on cognitive function and daily activities in patients with Parkinson’s disease. The study showed that diclofenac can restore behavioral motor impairment and neuronal loss caused by Parkinson’s disease. These effects may be related to its potent anti-inflammatory activity [14]. In a rat model of parkinsonism (chlorpromazine-induced), diclofenac was found to significantly reduce catalepsy, improve motor activity, and alleviate histological damage in the midbrain. It was found that dopamine and DOPAC levels were higher in the diclofenac-treated group than in the control group. The authors also observed improved neuronal preservation, indicating a neuroprotective effect, likely due to anti-inflammatory effects, antioxidant activity, and activation of PPARγ receptors [14]. After two months of treatment, the diclofenac group exhibited a significant improvement in cognitive function according to the MMSE and MoCA tests and activities of daily living (ADCS-ADL). The severity of Parkinson’s disease also significantly decreased, as measured by the UPDRS III and Hoehn & Yahr scales [14].

Another study established a tendency toward slowing the progression of Alzheimer’s disease with the use of diclofenac in combination with misoprostol [10]. In a study by the authors of [15], conducted In vivo on a rat model, diclofenac was shown to inhibit the activity of the hepatic enzyme TDO and increase the levels of the brain enzyme IDO. These enzymes were involved in the metabolism of tryptophan through the kynurenine pathway. This could potentially alter the levels of neuroactive metabolites, in particular kynurenic acid and quinolinic acid, which play a role in the pathogenesis of Alzheimer’s disease. The authors suggested that diclofenac may have a neuroprotective effect by modulating kynurenine metabolism in the brain [15]. In a retrospective clinical study [16], it was determined that the incidence of Alzheimer’s disease was 6–8 times lower among patients taking diclofenac compared to those taking naproxen or etodolac. The authors of this study attributed this effect to the ability of diclofenac to inhibit interleukin-1β (IL-1β) and reduce neuroinflammation, particularly by affecting NLRP3 inflammasomes. A study by American scientists [16] showed the potential of diclofenac in the treatment of Alzheimer’s disease due to its ability to suppress neuroinflammation and reduce microglia activity. It also decreased NLRP3 inflammasome expression and proinflammatory cytokines (IL-1β, IL-6, TNF-α).

In a joint study by German and Swedish researchers [17], the neuroprotective effect of diclofenac was evaluated in a model of penetrating traumatic brain injury In vivo in male rats. The study specifically assessed its impact on apoptosis, neuronal degeneration, and the size of brain damage. The authors found that COX-2 inhibition by diclofenac after penetrating traumatic brain injury was associated with lower levels of apoptosis and minor brain tissue damage. This study indicated the neuroprotective potential of diclofenac in the treatment or prevention of secondary brain damage after trauma [17].

The main results of neuroprotective study of diclofenac are summarized in

Table 1. The main results of neuroprotective study of diclofenac.

Research Method	Diclofenac Dose	Research Model	Effect of Diclofenac	Reference
Behavioral tests. Histopathological examination of midbrain region. Neurochemical assessment (dopamine and DOPAC levels in midbrain via HPLC-EC)	20 mg/kg per day (oral suspension)	In vivo (adult Wistar rats) with chlorpromazine (CPZ)-induced Parkinson’s-like symptoms In vitro models for neuroprotective effects	Ameliorated behavioral performances, including reduced motor impairment in open field and wire hanging tests, and a significant reduction in cataleptic scores. Improved midbrain architecture with reduced gliosis in histopathology. Increased dopamine and 3,4-Dihydroxyphenylacetic acid (DOPAC) levels in the midbrain. Mediated neuroprotection against CPZ-induced Parkinson’s disease by preventing dopaminergic neuronal cell death. Acted as an antioxidant and inhibited neuronal cyclooxygenase enzymes. Activated nuclear factor peroxisome proliferator-activated receptor (PPARγ), which is neuroprotective in Parkinson’s disease. Restored interferon (IFN)-alpha induced monoamine neurotransmitter turnover and dopamine levels. Increased dopamine levels to normal and decreased toxic metabolite concentration. Protected against oxidative stress in brain tissue and reduced the severity of CPZ-induced catalepsy.	[14]
Apoptosis detectionю Neuronal degeneration detection COX-2 immunofluorescence for protein expression	5 µg (intralesional, immediately following trauma)	In vivo (male Sprague–Dawley rats) with focal penetrating traumatic brain injury (TBI)	Decreased apoptosis (TUNEL staining) in the perilesional area by 54%. Did not affect neuronal degeneration or COX-2 protein expression levels. Suggested to mitigate inflammation primarily in cells other than neurons (e.g., glia cells). May be beneficial in preventing brain tissue damage and is a potential candidate for further clinical applications.	[17]
Retrospective cohort study (analysis of patient charts) Cross-sectional study; longitudinal retrospective stud.	Average of 131.3 mg daily (for at least 1 year) Dose not reported in one cross-sectional study	Clinical studies (US veteran populations)	Significantly decreased the frequency of Alzheimer’s disease (AD) compared to etodolac and naproxen. Hazard ratio for AD was significantly lower for diclofenac compared to naproxen after controlling for site, age, and comorbidities. Odds ratio for AD decreased significantly for patients receiving NSAIDs for more than 5 years, with diclofenac showing the greatest risk reduction compared to other NSAIDs. Associated with a significant reduction in cognitive decline over time based on MMSE scores. Actively transported into the central nervous system and reaches significant cerebrospinal fluid levels. Showed lower amyloid beta (Aβ) and lower interleukin 1 beta (IL-1β).	[16]
Randomized, double-blind, placebo-controlled pilot study	50 mg daily (in combination with misoprostol 200 mg)	Clinical studies (41 patients with mild-moderate Alzheimer’s disease)	No significant group differences observed in any outcome measures, but there were nonsignificant trends for the placebo group to have deteriorated more than the diclofenac/misoprostol-treated patients. A beneficial trend in Mini-Mental State Examination (MMSE) scores was noted.	[10]
Biochemical determinations activity in liver homogenates. HPLC for L-tryptophan and kynurenine; Indoleamine 2,3-dioxygenase (IDO) activity via Kynurenine/Tryptophan ratio)	2 mg/kg (intraperitoneal) daily for acute (3.5 h) or chronic (3, 5, and 7 days) treatment	In vivo (adult albino Wistar rats)	Inhibited hepatic TDO enzyme activity following chronic treatment, leading to increased liver tryptophan concentrations. Augmented brain IDO activity following both acute and chronic treatment. Increased brain kynurenine concentrations significantly. Initially decreased brain tryptophan (acute/short-term chronic) but significantly increased it after longer chronic treatment (5 and 7 days). May modulate IFN-alpha-induced neurochemical alterations and contribute to preventing IFN-alpha-induced depression. Proposed to increase brain kynurenic acid (KYNA), which has analgesic properties and has been implicated in the pathophysiology of schizophrenia. NSAIDs, including diclofenac, have anti-amyloidogenic effects for Alzheimer’s ß-amyloid fibrils In vitro	[15]

.

The reviewed studies suggest that diclofenac’s neuroprotective properties are multifaceted, encompassing direct anti-inflammatory effects in the brain and modulation of key neurodegenerative pathways. Additionally, it provides beneficial systemic effects, such as inflammation reduction and hematopoietic recovery. Diclofenac demonstrates compelling, albeit preliminary and sometimes controversial, evidence of neuroprotective properties in animal models of Alzheimer’s disease, Parkinson’s disease, and traumatic brain injury (TBI). However, significant gaps in the research remain. There is an urgent need for large, well-controlled randomized placebo-controlled trials to confirm the effect of diclofenac in humans. These trials should preferably focus on individuals with mild cognitive impairment or early stages of Alzheimer’s/Parkinson’s disease. Ideally, the studies should be conducted before disease onset. Patient selection should be based on a combination of clinical, blood, and imaging biomarkers for a homogeneous population, with serial biomarker testing throughout the study. A standard dose with careful monitoring for side effects is recommended [10,12,13,14,15,16]. For TBI, further studies should investigate the specific apoptotic pathways and the effects of diclofenac on neurons and glia. These studies should also assess diclofenac’s potential to restore axonal functionality. Additionally, exploring specific TBI subgroups, such as males, and evaluating the feasibility of local drug administration in clinical settings are warranted [12,13,14,15,16].

