Introduction

While a wide range of bacteria is inhibited by currently available antibiotics, many bacteria have been found to acquire resistance against the available antibiotics. Infectious diseases such as HIV/AIDS, tuberculosis, malaria, and influenza remain a global health concern.^[1] Therefore, there is an urgent need to find novel and cheap antibiotics.^[2] Developing new drugs using conventional drug discovery processes requires time and finance. According to reports in the period from 1995 to 2001, no new drug candidates were developed by Pfizer, GlaxoSmithKline, and AstraZeneca^[3-5], highlighting the challenge of drug discovery and development.

A solution to this issue is the repurposing of existing drugs for treating conditions other than the conditions they are normally used for.^[6]Repurposing is an alternative to conventional drug discovery and can be used to identify further applications of existing drugs.^[7-11] The best example of drug repurposing is the repurposing of thalidomide. Thalidomide was developed as a sedative-hypnotic agent against nausea and morning sickness in pregnant women in the 1950s. However, its use was later prohibited due to its teratogenic and anti-angiogenic effects.^[12,13] Although, further research found the drug to be effective in the treatment of multiple myeloma and other related malignancies.^[14,15] Currently, it is an FDA-approved drug for multiple myeloma.^[16]

Repurposing of commonly used or over-the-counter (OTC) drugs has already been attempted. For example, non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen have been recently proven to have anti-cryptococcal activity as an off-target effect.^[17] In addition, many OTC drugs such as aspirin, rapamycin, minocycline, celecoxib, valproic acid, and metformin have been reported to demonstrate anticancer activity.^[18] Furthermore, Viagra (sildenafil citrate) was originally used for chest pain and later reprofiled for male infertility.^[19] The most recent addition in the repurposing of drugs is for the COVID-19 infections where Remdesivir and Chloroquine were used against the novel coronavirus.^[20,21]

Deferoxamine B is an FDA-approved drug for the treatment of iron overload in thalassemia patients.^[22-25] It was found to demonstrate anti-tuberculosis activity and thus, could be used as an alternative therapy for treating infections caused by MDR Mycobacterium tuberculosis strains.^[26]

In the present study, we evaluated OTC drugs including diclofenac (NSAID), dimenhydrinate (antiemetic, antihistamine, and anticholinergic agent), Sinarest (Combination medicine), Rantac (Antacid), and an iron chelator Desifer for their antibacterial and anticancer activity as well as cytotoxicity. OTC drugs were evaluated alone and in combination with Desifer. Drugs included in the study are FDA-approved and have ​a long record of safety in patients.

The development of such an alternative therapy will be extremely useful in cancer chemotherapy or the treatment of infections caused by drug-resistant pathogenic bacteria. 

Materials and Methods

Determination of antibacterial activity

Determination of antibacterial activity of Draminate, Diclofenac, Rantac, Sinarest, and Desifer by ditch plate method

Tablet formulations of Draminate (Dimenhydrinate), Diclofenac(Diclofenac sodium IP), Rantac(Ranitidine hydrochloride IP), Sinarest (Paracetamol IP), and Desifer (Deferasirox) were used

Qualitative determination of the antibacterial activity of these drugs was conducted using the agar ditch method according to the protocol given by Rice et al.^[27]

The powdered drugs (Draminate, Diclofenac, Rantac, and Sinarest) were poured into a tube containing 4ml of molten Nutrient agar and this mixture was poured into the ditch. Two concentrations of each drug were used. The following 8 laboratory test cultures were used: Gram-negative organisms – Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa​, and gram-positive organisms – Staphylococcus aureus, Mycobacterium smegmatis, Streptococcus pyogenes, and Corynebacterium diphtheriae. Four test cultures were streaked on each plate. The plates were incubated at 37℃for 24 hours.

Determination of Antimicrobial activity of Drug pellet and Drug supernatant

The drugs Draminate, Diclofenac, Rantac, Sinarest, and Desifer (desferri-form), were dissolved in water, this suspension was then centrifuged at 10,000 rpm for 10 minutes. Next, the supernatant was collected and sterilized using a 0.2µm membrane syringe filter. Sterile nutrient agar plates were swabbed with test culture suspension (​E.coli, K. pneumoniae, S. typhi, P. aeruginosa, S. aureus, M. smegmatis, S.pyogenes, and C. diphtheriae ​), the turbidity of which was adjusted to 0.5 MacFarland standard​. The supernatant and pellet were spotted onto these plates and incubated at 37℃ for 24 hours. The plates were checked for a zone of inhibition after incubation.

