Antimicrobial activities of Diltiazem Hydrochloride: drug repurposing approach

Antimicrobial activity of Diltiazem HCl

The antimicrobial potential of Diltiazem HCl (Sigma-Aldrich, St. Louis, MO, USA) was performed against three pathogenic yeasts (Candida albicans AUMC 13415, Candida glabrata AUMC 13412, and Candida krusei AUMC 13420), and four pathogenic bacterial strains (Escherichia coli ATCC 8739, S. epidermidis ATCC 12228, S. aureus ATCC 6538, and Serratia marcescens AUMC B-58), using the well diffusion method as described by Valgas et al. (2007). Diltiazem HCl was used at concentrations of 50, 25, 12.5, and 6.32 mg mL⁻¹. Fluconazole and Chloramphenicol are among the most widely used drugs to treat fungal and bacterial infections, respectively (Russell, 2004; Abuhammour & Habte-Gabr, 2001), therefore, they were used as reference drugs at a concentration of 12.5 mg mL⁻¹. The examined organisms were cultivated in 24-hour-old cultures. Using sterile cotton swabs, the bacterial and fungal suspensions (10⁸ CFU/mL = 0.5 McFarland standard solution) were equally spread on sterile Petri plates filled with either nutrient agar for bacteria or Sabouraud’s dextrose agar for Candida species. Five wells (6 mm diameter holes cut in solid medium) were filled with 50 µL of the substance under test. The plates were incubated for 24 h at 37 °C to allow the bacteria and Candida species to grow. Following incubation, the suppressions of fungal and bacterial growth were measured in millimeters. The minimum bactericidal concentration (MBC) was determined. All tests were carried out in triplicate.

Microtitre plate assay for biofilm quantification

The effect of Diltiazem HCl on biofilm formation was evaluated as described by Mohanta et al. (2020). Briefly, using a 96-well polystyrene plate, 300 µL of fresh tryptone soy yeast broth inoculated with 10⁶ CFU mL⁻¹ was aliquoted into each well and cultured in the presence of 75%, 50%, and 25% of MBC which was determined in the previous step. Wells with bacteria but no drugs were used as a control, while those containing medium without bacteria were used as a blank. The plates were then incubated for 48 h at 37 °C. After the incubation period, the supernatant was disposed of and every well was meticulously cleaned using sterile distilled water to eliminate any floating cells. After 30 min of air drying, the biofilm formed was stained for 15 min at 24 °C using an aqueous solution of crystal violet (0.1%). Finally, 250 μL of 95% ethanol was added to each well to solubilize the dye. The plate was then incubated for 15 min and the optical density (OD) was recorded at 570 nm using a microplate reader (SN 18052112; USA). To calculate the biofilm inhibition ability of Diltiazem HCl, the following formula was used:

$$\mathrm{B}\mathrm{i}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{l}\mathrm{m}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{a}\mathrm{y}=1-{\displaystyle \frac{\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{D}\mathrm{i}\mathrm{l}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{z}\mathrm{e}\mathrm{m}\mathrm{H}\mathrm{C}\mathrm{l}}{\left(\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{n}\text{-}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}-\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}\times 100}$$

Antiviral activity

Statistical calculation

Statistical analyses were carried out using Microsoft Excel (XLSTAT 2018.3.16, Florida, USA) and GraphPad Prism version 7 software.

Antimicrobial activity of Diltiazem HCl

Unfortunately, many traditional antibiotics have failed to treat patients when used over extended periods or improperly, due to the antimicrobial resistance developed by several pathogenic microorganisms (Ramadan et al., 2024). Therefore, the search for alternative antibiotics rather than conventional antibiotics is urgently needed (Ramadan et al., 2023). Drug repurposing is a productive approach for the discovery of new therapeutic uses for already-available drugs that cut down the time and cost required to develop safe drugs (Singh et al., 2020). In this study, the antimicrobial activity of the commonly prescribed antihypertensive drug, Diltiazem HCl (Stepanovs et al., 2016) was preliminarily evaluated at concentrations of 50, 25, 12.5, 6.25, and 3.12 mg mL⁻¹ against three fungal pathogenic strains of Candida sp and four pathogenic bacterial strains (two Gram-positive and two Gram-negative) using the agar well diffusion method. The zones of inhibition were measured (Fig. 1), and the data is presented in ‘Table 1’. The results of the current study revealed that Diltiazem HCl efficiently suppressed the growth of tested Gram-positive bacteria. The highest zone of inhibition was recorded against S. epidermidis (20 ± 3 mm) at a concentration of 50 mg mL⁻¹. The MIC for Diltiazem HCl was 6.25 mg mL⁻¹ when employed against S. epidermidis with an inhibition zone (10 ± 2.0 mm). The inhibition zone for S. aureus was 12 ± 2 mm, and the minimum inhibitory concentration (MIC) was 50 mg mL⁻¹ (Table 1, Fig. 1), compared to the Chloramphenicol antibacterial standard. Diltiazem HCl showed a weak antifungal activity against C. glabrata with inhibition zone 12 ± 1.0 mm at 50 mg mL^- compared to the antifungal standard Fluconazole. However, the yeasts C. albicans and C. krusei as well as the tested Gram-negative bacteria (S. marcescens and E. coli) were unaffected by Diltiazem HCl (Table 1, Fig. 1).