3.2. Anti-Amyloidogenic Properties

A group of American scientists investigated the effect of diclofenac and its derivatives as potential transthyretin amyloid fibril formation inhibitors [18]. In 2016, Canadian researchers studied the ability of diclofenac to inhibit the aggregation of islet amyloid polypeptide, the formation of which is associated with neurodegenerative diseases such as Alzheimer’s [19]. In 2023, a group of Canadian investigators [20] examined the ability of disease-associated proteins, including β-amyloid, to aggregate in intranuclear amyloid bodies (A-bodies). The research specifically focused on these aggregation processes under stressful conditions. The authors also investigated the effect of diclofenac on inhibiting this aggregation, particularly in cellular conditions. Scientists determined that diclofenac significantly reduced the ability of β-amyloid (1–42) to form A-body aggregates in a cellular model (hypoxia + acidosis) [20]. The summarized data on the study of amyloid properties of diclofenac are presented in

Table 2. The summarized data on the study of amyloid properties of diclofenac.

Research Method	Diclofenac Dose	Research Model	Effect of Diclofenac	Reference
Transthyretin (TTR) amyloid studies:
Isothermal titration calorimetry	- Kd1: 60 nM, Kd2: 1200 nM (wild type TTR); - Kd1: 160 nM, Kd2: 3900 nM (V30M TTR); - Kd1: 380 nM, Kd2: 6180 nM (L55P TTR)	In vitro	Binds to TTR with high affinity; exhibits negative cooperativity; dissociation constants are comparable to flufenamic acid, a benchmark inhibitor	[18]
Stagnant fibril formation assay	- concentration sufficient to load one of the two TTR binding sites (e.g., 3.6 µM TTR + ~3.6 µM diclofenac) - concentration sufficient to load both TTR binding sites (e.g., 3.6 µM TTR + ~7.2 µM diclofenac)	In vitro	Reduced wild type TTR fibril formation by 65%; reduced wild type TTR fibril formation by 83%; diclofenac is highly effective as an inhibitor of amyloid formation by TTR	[18]
Plasma partitioning	10.8 µM	In vitro (human plasma)	Diclofenac itself did not partition effectively into TTR in human plasma	[18]
Islet amyloid polypeptide (IAPP) studies:
thioflavin T fluorescence assay	100 µmol/L (molar ratio 1:10 hIAPP: diclofenac, with 10 µmol/L hIAPP)	In vitro	Significantly reduced hIAPP fibrillization (fluorescence intensity)	[19]
Photo-induced cross-linking assay	1.25 mmol/L (molar ratio 1:5 hIAPP: diclofenac, with 0.25 mmol/L hIAPP)1920	In vitro	Abrogated the oligomerization of hIAPP1421; it was identified as the most potent inhibitor in this assay among those tested	[19]
β-amyloid (1–42) amyloid study: cellular amyloid body (A-bodies) targeting assay	100 µM	In vitro (MCF-7 cells)	Significantly impaired the targeting (aggregation) of β-amyloid (1–42) to A-bodies under hypoxic/acidotic conditions; impaired the targeting (aggregation) of immunoglobulin light chain to A-bodies	[20]

.

Diclofenac represents a multifaceted anti-amyloid agent [18,19,20]. Its direct protein-stabilizing role in TTR amyloidosis contrasts with a potentially indirect, cellular pathway-dependent mechanism for β-amyloid aggregation. The most critical insight is the stark contradiction between diclofenac’s performance in In vitro Thioflavin T assays versus cellular A-body assays for β-amyloid aggregation [20]. This emphasizes that In vitro assays may not fully capture the complex biological environment and indirect mechanisms relevant to disease [20]. Diclofenac directly binds to TTR, but its effect on β-amyloid appears to be indirect, mediated through modulation of cellular pathways such as the COX pathway. This suggests that diclofenac may not act as a “universal amyloid inhibitor” by directly targeting fibril structure across all proteins. Instead, it likely influences upstream cellular processes that affect amyloid formation or sequestration. Diclofenac shows promise for TTR and IAPP amyloidosis [18,19]. However, issues like plasma partitioning selectivity (for TTR) and potential toxicity (for NSAIDs in general) need to be carefully considered for drug development. For Alzheimer’s disease, the cellular A-body model supports an epidemiological link to reduced risk with diclofenac, but In vitro assays of fibril inhibition do not confirm this association. This implies that its protective effects might stem from modulating inflammatory pathways or other cellular processes [18,19].

3.3. Anticancer Properties

Research on diclofenac as a potential anticancer agent encompasses several complementary approaches. In silico methods are extensively employed to elucidate its molecular mechanisms of action [21]. A broad spectrum of In vitro experiments was conducted to demonstrate its anticancer activity in various cancer cell lines. Numerous In vivo studies using animal models provided additional evidence supporting its anticancer potential. The discussion of diclofenac as a potential anticancer agent is presented in works [8,22,23]. Its efficacy was demonstrated in various types of tumors, including fibrosarcoma, colorectal cancer, neuroblastoma, glioblastoma, melanoma, pancreatic cancer, and more.

As shown in

Table 3. The results of studies on the antitumor properties of diclofenac on some types of cancer.

Type of Cancer	Key effects of Diclofenac	Mechanisms of Action	Type of Research	References
Human glioma	Restricts migration and proliferation	Inhibits the β-catenin/tcf signaling pathway	In vitro	[22,27,28,29]
Mouse glioma	Inhibits the formation of lactate and counteracts local immune suppression, reduces the accumulation and activation of Tregs in the tumor	Effect on lactate metabolism and the immune microenvironment of the tumor	In vivo	[29]
Colorectal cancer	Inhibits tumorigenesis induced by 1,2-dimethylhydrazine	Inhibiting MCP-1, MIP-1α and VEGF	In vivo	[30]
Human neuroblastoma	Induces apoptosis	Affects the mitochondrial superoxide dismutase	In vitro	[31]
Lung cancer	Enhances apoptosis induced by docosahexaenoic acid	The combination of docosahexaenoic acid and Diclofenac increases their anticancer activity by changing the expression of critical proteins in the RAS/MEK/ERK and PI3K/Akt pathways	In vitro	[32]
Cervical cancer	Cytotoxic effect on the proliferation of cancer cells	Inhibiting caspase-8 and caspase-9	In vitro	[33,34]
Breast, lung and kidney cancer	Prognostic factors in different cohorts of patients who underwent surgery	Relationship with diclofenac use and neutrophil-lymphocyte ratio	Clinical trial	[35]
Breast cancer	Affects the proliferation of cancer cells, decreased expression of GLUT1 and c-Myc	Inhibiting cellular glycolysis and suppression of cancer cell growth by decreasing GLUT1 protein expression and HK activity through the c-Myc pathway	In vitro	[36]
Melanoma, leukemia, and carcinoma	Significantly reduces the expression of glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) genes following a decrease in glucose uptake and lactate secretion	Inhibiting MYC and lactate transport	In vitro	[37]
Melanoma	Releases intracellular reactive oxygen species (ROS), leading to a decrease in transmembrane potential, promoting mitochondrial apoptosis, and activating ROS through the p38/p53 signaling pathway	Sensitizing BRAF-resistant melanoma cells to BRAF, increasing the release of ROS, and activating the p38/p53 signaling pathway	In vitro	[38]
Prostate cancer	Induces apoptosis and Epithelial–Mesenchymal transition in cancer cells, affects the oxidative stress independently of p53	Inducing the process of Epithelial–Mesenchymal transition through increased generation of ROS independent of p53	In vitro	[39]
	Increases radio sensitivity (when applied topically)	Enhancing TRAIL, inhibition of COX-2 expression	In vitro In vivo	[40]
Ovarian cancer	Reduces cancer cell growth, induces cell cycle arrest and apoptosis	Inhibiting E2F1 regulation	In vitro In vivo	[41]

, diclofenac may exert its effects through a variety of mechanisms. The primary mechanisms of diclofenac’s action are diverse. One of the key mechanisms involves the inhibition of cyclooxygenase-2 (COX-2) and the reduction in prostaglandin E2 (PGE2) synthesis. This contributes to decreased angiogenesis and immunosuppression. Additionally, diclofenac suppresses angiogenesis, induces apoptosis, and causes cell cycle arrest. It also destabilizes microtubules and disrupts autophagy. Furthermore, it exerts significant effects on tumor metabolism [8,23,24]. In addition, diclofenac has been found to act on DNA (apurinic/apyrimidinic) lyase, ADAM10, and tyrosine-protein kinase, which are involved in the development of various types of cancer [8,23,24]. Diclofenac also found its place in studies of combinations with other drugs for the treatment of osteosarcoma, breast cancer, ovarian cancer, gastrointestinal cancer, and non-small cell lung cancer [25]. In 2024, a group of researchers [26] conducted a promising study on the production of diclofenac-based prodrugs. They studied their anticancer effect on mouse colon adenocarcinoma, human colorectal carcinoma, and human colon adenocarcinoma.

The results of studies on the anticancer properties of diclofenac on some types of cancer are presented in Table 3.