Determination of the inhibitory effect of different concentrations of OTC drugs (Draminate, Diclofenac, Rantac, Sinarest), and the iron-chelator Desifer by agar cup diffusion method

The Agar cup diffusion method was performed as per the protocol given by Rose and Miller.^[28]Culture suspensions​(turbidity adjusted to 0.5 MacFarland standard) ​of the eight laboratory cultures ​(​E. coli, S. aureus, M. smegmatis, S. pyogenes, C. diphtheriae, K. pneumoniae, S. typhi, and P. aeruginosa​) were pour plated using sterile iron-deficient Mueller Hinton (MH) agar medium. Aqueous supernatants(100µl aliquots) of Diclofenac (0.5, 1, 5, 10, 15, and 20 mg/ml), Draminate (15, 20, 25, 30, 35, 40, 45, and 50 mg/ml), Rantac (25, 50, 75, and 100 mg/ml), Sinarest (125 and 250 mg/ml) and Desifer (0.5, 1,5,15,25, and 40 mg/ml) were added in cups​. The plates were incubated at 37℃ for 24 hours and checked for a zone of inhibition.

Determination of the antibacterial activity of Draminate, Diclofenac, Rantac, and Sinarest​ each in combination with Desifer.

Agar cup diffusion method

Culture suspensions (​turbidity adjusted to 0.5 MacFarland standard​) of the eight laboratory cultures ​(​E. coli, K. pneumoniae, S. typhi, P. aeruginosa, S.aureus, M. smegmatis, S. pyogenes, and C. diphtheriae) were seeded in sterile iron deficient MH agar medium. Cups were bored in these plates. Aqueous supernatants of the 4 drugs (Draminate, Diclofenac, Rantac, and Sinarest) were diluted and 50µl of these dilutions were added in combination with 50µl of Desifer (40 mg/ml). Desifer-only controls were applied by adding 50 µl each of Desifer stock and distilled water to the ​cup. The plates were incubated at 37​℃ ​for 24 hours and checked for a zone of inhibition. ​

Micro broth dilution method

Culture suspensions (​turbidity adjusted to ​0.5 MacFarland standard​) of the 8 laboratory cultures ​(E. coli, K. pneumoniae, S. typhi, P. aeruginosa, S.aureus, M. smegmatis, S. pyogenes, andC. diphtheriae​) were prepared in sterile iron-deficient double-strength MH (Mueller-Hinton) broth. These suspensions (100 µl aliquots) were added to a sterile 96-well microtiter plate. Dilutions of the four drugs (Draminate, Diclofenac, Rantac, and Sinarest) (50µl) were added individually and in combination with 50µl of Desifer (40mg/ml). Drug-only controls were applied by adding 50 µl each of drug stock and distilled water in the culture-containing wells. Appropriate negative controls were used. The microtiter plate was incubated at 37℃ for 24 hours. Furthermore, a growth curve was obtained by measuring the turbidity at 30-minute intervals for 24 hours. Absorbance was measured at 600nm using a Microplate reader (Epoch 2, BioTek). Bactericidal activity of these combinations was determined by spotting 10µl samples from wells showing no growth onto sterile MH agar plates, which were incubated at 37℃ for 24 hours.

Determination of anticancer and cytotoxic activity.

Anticancer activity and cytotoxicity of the drugs under study were checked (individually and in combination with Desifer) against human lung cancer cell line A549 and human embryonic kidney (HEK293) cell lines, respectively. MTT assay was performed for checking the anticancer activity and cytotoxicity of these drugs. Absorbance was measured at 590 nm using an Epoch 2 plate reader and percentage survival rates were calculated using Sigma Stat Software.

Analysis of aqueous supernatants of Draminate, Diclofenac, Rantac, Sinarest, and Desifer.

Detection of active components in aqueous supernatants of the selected drugs using Thin Layer Chromatography

A chromatography chamber was saturated with Methanol: Acetone (8.5:6.5) solvent vapors. A silica gel plate was loaded with aqueous supernatants of Draminate, Diclofenac, Rantac, Sinarest, and Desifer, this was placed in the saturated chamber and allowed to run until the solvent front had reached an appropriate length. The chromatogram was baked at 60℃ for 10 minutes and then developed using saturated iodine vapors.