A variety of “non-antibiotics” which are involved in the management of diseases of non-infectious etiology, have been reported to exhibit antimicrobial activity against bacteria and fungi (Glajzner, Bernat & Jasińska-Stroschein, 2024; Kruszewska, Zareba & Tyski, 2008). 1,5-Benzothiazepine moiety itself has no antimicrobial activity; however, many derivatives have been reported to have variable antibacterial and antifungal activities according to the different substitutions of the main nucleus (Pant, Avinash & Yadav, 2014; Dandia et al., 2010; Saini, Joshi & Joshi, 2008). Calcium channel blockers, such as Diltiazem HCl, at their therapeutic dosage, reduce the elevated blood pressure of hypertensive patients but do not affect the blood pressure of normotensive individuals because they have little effect on the peripheral vasculature (Godfraind, 2017). So, they can be used safely as anti-infectious agents for both hypertensive and non-hypertensive patients. The observed results demonstrated the noticeable efficacy of Diltiazem HCl, a 1,5-Benzothiazepines derivative, against S. epidermidis, and moderate activity against S. aureus. To determine how Diltiazem HCl affects the S. aureus and S. epidermidis growth, the reduction of their ability to form biofilms was carried out as illustrated in the following section.

Antibiofilm formation

Bacterial biofilms play an important role in the protection of pathogenic bacteria from conventional antibiotics (Salem et al., 2022). It is the root reason behind delayed growth, nutritional constraint, poor antibiotic penetration, adaptive stress responses, and the development of persistent cells that serve as a multi-layered defense against antibiotics (Salem et al., 2022). Disrupting biofilm formation may enhance the efficacy of antibacterial medications to clear infections involving biofilms that are refractory to current therapies (Stewart, 2002). Determining Diltiazem HCl’s capacity to prevent S. aureus and S. epidermidis from forming biofilms was thus carried out.

The present findings showed that at 75%, 50%, and 25% of MBC (50 mg mL⁻¹), Diltiazem HCl decreased the formation of S. aureus biofilm by 90.7%, 86.0%, and 81.5%, respectively (Table 2). Furthermore, it decreased the production of S. epidermidis biofilm by 95.1%, 91.2%, and 85.0% at 75%, 50%, and 25% of its MBC (6.25 mg mL⁻¹), respectively (Table 2). The categorization of bacteria according to their specific biofilm formation’s (SBF) ability was published by Mittal et al. (2010). The groups included weak biofilm producers (SBF index < 1.00), intermediate biofilm producers (SBF index < 2.00), and strong biofilm producers (SBF index > 2.00). Accordingly, the ability of Diltiazem HCl to suppress the formation of both bacteria’s biofilms was demonstrated by the significant reduction in SBF indices of S. aureus and S. epidermidis from SBF 1.338 and 1.433, respectively, to SBF indices <1.0 at varied concentrations (Table 2).

The drugs that impact calcium binding may also affect biofilm development because divalent cations, in particular calcium, are crucial bridging ions for bacterial polysaccharides, and biofilm formation, and they also play regulatory functions in bacterial gene expression (Sarkisova et al., 2005). While Diltiazem HCl was shown in the current study to be effective in decreasing the formation of biofilms in tested Gram-positive bacteria, specifically S. aureus and S. epidermidis, it was found to have the opposite effect on the inhibition of biofilms in Gram-negative bacteria, Pseudomonas aeruginosa as reported by Elkhatib, Haynes & Noreddin (2009). Its antagonist effect on Gram-negative bacteria was in contrast to other calcium channel blockers which showed an agonistic effect in the presence of conventional antibiotics (Elkhatib, Haynes & Noreddin, 2009). It seems that the specific medicine employed and the bacterial Gram stain types influence how different calcium channel blockers affect the production of biofilms (Elkhatib, Haynes & Noreddin, 2009). Further research is recommended to explore whether Diltiazem HCl’s biofilm suppression of S. aureus and S. epidermidis works synergistically or antagonistically with conventional antibiotics.

Antiviral activity

Despite the efficacy of conventional antivirals, treatment failures brought on by antiviral resistance are on the rise (Abdelkarem et al., 2022). Drug repurposing is a useful tool for investigating the antiviral potential of “non-antibiotic” drugs (Singh et al., 2020). The efficacy of 1,5-benzothiazepine moiety as the primary nucleus for antiviral derivatives was demonstrated (Li et al., 2017). Therefore, by using the MTT assay procedure, the antiviral activity of Diltiazem HCl was tested against the CoxB4 viruses compared to Acyclovir, a standard antiviral drug.