Although the evidence of diclofenac’s antitumor activity is promising, particularly at the preclinical level, the vast majority of these studies remain at the preclinical stage. This represents the primary limitation in the current assessment of its full antitumor potential. There is clear evidence of diclofenac’s ability to influence key cancer processes, such as proliferation, apoptosis, angiogenesis, and immune response [22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42]. Researchers are making significant efforts to reduce the toxicity of diclofenac and enhance its efficacy through chemical modification [24] and prodrugs development [26]. Overcoming challenges related to its side effects and bioavailability through novel structural solutions and combination therapies is critical for further development.

3.4. Antiviral Properties

Reports on the repositioning of diclofenac as a promising antiviral agent are relatively limited in the literature. The available studies of antiviral properties are represented by laboratory (In vitro, In vivo) or computational (in silico) results. Diclofenac was first studied in 1998 by Y. Gordon and co-authors [43] In vitro and In vivo for its effect on adenovirus replication, but no significant effect was found.

In 2001, Y. Gordon and E. Romanowski studied the effect of diclofenac on the inhibitory activity of cidofovir on adenovirus replication in the Ad5/NZW rabbit model [44]. The authors found that the simultaneous use of diclofenac with the antiviral drug did not reduce its antiviral inhibitory activity against adenovirus replication. Additionally, it did not prevent the formation of subepithelial infiltrates in the In vivo model [44].

A study on rabbit eyes [45] with acute herpetic keratitis (herpes simplex viruse HSV-1) found that topical 0.1% diclofenac sodium did not exacerbate the disease. Lesions were less severe or no more severe than those treated with other anti-inflammatory agents. Crucially, virus shedding was not prolonged.

In 2011, Colombian researchers tested diclofenac for its effect on ECwt (wild-type) rotavirus in ICR mice [46]. The study showed that treatment of mice infected with this virus with diclofenac led to a decrease in cell infection. In 2017, Turkish scientists proposed a synthetic approach to structurally modify diclofenac, resulting in its hydrazone and spirothiazolidinone derivatives [47]. These derivatives demonstrated good antiviral activity against herpes simplex viruses (HSV-1, HSV-2, and HSV-1 TK-), vaccinia virus, and Coxsackie B4 virus.

The main results of antiviral study of diclofenac are summarized in

Table 4. The main results of antiviral study of diclofenac.

Virus Type	Diclofenac Dose	Research Model	Effect of Diclofenac	Reference
Adenovirus	3.5 to >10.0 µg/mL	In vitro	Showed weak/minimal direct antiviral inhibitory effect against various adenoviral serotypes, with IC50 values ranging from 2.8 to 86 µg/mL.	[43]
	0.1% topical	In vivo (rabbit ocular model, infected with adenovirus type 5 Ad5)	No significant inhibitory effect on adenoviral replication, no prolongation of viral shedding, and no significant effect on subepithelial immune infiltrate formation. Did not diminish Cidofovir’s antiviral inhibitory activity on adenovirus replication and did not prevent subepithelial infiltrate formation.	[43,44]
Herpesvirus	0.1% topical	In vivo (rabbit eyes with acute herpes simplex virus type 1 (HSV-1) keratitis)	Inhibited herpesvirus activity, reducing lesion size, lesion number, and viral titer. Did not exacerbate acute herpes keratitis or prolong virus shedding.	[45]
Rotavirus	1 mg/kg/day (oral)	In vivo (ICR suckling mice infected with rotavirus ECwt)	Reduced rotavirus ECwt infectivity by about 35%. Reduced cells positive for rotavirus structural proteins. Reduced Hsc70 and PDI expression to levels similar to uninfected mice. Demonstrated inhibitory capacity on ECwt infection.	[46]

.

Analysis of works [43,44,45,46,47] revealed that diclofenac’s antiviral research journey reflects both challenges and opportunities. Diclofenac is not consistently a direct and potent antiviral agent. However, research highlights its significant role as a safe and effective anti-inflammatory adjunct therapy in various viral infections. Additionally, it serves as an effective immunomodulatory therapy in these contexts. Its ability to reduce inflammation and manage severe immune responses without exacerbating viral replication or interfering with primary antiviral treatments makes it a valuable tool. Diclofenac, in its current form, may not serve as a stand-alone antiviral solution. However, its incorporation into combination treatment regimens [44] shows promise for developing adjunctive therapies. Additionally, further structural optimization [47] could enhance its potential. These therapies aim to address both viral replication and the damaging host’s inflammatory response.

3.5. Immunomodulatory Properties

Diclofenac, in addition to its main anti-inflammatory properties, may exert immunomodulatory effects by affecting various immune system components. These properties are indirect and associated with its effect on inflammatory cascades and metabolic pathways. In particular, the review [48] highlighted In vitro and In vivo studies on diclofenac, demonstrating its ability to inhibit the activation of the NLRP3 inflammasome. Additionally, it showed diclofenac’s immunomodulatory activity through the suppression of PPAR-γ expression. This mechanism may contribute to a reduction in the production of proinflammatory cytokines in COVID-19 disease. The study [49] found that diclofenac and its metabolite, 4-hydroxydiclofenac, modulate the production of proinflammatory cytokines in lymphoblastoid cell lines, indicating immunomodulatory effects. Villalonga et al. [50] found that diclofenac may inhibit the activity of Kv1.3 potassium channels, which play a key role in the activation and proliferation of T-lymphocytes and macrophages. Blocking these channels can reduce the immune response, which is potentially valuable for autoimmune diseases.

Diclofenac also inhibited the differentiation of monocytes into mature dendritic cells under the influence of 27-hydroxycholesterol. This may reduce T-cell activation and modulate the adaptive immune response [51]. In addition, it is considered a potential treatment for autoimmune diseases and in combination with other therapies, such as metronomic chemotherapy, to enhance the antitumor response [25].

Thus, diclofenac has a complex effect on the immune system. On the one hand, it may inhibit certain aspects of the immune response, such as macrophage activation and migration and IL-2 production in T lymphocytes. It may be helpful in the treatment of autoimmune diseases, including rheumatoid arthritis [50]. On the other hand, in different experimental conditions, it can modulate cytokine production, particularly by increasing IL-2 levels [49]. The effect of diclofenac on Kv1.3 channels in leukocytes is an essential mechanism of its immunomodulatory effect [49,50].

The main results of immunomodulatory study of diclofenac are summarized in

Table 5. The main results of immunomodulatory study of diclofenac.

Research Method	Diclofenac Dose	Research Model	Effect of Diclofenac	Reference
Whole-cell patch-clamp, RT-PCR, migration assays, IL-2 ELISA	1.5 mM, 15 mM	In vitro (Raw 264.7 macrophages, Jurkat T-lymphocytes, murine bone marrow-derived macrophages)	Impaired immune response via Kv1.3 by inhibiting its expression in activated macrophages and T-lymphocytes. Decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to LPS221. Blocked LPS-induced macrophage migration. Blocked IL-2 production in stimulated Jurkat T-cells. Reduced voltage-dependent K+ currents in LPS-activated macrophages (at 15 mM).	[50]
Luciferase reporter gene assays, XENoscreen YES/YAS assay (ER, AR), polarscreen GR/AR competitor assay, cytokine assay	various EC50/IC50 concentrations (e.g., 4.6 × 10−8 M for GR agonist, 1.9 × 10−8- M for AR antagonist)	In vitro (Human lymphoblastoid cell lines, MDA-kb2, GH3.TRE-Luc cell lines)	Showed direct binding to the Glucocorticoid Receptor (GR)437. Significantly modulated proinflammatory (TNFα, IL-6, IL-2) and immunoregulatory (IL-10) cytokine production in lymphoblastoid cell lines. Decreased TNFα, IL-6, and IL-2 at low glucocorticoid effect concentration.	[49]
Morphological assessment, endocytosis assay, real-time PCR, flow cytometric analysis	5 mg/mL, 10 mg/mL, 25 mg/m	In vitro (Human THP-1 monocytic cells)	Inhibited 27-hydroxycholesterol (27OHChol)-induced differentiation of monocytic cells into mature dendritic cells (mDCs). Attenuated dendrite formation and cell attachment. Promoted endocytic function (restored impaired activity). Inhibited transcription and surface expression of mDC markers (CD80, CD83, CD88)1243. Reduced 27OHChol-induced elevation of surface levels of MHC class I and II molecules. Reduced expression of CD197. Can negatively regulate the activation of T and B lymphocytes.	[51]

.

The immunomodulatory effects of diclofenac are often dose-dependent, with higher concentrations (e.g., 15 mM) demonstrating significant effects. In contrast, lower concentrations (e.g., 1.5 mM) do not exhibit these effects [50]. The effects also vary based on the cell type and its activation state [50]. Its ability to inhibit macrophage activation and migration underscores its potential to attenuate overactive immune responses relevant in autoimmune diseases [50]. Additionally, it suppresses T-lymphocyte IL-2 production, further contributing to immune modulation [50]. Furthermore, its interference with dendritic cell differentiation supports this therapeutic potential in autoimmune conditions [50]. Diclofenac role in acute infectious diseases, especially viral ones like SARS-CoV-2, presents a more nuanced picture. While concerns about potential adverse effects were raised, current evidence generally suggests that diclofenac does not worsen outcomes in COVID-19 [48]. Future research should focus on optimizing its use in combination therapies. It is also important to explore its impact on host metabolic responses during infections. These efforts will help fully leverage its therapeutic potential while mitigating potential risks.

3.6. Antibacterial Properties

Diclofenac demonstrates a wide range of antibacterial activity against both Gram-positive and Gram-negative bacteria [9,52]. There are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Bacillus pumilus, Bacillus subtilis, Listeria monocytogenes, Mycobacterium tuberculosis, Stenotrophomonas maltophilia, Acinetobacter baumannii, Aeromonas trota, Streptococcus suis, Paenibacillus lactis, Pasteurella canis, Salmonella typhimurium, Salmonella virchow and Staphylococcus equorum.

The mechanisms of antibacterial action of diclofenac could be manifested through inhibition of DNA synthesis [9], disruption of membrane activity and inhibition of enzymes [53], effect on biofilm formation [54,55], synergy with antibiotics [9,53,56,57,58].

A study by Salem Milani et al. showed that diclofenac has a pronounced antibacterial activity against E. faecalis, but less than the antibacterial activity of conventional antibiotics (amoxicillin, gentamicin, etc.) [59]. In combination with antibiotics, diclofenac reduced the inflammatory response and improve the patient’s condition, for example, in treating urinary system diseases [56,58]. Diclofenac also inhibited some mechanisms of bacterial resistance [60]. Studies in mice with infections caused by methicillin-resistant S. aureus (MRSA) showed that combined treatment with diclofenac and oxacillin significantly reduces biofilm formation and bacterial load [54].

Studies [53,55,61] explored the potential use of diclofenac as an individual antibacterial agent, as it inhibits the growth of bacteria of different strains, such as B. subtilis, E. coli, P. aeruginosa, and S. typhi.

The main results of antibacterial study of diclofenac are summarized in

Table 6. The main results of antibacterial study of diclofenac.

Bacterial Strain	Diclofenac Concentration	Model Study	Diclofenac Effect	Reference
Escherichia coli	5–50 µg/mL	Clinical	Antibacterial effect; effective in treating urinary tract infections	[58]
	25–100 µg/mL	In vitro (agar diffusion)	Showed 24–25 mm zone of inhibition	[53]
	50 µg/mL	In vitro (agar dilution)	Inhibited growth	[9]
	0.25–2500 µg/mL	In vitro (agar dilution)	Inhibitory activity	[52]
Escherichia coli O157 H7	25,600 µg/mL	In vitro	Began to reduce bacterial counting; killing bacteria after 40 h. Destroyed formed biological membranes; caused deformities, core holes, cell surface damage, spurting of cellular contents, loss of form, central hollowing, and leaking of cytoplasmic contents.
	400 µg/mL	In vitro (with ciprofloxacin 0.48 µg/mL)	Significant antibacterial activity, decreased ciprofloxacin MIC (from 0.96 µg/mL alone).	[58]
	1 mg/kg	In vivo (Rabbits)	No significant difference in pus cell concentration after 14 days compared to positive control. Increased pus and epithelium cell concentration after 3 days.
	1 mg/kg (in combination with ciprofloxacin 3.5 or 1.75 mg/kg)	In vivo (Rabbits)	Decreased pus and epithelium cell concentration after 7 and 14 days
Klebsiella pneumoniae	2–1024 µg/mL	In vitro (agar dilution)	Inhibitory activity	[9,52]
	≥160 µg/mL (in combination with doxycycline)	In vitro	Synergy with doxycycline (MIC from 8 µg/mL to 1 µg/mL)
Pseudomonas aeruginosa	25–3125 µg/mL	In vitro	Inhibitory activity	[52,53]
Salmonella typhi	25–2500 µg/mL	In vitro	Inhibitory activity	[52,53]
	1.5 and 3.0 µg/g body weight	In vivo (mice)	Significantly protected animals from death after experimental infection	[52]
Staphylococcus aureus	5–100 µg/mL	In vitro (MIC range)	Inhibitory activity	[53]
	125 µg/mL (MIC for MRSA, MSSA, MRSE)	In vitro	Inhibited growth; bacterial viability significantly declined. Prevented biofilm formation.
	80 mg/kg (in combination with Oxacillin 200 mg/kg)	In vivo (Murine skin/soft tissue infection model)	Significantly restricted dermonecrosis area and abscess volume, resulted in fewer MRSA micro abscesses
Staphylococcus epidermidis	0.4 mM	In vitro	Significantly inhibited biofilm formation in MSSE and MRSE isolates; reduced metabolic activity of biofilms. Decreased initial bacterial adhesion. Downregulated biofilm-associated genes and upregulated negative regulatory genes.	[54]
Enterococcus faecalis	50 µg/mL and above	In vitro (agar diffusion test, dilution method)	Inhibitory activity	[52,53,59]
Bacillus subtilis	0.315–100 µg/mL	In vitro (agar diffusion test, dilution method)	Inhibitory activity	[52,53]
Listeria monocytogenes	25–100 µg/mL	In vitro (agar diffusion test, dilution method)	Inhibitory activity	[52,53]
	50 µg/mL in combination with Gentamicin)	In vitro	Synergistic effect	[9]
	2.5 mg/kg/day	In vivo (Murine)	Significantly reduced bacterial counts in liver and spleen; decreased hepatic colonization and necrosis; upregulated inflammatory cytokines	[52,53]
Mycobacterium tuberculosis	10–40 µg/mL	In vitro	Remarkable inhibitory action against drug-sensitive and drug-resistant clinical isolates.	[9,53]
	10 mg/kg body weight/day	In vivo (Murine)	Significantly lowered bacterial counts and reduced mean spleen weight	[9]
	2.5 µg/mL (in combination with Streptomycin)	In vitro	Synergistic effect, lowered streptomycin MIC from 2 µg/mL to 0.25 µg/mL	[9,52]
Acinetobacter baumannii	100 µM (approximately 31.8 µg/mL) (in combination with colistin)	In vitro	Alone did not have an effect on growth. In combination with Colistin sensitizes colistin-resistant strains to colistin. Enhanced bactericidal activity of colistin, leading to a decrease in MIC values (≥4-fold decrease in colistin-resistant strains).	[53,56,57,58]
	1.25 mg/kg (in combination with Colistin 2.5 mg/kg)	In vivo (Murine pneumonia model	Produced a significant reduction in bacterial burden in lungs, kidneys, and spleens in a murine pneumonia model.

.

The existing research strongly suggests that diclofenac holds significant promise as an agent with notable antibacterial and anti-biofilm properties. This potential is particularly evident when diclofenac is used in synergistic combination with conventional antibiotics [9,52,53,54,55,56,57,58,59,60,61]. This could be a valuable strategy for combating multi-drug resistant pathogens and managing biofilm-associated infections. A major concern is that the effective in vitro concentrations of diclofenac can be significantly higher (25,600 µg/mL) than clinically achievable human plasma concentrations (0.5–2.5 µg/mL). However, for its clinical application as an antibacterial agent, rigorous in vivo and clinical trials are essential to determine optimal, safe, and effective dosing strategies. This is especially important considering its known toxicological profile at high concentrations.

3.7. Antifungal Properties

Research on the antifungal activity of diclofenac is represented by a relatively limited number of scientific studies. However, based on the available evidence, diclofenac demonstrates considerable antifungal activity in vitro and in vivo against pathogenic fungi, particularly Aspergillus fumigatus and various Candida species, including C. albicans and C. tropicalis. The antifungal effect is provided by inhibition of growth, hyphal formation, biofilm formation, and the potential for synergy with other antifungal drugs. In particular, a study [62] reported that diclofenac sodium significantly reduced both the growth of A. fumigatus mycelium growth and Ef-1 gene expression in a dose-dependent manner. Studies [63,64,65] showed that diclofenac inhibits the growth and morphogenetic processes of C. albicans, particularly the formation of hyphae and biofilms. A significant inhibitory effects of diclofenac on the adhesion and formation of C. albicans biofilms [63,66,67] and an increase in the permeability of biofilm cell membranes [64,68,69] were established.

Diclofenac in combination with essential oils, such as Mentha piperita, Melaleuca alternifolia, and Pelargonium graveolens, had a synergistic effect, significantly reducing the formation of biofilms of various Candida strains [70].