Cytotoxicity against Vero cells

At first, the cytotoxicity of Diltiazem HCl and Acyclovir on Vero cells was assessed. The data analysis revealed dose-dependent effects, with higher cytotoxicity observed at higher drug concentrations and decreased cytotoxicity at lower concentrations (Fig. 2). To ensure accurate antiviral screening, non-cytotoxic doses were selected, and the half-maximal cytotoxic concentrations (CC₅₀) of Diltiazem HCl and Acyclovir, were assessed on Vero cells using the MTT assay (Table 3). Diltiazem HCl showed a CC₅₀ value of 310.37 ± 0.067 µg mL⁻¹ while Acyclovir exhibited a CC₅₀ value of 320.21 ± 0.021 µg mL⁻¹ which indicated that Diltiazem HCl has less toxicity than Acyclovir against the Vero cell line. The MNTC on Vero cells was 62.5 µg mL⁻¹ for both drugs; this concentration was used later for testing the antiviral activities.

The morphological traits of Vero cells treated with various Diltiazem HCl concentrations

The results of this study showed that following Diltiazem HCl treatment, Vero cell morphology altered in a concentration-dependent way, resulting in either whole or partial monolayer loss (Fig. 3). In comparison to untreated cells, higher doses caused morphological abnormalities for some treated cells such as rounding, detachment, shrinking, and granulation. Lowering the concentrations below 62.5 µg mL⁻¹ resulted in a decrease in or elimination of these morphological changes without endangering the cell monolayer (Fig. 3).

Assessment of the cell viability of the MNTC of Diltiazem HCl

The cell viability of control Vero cells and CoxB4-infected Vero cells were evaluated using an MTT assay. Vero cells were infected with the CoxB4 virus, and treated with and without Diltiazem HCl or Acyclovir for 48 h. The results showed that 99.99% of the control Vero cells were viable. However, Vero cell viability dramatically dropped to 40.44% in the presence of the CoxB4. The cell viability percent of CoxB4-infected cells treated with 62.55 µg mL⁻¹ of Acyclovir was 81.35%, and that of Diltiazem HCl was 93.72% (Table 4) indicating a markedly higher rate of cell survival compared to the untreated CoxB4-infected cells. These findings show that Diltiazem HCl administration partially restored the cell viability that had been considerably reduced by CoxB4 infection, suggesting that the drug has potential as an effective therapeutic agent for treating CoxB4 infection.

Antiviral activity of Diltiazem HCl using the MTT assay technique

The reported bioactivities of Diltiazem HCl against Alzheimer’s disease, influenza infection, and antitumor effects, make it an excellent model for drug repurposing against other infectious diseases such as CoxB4 infection (Alluri et al., 2023; Kaur et al., 2023; Ribeiro et al., 2023). Using the MTT assay, the antiviral activity of Diltiazem HCl against the CoxB4 virus was assessed at nontoxic doses (<62.5 µg mL⁻¹). It showed a significant antiviral efficacy against the CoxB4 viruses where its IC₅₀ (35.8 ± 0.54 μg mL⁻¹) was lower than that of the reference antiviral, Acyclovir (IC₅₀ 42.71 ± 0.43 μg mL⁻¹). The selectivity indices (SIs) of Diltiazem HCl and Acyclovir against the CoxB4 were calculated. The SIs were found to be 8.66 and 7.49 for Diltiazem HCl and Acyclovir, respectively, suggesting the potential of Diltiazem HCl as a promising antiviral agent against CoxB4.

Considering the role of calcium ion in the replication and morphogenesis of numerous viruses, these findings are corroborated by earlier research showing Diltiazem HCl’s antiviral effectiveness against rotaviruses (Khales et al., 2023), rhinoviruses (Gazina et al., 2005), and influenza viruses (Fujioka et al., 2018). Also, Diltiazem did not exhibit antiviral activity against SARS-CoV-2; however, it strongly synergized the activity of the conventional drug Remdesivir (Pizzorno et al., 2020). It is worth noting that, not all Ca₂⁺ channel inhibitors have appreciable antiviral activity (Pizzorno et al., 2019). Therefore, the observed antiviral activity of Diltiazem HCl may not be entirely explained by its induced modification of Ca₂⁺ channel activity (Pizzorno et al., 2020). According to Fujioka’s study, Diltiazem’s antiviral effects are mostly due to its ability to control the expression of particular genes linked to cholesterol metabolism, the host antiviral response, and the induction of interferon-antiviral responses (Fujioka et al., 2018). The current study suggests that Diltiazem HCl could potentially be used to treat the CoxB4 virus infection, which would provide it an additional off-target effect.