Another study found that diclofenac increases the sensitivity of C. albicans biofilms to caspofungin, which is promising in the combination therapy of candidiasis [68]. Diclofenac also exhibited synergistic effects with fluconazole and voriconazole against biofilms of resistant strains of C. tropicalis [66,71] and fluconazole against C. albicans strains.

The main results of antifungal study of diclofenac are summarized in

Table 7. The main results of antifungal study of diclofenac.

Fungal Strain	Diclofenac Concentration	Model Study	Diclofenac Effect	Reference
Aspergillus fumigatus	500, 700, 900 µg/mL	In vitro	Significant inhibitory effect on A. fumigatus growth at 500 µg/mL and higher. By increasing concentration, mycelium production decreased and normal shape deviation was observed3. Decreased EF-1 gene expression in a dose-dependent manner (27.7 in untreated vs. 0.672 with 900 µg/mL)	[62]
Candida albicans	50, 100, 200, 500 µg/mL	In vitro	Inhibited filamentation in C. albicans in a dose-dependent manner. Preincubation with 500 µg/mL completely inhibited hypha formation. Decreased expression levels of ALS3, RAS1, EFG1 mRNA (regulated by cAMP-EFG1 pathway) and hypha-specific genes (ALS1, ECE1, HWP1).	[63]
Candida albicans, Candida glabrata, Candida tropicalis, Candida kefyr, Candida krusei, Candida parapsilosis	1.02 to 2.05 µg/mL	In vitro	Inhibitory activity ranged from 1.02 to 2.05 µg/mL against various Candida strains	[70]
Candida albicans, Candida glabrata, Candida krusei	52.6 to 1000.5 µg/mL; 0.5×MIC, 2×MIC.	In vitro	Showed lower MIC against C. albicans (52.6 µg/mL for ATCC 10231; 166.5 µg/mL for clinical isolate) and C. glabrata (147.6 µg/mL). Inhibited dimorphic transition of C. albicans at 500 µg/mL. Showed highest inhibitory effect on adherence of C. albicans and C. glabrata. Showed highest disruptive effect on mature biofilms formed by all tested Candida spp.	[65]
Candida tropicalis  (clinical strains)	256–2048 µg/mL	In vitro	Showed MIC of 1024 µg/mL against all C. tropicalis planktonic isolates. At 2048 µg/mL, decreased C. tropicalis biofilm formation by 50%. Suggested to inhibit hyphae formation and cause alterations in cell membrane permeability. May lead to diminished biofilm forming ability possibly.	[66]
Candida albicans (clinical strains)	31.8 µg/mL	In vitro	Diclofenac reduced filamentation and registred an important inhibition effect on C. albicans cells	[67]
	up to 2 mM (636.3 µg/mL) for planktonic cells; 6 mM (1908.8 µg/mL) for pretreated biofilms; 10 mM (3181.3 µg/mL) for biofilms); 0.25–4 mM (79.5–1272.5 µg/mL) for membrane permeability.	In vitro	No inhibitory activity up to 2 mM against planktonic cells. Inhibited yeast-to-hyphae transition at 2 mM. Showed a moderate antibiofilm effect 6 mM when pretreated, 10 mM against mature biofilms. involvement in fungal prostaglandin biosynthesis.	[68]
	3mg/kg/day	In vivo (rat model)	Did not affect biofilm formation when used alone. Significantly increased membrane permeability of C. albicans biofilm cells.	[68]

.

Therefore, diclofenac exhibits significant antifungal activity, particularly through its ability to inhibit fungal morphogenesis (hyphae/germ tube formation) and reduce biofilm production. A critical observation is the wide range of effective diclofenac concentrations reported across studies and even within a single study depending on the model [62,63,64,65,66,67,68,69,70,71]. While the In vitro results are encouraging, the number of In vivo studies is limited. Some In vivo animal model data showed synergistic activity (e.g., with caspofungin against C. albicans biofilms) [68]. Crucially, diclofenac demonstrates strong synergistic effects when combined with conventional antifungals and essential oils, which is a promising strategy to overcome antifungal resistance and manage biofilm-associated infections by allowing for lower, potentially less toxic, effective concentrations [66,68,71]. The precise molecular mechanisms by which diclofenac exerts its antifungal and synergistic effects, especially in complex combination therapies, require deeper investigation.

3.8. Anticonvulsant and Proconvulsant Properties

Diclofenac is also the subject of some In vitro studies investigating its potential anticonvulsant effect, which is associated with its influence on inflammatory and neural mechanisms. The first studies in this area were conducted in the early 1980s. In particular, an In vivo study in mice conducted in 1981 [72] showed that diclofenac reduces the seizure threshold. The authors found that mice administered with the convulsant pentylenetetrazol exhibited a significant increase in immunoreactive material resembling prostaglandins (PGs) and thromboxane B2 (TXB2) in brain extracts. Diclofenac was shown to inhibit the pentetrazole-induced elevation in immunoreactive PGF2α, PGE2, and TXB2 by more than 90%.

Studies in 2005 and 2018 found that diclofenac may act as a KCNQ2/Q3 potassium channel opener [73,74,75]. In particular, In vivo, the combined administration of retigabine (a KCNQ potassium channel activator) and diclofenac significantly increased the percentage of protection against seizures induced by picrotoxin and maximum electroshock. They prolonged the average latency period of seizures in pilocarpine-induced persistent epilepsy [73]. The efficacy of diclofenac in the maximum electroshock test was blocked by a KV-channel blocker, which confirmed its possible action as a KCNQ2/3 potassium channel opener.

In two studies conducted in 2016 and 2022 [76,77] on rat models of seizures induced by pentylenetetrazole, administration of diclofenac sodium led to a significant reduction in seizure severity according to the Racine seizure scale. This effect was accompanied by a decrease in the levels of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the hippocampus [76]. There was also a significant decrease in MDA and TNF-α levels and an increase in SOD levels, suggesting a possible role for the antioxidant and anti-inflammatory effects of diclofenac in reducing seizure activity [77].

A study in 2023 [75] reported that the combined administration of diclofenac potassium and diazepam was more effective in reducing the amplitude of spikes than diclofenac potassium alone. This study also noted that the effects of diclofenac potassium on epileptic seizures may vary as either proconvulsant or anticonvulsant. These effects depended on the form of diclofenac, dose, type of experimental model, and study protocol.

The main results of antiseizure study of diclofenac are summarized in

Table 8. The main results of antiseizure study of diclofenac.

Seizure Model	Diclofenac Dose	Study Model	Diclofenac Effect	Reference
Pentylenetetrazole (PTZ)-induced seizures	10 mg/kg	In vivo (mice)	Accelerated the onset of tonic seizures. Inhibited the convulsion-induced increase in prostaglandins (PGF2α, PGE2) and thromboxane B2 (TXB2) in mouse brain	[72]
	25 mg/kg, 50 mg/kg, 75 mg/kg	In vivo (Sprague–Dawley rats)	Dose-dependently lowered spike percentages on electroencephalography, reduced racine convulsion scale scores, and prolonged the duration of the first myoclonic jerk. Significantly decreased MDA (lipid peroxidation marker), TNF-α, IL-1β, and PGE2 levels in brain tissue. Increased superoxide dismutase levels in brain tissue.	[77]
PTZ-induced kindling model	5 mg/kg, 10 mg/kg	In vivo (Wistar rats)	Decreased the severity of seizures. Reduced hippocampal TNF-α levels (both doses) and IL-6 levels (at 5 mg/kg). No significant difference in seizure intensity compared to diazepam (positive control). Increased serum TNF-α levels at 10 mg/kg, which was an unexpected finding discussed as a potential compensatory mechanism.	[76]
Picrotoxin (PTX)-induced convulsions	5 mg/kg, 10 mg/kg, 20 mg/kg	In vivo (albino mice)	Reduced mortality rate (from 37.5% to 25%). It had no effect on reducing episodes compared to diazepam in this model alone.	[73]
	10 mg/kg (in combination with diazepam)	In vivo (albino mice)	Potentiated the anticonvulsant effect of diazepam (significantly prolonged onset time, decreased episode number, and complete inhibition of death).	[73]
	10 mg/kg (in combination with retigabine)	In vivo (albino mice)	Significantly increased percentage of protection (100% protection) and completely inhibited death.	[73]
Pilocarpine-induced sustained epilepsy	5 mg/kg, 10 mg/kg, 20 mg/kg	In vivo (albino mice)	Non-significant increase in mean latency period. Non-significant change in death rate.	[73]
	10 mg/kg (in combination with retigabine)	In vivo (albino mice)	Significantly increased the mean latency period and reduced the frequency of convulsion to 50%.	[73]
Maximal electroshock seizure (MES) test	25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg	In vivo (ICR mice)	Dose-dependently suppressed the tonic extension (ED50 of 43 mg/kg).	[74]
Maximal electroshock seizure threshold (MEST)	10 mg/kg	In vivo (albino mice)	Efficacy was blocked in a dose-dependent manner by the Kv channel blocker 4-aminopyridine, supporting its KCNQ channel opening mechanism.	[73]
	10 mg/kg (in combination with retigabine)	In vivo (albino mice)	Produced complete protection from convulsions (100% protection). This potentiation is due to diclofenac acting as a KCNQ2/3 channel opener, similar to retigabine.	[73]
Rotenone corneal kindling model of drug-resistant epilepsy (DRE)	5 mg/kg, 10 mg/kg, 20 mg/kg (as adjunctive therapy)	In vivo (albino mice)	Significantly decreased drug resistance to antiseizure medications, restoring their antiseizure potential and reducing seizure se-verity (dose-dependent effect, 20 mg/kg most significant). Restored serotonin (5-HT) to kynurenine (Kyn) ratio. Restored kynurenic acid (KYNA) levels. Reduced quinolinic acid levels.	[73]
Penicillin- induced epileptiform	10 mg/kg (diclofenac potassium)	In vivo (Wistar rats)	Did not show an anti-inflammatory effect in serum. Not more effective than diazepam in reducing spike fre-quency or amplitude.	[75]
	10 mg/kg (diclofenac potassium in combination with diazepam)	In vivo (Wistar rats)	More effective in reducing spike amplitude compared to diclofenac potassium alone and more effective as an anticonvulsant than diazepam alone.	[75]

.

Therefore, the initial interpretation of diclofenac lowering the convulsive threshold contrasts with later studies showing broader anticonvulsant effects. This may reflect a developing understanding of the complex interplay between prostaglandins and seizure activity. Additionally, the discovery of mechanisms such as KCNQ channel opening and KMO inhibition directly contributes to the anticonvulsant effects [73]. Diclofenac’s effects are highly dependent on the dose administered and the experimental model used. For instance, its effectiveness in MES and PTZ models appears stronger than in the pilocarpine model when used alone [72,73,74,75,76,77]. While early research presented some proconvulsant indications in specific acute contexts, the bulk of the evidence points to diclofenac having significant anticonvulsant properties. This is particularly supported by more recent studies and findings from combination therapies. Its multifaceted actions, including COX and kynurenine 3-monooxygenase inhibition, make it a promising candidate. Additionally, its KCNQ potassium channel opening and general anti-inflammatory/anti-oxidative effects further enhance its therapeutic potential. Diclofenac appears particularly effective as an adjunctive therapy, enhancing the effects of established anticonvulsants like diazepam and retigabine. Importantly, it also combats drug resistance in epilepsy models. Further investigations, especially in chronic epilepsy models and clinical trials, are warranted to fully establish its therapeutic role in epilepsy management.

3.9. Radioprotective Properties

In vitro and In vivo studies of diclofenac as a potential radioprotective agent began in the 1980s, and their results indicated the possibility of protecting normal tissues from radiation damage. In particular, in 1985, the effect of radiation exposure on the inflammatory process and the effect of diclofenac on it were studied. It was found that it effectively suppressed inflammatory reactions in both normal and irradiated animals. This effect may be due to its inhibitory action on prostaglandin synthetase and stabilization of lysosomal membranes [78]. In 1992 and 1993 [79,80], it was determined that the combination of diclofenac and carboxymethyl glucan (an immunomodulator and hematopoietic stimulant) enhanced the recovery of hematopoiesis in gamma-irradiated mice.

In another study [81], repeated administration of diclofenac to mice before each irradiation resulted in a statistically significant increase in leukopoiesis compared to the control group. However, after a lethal total radiation dose accumulated, a slight decrease in survival rate was observed in diclofenac-treated mice [81].

Studies on cultured human peripheral blood lymphocytes showed that both pre- and post-irradiation with diclofenac sodium reduced the formation of gamma radiation-induced dicentric chromosomes, cytochalasin-blocked micronuclei, and γ-H2AX foci [82].

In vitro and in vivo studies were conducted to investigate the effects of diclofenac on cancer cells’ sensitivity to radiation and chemotherapy [83]. They demonstrated radio- and chemosensitizing effects on some cancer cells [83], particularly on colorectal adenocarcinoma cell lines (LS174T and LoVo). The combined use of diclofenac and radiation significantly increased the production of reactive oxygen species (ROS) in LS174T cells but not in A549 lung cancer cells. In contrast to colorectal cancer cells, the radiosensitizing effect of diclofenac was not observed in lung cancer (A549), breast cancer (MDA-MB-231), and pancreatic cancer (COLO357) cells, where diclofenac did not induce changes in lactate metabolism and stress response. A study in the LS174T colorectal adenocarcinoma mouse model showed that diclofenac also enhances the effectiveness of radiotherapy, leading to a reduction in tumor size, especially when used diclofenac and radiation treatment [83].

Possible mechanisms of diclofenac’s radioprotective properties may be realized by reducing prostaglandin levels [81] and decreasing radiation-induced inflammatory reactions [79]. It may also act by reducing free radicals [83]. Additionally, when combined with radiation, diclofenac can increase ROS production, affect cancer cell metabolism, and modulate the stress response [83].

The main results of radioprotective study of diclofenac are summarized in

Table 9. The main results of radioprotective activity study of diclofenac.

Research Method	Diclofenac Dose	Radiation Dose	Type of Study	Effect on Radiation Response	Result	Reference
Cytogenetic study (dicentric chromosome, micronucleus, γ-H2AX assays)	10 µM, 100 µM, 1 mM, 5 mM	2 Gy, 5 Gy	In vitro (human peripheral blood lymphocytes)	Radioprotection/radiomitigation	Reduced radiation-induced chromosomal aberrations (dicentrics, micronuclei) and DNA damage foci (γ-H2AX) both pre- and post-irradiation.	[82]
Carrageenan-induced paw oedema and adjuvant-induced arthritis tests	1–5 mg/kg; 0.3 mg/kg	0.5, 1, 2 Gy (whole-body gamma)	In vivo (rats)	Radioprotection	Reduced exaggerated inflammatory response in irradiated animals. Showed a prophylactic value against radiation damage-induced inflammation.	[78]
Hematopoietic recovery (GM-CFC, bone marrow cellularity, spleen weight, peripheral blood counts, CFU-S) and Survival studies	0.6 mg per mouse	Fractionated: 5 × 2 Gy, 3 × 3 Gy, 4 × 3 Gy, 5 × 3 Gy (sublethal); 5 × 3 Gy + 3.5 Gy (lethal “top-up”)	In vivo (mice)	Radioprotection	Significantly enhanced hematopoietic recovery (endogenous spleen colony formation, leukopoiesis) at sublethal radiation doses, with a Dose Modification Factor (DMF) of 1.4 for CFU-S. Slightly impaired survival was observed at lethal accumulated doses, possibly due to gastrointestinal side effects. Erythrocyte counts were sometimes decreased.	[81]
Hematopoietic recovery (GM-CFC, cellularity, spleen weight, peripheral blood counts, serum CSA)	0.12 mg per mouse	6 × 2 Gy (total 12 Gy, repeated gamma)	In vivo (mice)	Radioprotection	Repeated combined administration of diclofenac and glucan enhanced granulopoiesis and lymphopoiesis in repeatedly irradiated mice. The protective effect was attributed to the cumulation of single protective effects and additive/synergistic action on cell proliferation. Diclofenac dose was well below toxicity limits.	[81]
Clonogenic cell survival (V79 cells), pBR322 plasmid DNA relaxation assay, ABTS and DPPH radical scavenging assays, whole-body animal survival study	V79: 100 µM; plasmid DNA: 100 µM, 250 µM, 5960; mice: 25, 50, 75 mg/kg	V79: 0–10 Gy; plasmid DNA: 20–100 Gy; mice: 9 Gy (lethal)	In vitro (V79 cells, plasmid DNA), In vivo (mice).	Radioprotection	Showed radioprotective efficacy in V79 cells. Restored supercoiled plasmid DNA by scavenging free radicals. Demonstrated free radical scavenging activity. Protected 45.5% of mice at a lethal 9 Gy dose (at 75 mg/kg) in a dose-dependent manner.	[84]
Clonogenic cell survival, flow cytometry	In vitro: 0.1 mM (non-lethal); In vivo: 40 mg/kg	In vitro: 1, 2, 4, 6 Gy (single dose); In vivo: 6 Gy (local tumor), 3 Gy (whole-body pre-treatment)	In vitro (human colorectal, lung, breast, pancreatic cancer cells) In vivo (murine xenograft tumor model)	radiosensitization (in specific cancer cells); radioprotection (in normal cells—proposed dual function)	Significantly increased sensitivity to radiation and chemotherapy (5-FU) in LS174T and LoVo colorectal cancer cells. This radiosensitizing effect was not observed in lung, breast, or pancreatic cancer cells. In a murine colorectal cancer xenograft model, diclofenac caused radiosensitization and improved tumor control. Diclofenac may have a dual function, radioprotective in normal cells (anti-oxidative) and radiosensitizing in tumor cells.	[83]

.

Therefore, diclofenac demonstrates a complex and context-dependent role in radiation exposure (Table 9). As a radioprotector in normal cells and tissues, particularly the hematopoietic system and DNA, its efficacy is supported by strong In vitro and In vivo evidence. However, a critical perspective highlights its capacity as a radiosensitizer in specific cancer cells, particularly colorectal cancer. This effect is achieved by disrupting tumor metabolism and stress response and potentially increasing ROS production. This dual effect suggests diclofenac might be beneficial in cancer therapy, potentially enhancing tumor cell killing while simultaneously offering some protection to surrounding normal tissues [83]. Despite its potential, diclofenac’s known gastrointestinal and other systemic side effects remain a significant concern. This is particularly evident with repeated administration at high radiation doses. These safety issues limit its broad application as a general radioprotector in accidental exposure scenarios [81]. Repurposing diclofenac as a radiation countermeasure agent is a promising avenue, benefiting from its wide availability and well-known pharmacokinetics. However, its precise application must carefully consider the specific context, including the type of cells or tissue (normal vs. cancerous) and the radiation dose. The treatment regimen should also be optimized to enhance beneficial effects while mitigating potential adverse outcomes [82,84].

3.10. Antioxidant and Prooxidant Properties

The literature sources indicated that diclofenac and its derivatives are capable of exhibiting a variety of antioxidant properties. These include the ability to scavenge different types of free radicals (such as DPPH, ABTS, hydroxyl, and peroxyl radicals), protect lipids and DNA from oxidative damage, and modulate the activity of antioxidant enzymes. In particular, a study [85] found that diclofenac sodium in rat liver model (In vivo) can bind the stable free radical DPPH but does not show the ability to bind superoxide anion (O₂-) or hydroxyl radicals (-OH). The same study showed that diclofenac sodium prevented lipid peroxidation in the liver of rats subjected to ischemia–reperfusion by reducing the level of phosphatidylcholine hydroperoxide in plasma and suppressing the increase in serum enzymes.

In work [86], the authors developed an In vitro assay (membrane model) to test the ability of diclofenac to scavenge free radicals using phosphatidylcholine liposomes as a membrane model. They found that diclofenac demonstrated significant oxygen and lipid radical scavenging activity.

The authors of the study [87] showed that diclofenac exhibits In vitro dose-dependent antioxidant activity in preventing human erythrocyte hemolysis induced by free radicals generated during the decomposition of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH). They also suggested that diclofenac may recycle radicals from other antioxidants, such as vitamins E and C.

In a work by Brazilian researchers [88], it was shown that diclofenac sodium led to a decrease in oxidized low-density lipoprotein and lipid hydroperoxides In vivo (male Wistar rats). It also resulted in an increase in total serum antioxidant capacity and superoxide dismutase activity. A subsequent study in 2009 [89] found that diclofenac and its sodium salt protected DNA from oxidation induced by AAPH in a concentration-dependent manner and effectively scavenged ABTS+ and DPPH radicals in the brain, liver, gill and blood of common carp.

Some studies suggested that diclofenac can cause oxidative stress, disrupting the body’s balance between free radicals and antioxidant systems in the brain, liver, gill, and blood of common carp and male Wistar rats [90,91]. It can generate excess free radicals and inhibit the activity of antioxidant enzymes such as superoxide dismutase and catalase. This leads to disruption of cellular redox homeostasis and potential tissue damage (male albino rats) [92,93]. However, when combined with certain drugs, diclofenac may contribute to the reduction in oxidative stress [93].

The main results for study of antioxidant and prooxidant properties of diclofenac are summarized in

Table 10. The main results for study of antioxidant and prooxidant properties of diclofenac.

Method	Dose of DC (DCH/DCNa)	Type Study	Effect	Reference
Hydroxyl radical scavenging	1.48, 0.74, 0.37 mg/mL	In vitro	Antioxidant: DC exhibited concentration-dependent scavenging activity against hydroxyl radicals, significantly reducing the DMPO-OH adduct formation (from 94.57 ± 0.51% to 76.56 ± 1.36%).	[94]
Superoxide anion scavenging	1.48, 0.74, 0.37 mg/mL	In vitro	Not Antioxidant: DC demonstrated no activity on superoxide anion. This is attributed to its molecular structure lacking typical hydrogen-donating moieties.	[94]
DPPH radical scavenging	1.48, 0.74, 0.37 mg/mL	In vitro (spectrometry)	Antioxidant: DC showed concentration-dependent scavenging activity against DPPH radicals, inducing a significant decrease in the intensity of the DPPH• spectrum.	[94]
ABTS radical cation scavenging	1.48, 0.74, 0.37 mg/mL	In vitro	Antioxidant: DC exhibited concentration-dependent scavenging activity against ABTS radicals; all concentrations caused a significant decrease.	[94,95]
Inhibition of neutrophil respiratory bursts (LACL)	1.48, 0.74, 0.37 mg/mL	In vitro (human neutrophils)	Antioxidant: DC showed a statistically significant inhibitory effect on human neutrophil respiratory bursts after only 2 min of incubation, which was concentration-dependent.	[94]
Protection against peroxyl radicals (hemolysis)	Concentrations 5.0–25.0 µM for DaH/DaNa	In vitro (human erythrocytes)	Antioxidant: Diclofenac acid (DaH) and its sodium salt (DaNa) protected human erythrocytes against AAPH-induced hemolysis in a dose-dependent manner. DaH (n = 4.96) and DaNa (n = 3.60) trapped significantly more radicals than traditional antioxidants like Trolox (n = 0.30).	[87]
Radical repair in mixed antioxidant systems (hemolysis)	Concentrations 5.0–25.0 µM for DaH/DaNa	In vitro (human erythrocytes)	Antioxidant (Synergistic): DaH/DaNa could “repair” the radicals of other antioxidants (e.g., Vitamin E, Vitamin C, Trolox), prolonging their lifespan and enhancing their total antioxidant effect.	[87]
Protection against DNA oxidative damage (TBARS)	100 µM–300 µM (DaH/DaNa)	In vitro (naked DNA)	Antioxidant: DaH and DaNa produced a concentration-dependent protection of DNA against AAPH-induced oxidative damage by trapping 3–4 radicals (nDaH ≈ 3.86, nDaNaH ≈ 3.56).	[89]
Protection against liver injury and LPO (PCOOH, GOT, LDH)	3 or 10 mg/kg (DCNa)	In vivo (male wistar rats)	Antioxidant: DCNa prevented lipid peroxidation by decreasing plasma PCOOH levels and significantly suppressed the elevation of serum GOT and LDH in ischemia-reperfused rats in a dose-dependent manner.	[85]
DPPH radical scavenging	10 µM, 0.1 mM, 1 mM (DCNa)	In vitro (spectrometry)	Antioxidant: DCNa showed a concentration-dependent radical-trapping ability for DPPH, weaker than α-tocopherol, but stronger than coenzyme Q10.	[85]
Superoxide anion scavenging	Up to 2.50 mM (DCNa)	In vitro (spectrometry)	Not Antioxidant: DCNa did not show radical-trapping ability for superoxide anion.	[85]
Hydroxyl radical scavenging	Up to 2.50 mM (DCNa)	In vitro (spectrometry)	Not Antioxidant: DCNa did not show radical-trapping ability for hydroxyl radicals.	[85]
NADPH-dependent LPO of Liver Microsomes	Up to 10 mM (DCNa)	In vitro (liver microsomes)	Not Antioxidant: DCNa had no suppressive effect against NADPH-dependent lipid peroxidation of liver microsomes.	[85]

DC—diclofenac, DaH—diclofenac acid, DaNa—sodium diclofenac.

.

Therefore, studies on antioxidant activity are conducted using both In vitro models (in test tubes, on chemical or cellular systems) and In vivo models (on living organisms, primarily animals, as well as on human cells). The In vitro models are chemical or cellular systems that, while allowing for the evaluation of certain aspects of antioxidant activity, cannot fully replicate the complexity of a living organism. In vitro studies, such as interactions with DNA and serum albumin, are considered promising for further biological studies and potential applications [89]. In vivo studies are conducted on whole organisms, such as rats or human cells (neutrophils, erythrocytes), allowing for the assessment of more complex biological interactions. Even the most complex In vivo animal models do not fully reflect human physiology and pathophysiology. This makes translating results to clinical studies a challenging task and requires caution when extrapolating conclusions. For example, studies using human neutrophils [94] and other cells are more relevant for local drug application in humans.

3.11. Diclofenac’s Safety and Repositioning

The discussion over diclofenac’s safety is emblematic of the broader challenges inherent in balancing therapeutic efficacy with adverse risk in pharmacotherapy. On the one hand, diclofenac’s potent anti-inflammatory and analgesic properties have made it a mainstay in the treatment of various painful conditions. On the other hand, its association with significant hepatotoxic and cardiovascular adverse events cannot be ignored. Accumulating evidence from clinical trials and epidemiological studies has revealed that diclofenac has a compromised safety profile due to two major categories of adverse effects: hepatotoxicity and cardiovascular risks. Clinical evidence suggests that the risk of both hepatotoxicity and cardiovascular events increases in a dose- and duration-dependent manner [96,97,98]. Advancements in pharmaceutical formulation led to the development of alternative diclofenac preparations that aim to reduce systemic exposure and, by extension, toxicity. The main strategies and pathways to mitigate hepatotoxicity and cardiovascular risks of diclofenac are presented in Figure 3.

In particular, one promising approach to counter diclofenac’s toxicity involves the development of nanodrug delivery systems that alter its pharmacokinetics and biodistribution. Polymeric micelle formulations reduced cardiac exposure by preferentially modulating the drug’s tissue distribution. This effectively normalizes CYP-mediated metabolism, thereby diminishing systemic cardiovascular injury [99]. Liposomal and nanosuspension carriers were explored to reduce peak plasma concentrations. These carriers ensured that sufficient local concentrations were achieved with minimal systemic toxicity [100].

Careful dose management is another key pathway for limiting both hepatotoxic and cardiovascular events. Research suggested that administering the lowest effective dose of diclofenac, ideally below threshold levels that triggered significant COX-2 inhibition, could reduce the risk of thrombotic events and liver injury. This approach also preserved analgesic efficacy [8,23]. Controlled-release formulations were designed to minimize peak plasma levels, thereby reducing off-target exposures and mitigating adverse effects associated with rapid spikes in drug concentration [23].

Chemical modification of diclofenac was employed as a molecular-level strategy to reduce toxicity. For instance, the synthesis of diclofenac methyl ester and metal salt derivatives, such as diclofenac zinc, copper, or choline salts, offered enhanced pharmacokinetic properties with potentially lower hepatic and cardiovascular risks [23]. Advances in computer-aided drug design allowed for the selective modification of molecular structures to attenuate toxic metabolite formation while retaining the desired anti-inflammatory actions [23].

Co-administration of hepatoprotective agents represents another viable strategy. Agents such as silymarin demonstrated significant efficacy in ameliorating diclofenac-induced liver injury, as evidenced by reductions in elevated liver enzymes and improvements in histopathological liver architecture [23,101]. Adjunctive therapies using omega-3 fatty acids, vitamin B₁₂, or curcuminoid complexes explored to reduce diclofenac-induced oxidative stress and inflammatory damage. These approaches have the potential to reduce the risk of adverse outcomes in both the liver and cardiovascular systems [23].

The adoption of individualized treatment strategies based on patient-specific risk factors, including pharmacogenomic profiling, offers a path to safer diclofenac therapy. Identifying individuals with genetic variants in CYP enzymes (e.g., CYP2C9 metabolizer status) could assist clinicians in predicting hepatic metabolism rates and subsequent risk of hepatotoxicity, thereby allowing for tailored dosing regimens or alternative treatment selections [102]. This approach also enables stratification based on cardiovascular risk factors, ensuring that high-risk patients are either excluded from diclofenac therapy or managed with enhanced monitoring protocols [23].

Employing non-oral routes, such as topical formulations, is another effective method to reduce systemic exposure. Topical diclofenac administration has proven effective in treating localized conditions, such as osteoarthritis. This approach has been shown to exhibit a substantially lower risk of systemic toxicity, including both cardiovascular and hepatic adverse effects [102,103]. This strategy minimizes drug plasma levels, thereby reducing the burden on the liver and cardiovascular system.

Leveraging modern data analytics and machine learning can further enhance patient safety by enabling early detection and prediction of adverse drug events. Recent studies employed machine learning models to analyze electronic health record data. These studies identified key drug interactions and risk factors associated with diclofenac-induced liver injury. The findings provided clinicians with actionable insights to prevent toxicity [104]. Such predictive approaches can be integrated into clinical decision support systems to facilitate real-time monitoring and risk mitigation during diclofenac therapy.

Therefore, each of these strategies has been supported by research demonstrating the potential to reduce systemic exposure and adverse effects while maintaining the drug’s therapeutic efficacy. By carefully addressing these risk factors, it is feasible to repurpose diclofenac for the treatment of other complex diseases, thereby expanding its clinical utility beyond its current indications.

The safety concerns associated with diclofenac, particularly hepatotoxicity and cardiovascular risks, are well established and supported by a robust body of evidence ranging from research studies and clinical trials. However, the convergence of advanced drug delivery systems, structural modifications, combination protective therapies, and personalized dosing regimens fosters a promising environment for repurposing diclofenac. By overcoming its hepatotoxic and cardiovascular risks, diclofenac could be repositioned to leverage its off-target pharmacological effects. These effects include modulation of voltage-gated potassium channels and inhibition of acid-sensing ion channels. Such mechanisms may offer benefits in treating neurological disorders, certain cancers, and autoimmune conditions [23,105]. The repositioning of diclofenac for oncological applications has been driven by its emerging role in inducing cell cycle arrest and promoting apoptosis in malignant cells. However, ensuring a favorable safety profile remains paramount [101]. Such repurposing strategies necessitate robust benefit–risk evaluations and the incorporation of risk mitigation techniques as described above [23].

Despite these promising strategies, several challenges remain in the safe repurposing of diclofenac. Comprehensive clinical validation through rigorously designed trials is required to confirm the safety and efficacy of novel diclofenac formulations and combination therapies. These trials should demonstrate that such approaches could reduce hepatotoxic and cardiovascular risks. At the same time, they should maintain therapeutic efficacy in new disease contexts [8,106]. The heterogeneity in patient populations and the influence of comorbid conditions underscore the need for individualized dosing and enhanced monitoring, which could complicate large-scale clinical implementation [102,103]. Structural modifications of diclofenac show promise but require extensive preclinical and clinical evaluation. These evaluations are necessary to ensure that new derivatives do not introduce unforeseen toxicities. They must also confirm that the pharmacodynamic properties making diclofenac effective are preserved [23]. Furthermore, the implementation of advanced predictive analytics, such as machine learning-based risk stratification, holds significant promise. However, integrating these tools into everyday clinical practice remains challenging due to limitations in data interoperability and the need for constant model validation against evolving clinical data sets [104].

Future research should focus on multi-center collaborative studies to better identify the risk factors associated with diclofenac use. These studies should also refine approaches for mitigating these risks while exploring diclofenac’s repositioning potential. Additionally, regulatory hurdles and the need for post-marketing surveillance are critical considerations. The approval of modified diclofenac formulations or combination therapy regimens will require robust evidence demonstrating both efficacy and significantly improved safety outcomes compared to conventional formulations [106,107].

4. Conclusions

As a result of the conducted analysis of the scientific literature, it was established that diclofenac possesses not only well-known anti-inflammatory and analgesic properties. It also demonstrates a range of non-traditional pharmacological effects. These effects may hold potential for therapeutic application within the framework of drug repositioning.

Particular interest lies in the potential use of diclofenac in the treatment of neurodegenerative diseases (such as Parkinson’s and Alzheimer’s diseases), epilepsy, various types of cancer, bacterial and fungal infections, as well as protection against radiation-induced damage. Its reported antiviral, antioxidant, and immunomodulatory effects also merit attention. Equally important is the identification of synergistic effects of diclofenac or the use in combination with conventional therapeutic agents for the treatment of the aforementioned diseases.

Thus, repositioning diclofenac is a promising strategy for the pharmaceutical industry and may lead to the creation of novel therapeutic approaches based on a well-established generic drug. However, a major barrier to diclofenac repositioning lies in the fact that existing evidence is the data supported mainly by preclinical In vitro and In vivo studies and a limited number of clinical trials.

Therefore, further research on the non-traditional pharmacological effects of diclofenac (Figure 4) should primarily focus on conducting in-depth preclinical studies to verify the molecular mechanisms of action. In addition, exploring the combination of diclofenac with other therapeutic agents—such as antibiotics, antiviral, or anticancer drugs—as part of combination therapies, as well as optimizing its dosage regimens, appears to be a promising direction. It is also necessary to evaluate the toxicological profile and develop strategies to reduce side effects under new therapeutic conditions of diclofenac use to ensure patient safety. Ultimately, to expand the clinical application of diclofenac and enable the development of effective new treatments for various diseases, a wide range of randomized clinical trials targeting novel indications is required.