Qualitative chrome azurol S (CAS) assay for detection of active siderophores in the aqueous supernatant of Desifer

Chrome azurol S (CAS) assay is the universal method for detection of iron-chelators/siderophores and was used to detect the presence of an active component (deferasirox) in the aqueous supernatant of Desifer. CAS dye changes from blue to orange in the presence of iron chelators.^[29]

Quantitative CAS assay for measuring the concentration of active siderophores aqueous supernatant of Desifer

CAS dye was used for quantitative estimation of deferasirox in the aqueous supernatant of Desifer. DFO-B (injectible) was used as the standard iron-chelator. A standard curve of DFO-B concentration (mg/ml) v/s absorbance at 630 nm ​was prepared. Deferasirox concentration in the aqueous supernatant was determined using this standard curve. To 100 µl aliquots of drug different drug concentrations, 10 µl CAS dye was added. Absorbance was determined using an Epoch 2 plate reader.

Results

Determination of antibacterial activity

Determination of the antibacterial activity of Draminate, Diclofenac, Rantac, Sinarest, and Desifer by ditch plate method

Draminate and Diclofenac showed antibacterial activity against all laboratory cultures except P. aeruginosa. However, selected concentrations of Rantac and Sinarest did not show any antibacterial activity against any of the laboratory cultures.

Figure 1: a,b and c represent the antimicrobial activity of Draminate (50mg), Diclofenac (50mg), and Rantac (37.5mg), respectively, against laboratory strains. Click here to view figure

Figure 1a,1b, and 1c represent the antimicrobial activity of Draminate (50mg), Diclofenac (50mg), and Rantac (37.5mg), respectively, against laboratory strains.

Determination of the antimicrobial activity of the drug pellet and drug supernatant

The supernatants of all drugs showed antimicrobial activity against at least one

laboratory culture. Thus, drug supernatants were used for further experiments.

Table 1 represents the antimicrobial activity of drug supernatants against laboratory strains. 

Table 1: Determination of antimicrobial activities of drug supernatants.

Key

(+) Clear Zone of Inhibition

(-) No Zone of Inhibition

(±) Partial Inhibition

Determination of the inhibitory effect of different concentrations of Draminate, Diclofenac, Rantac, Sinarest, and Desifer by agar cup diffusion method

Table 2 shows the inhibitory effect of Desifer and the OTC drugs, Draminate, Diclofenac, Rantac, and Sinarest, on laboratory cultures. 

Table 2: Determination of minimum inhibitory concentration of Desifer and OTC drugs (Draminate, Diclofenac, Rantac, and Sinarest) using agar cup diffusion method.

Key: (-) No inhibition, (*) Partial inhibition. 

Figure 2: shows examples of inhibition zones of S.aureus and E.coli by different concentrations of Draminate. Click here to view figure

The minimum concentrations of selected drugs at which respective microorganisms were inhibited are given in Table 3.No antimicrobial activity was observed against P. aeruginosa and K.pneumoniae. Further, individual activities of Draminate, Rantac, Sinarest, and Diclofenac and their synergistic activities in combination with Desifer were evaluated. 

Table 3: MIC of drugs (Draminate, Diclofenac, Rantac, Sinarest, and Desifer) using agar cup diffusion method.

 Determination of the synergistic antibacterial activity of ​Draminate, Diclofenac, Rantac, and Sinarest​ in combination with Desifer

Agar cup diffusion method

Table 4 shows the antibacterial effect of ​Draminate, Diclofenac, Rantac, and Sinarest​ each in combination with Desifer against laboratory cultures. 

Table 4: Determination of the antibacterial effect of ​Draminate, Diclofenac, Rantac, and Sinarest​ each in combination with Desifer. 

Key: (-) No inhibition (*) Partial inhibition.

Figure 3: shows representative examples of zones of inhibition of S.aureuscaused by Desifer alone and in combination with Sinarest as well as no inhibition by Sinarest alone, zone of inhibition of M.smegmatis caused by Desifer and Rantac alone as well as in combination. Click here to view figure

Micro broth dilution method

Figure 4 represents the effect of drug combinations on bacterial growth. Figure 4(a) shows that in presence of Desifer, the lag phase was tremendously prolonged till 16hr 40min. At 16hr 40 min., S. pyogenes resumes growth as Desifer (desferri form) gets saturated with ferric ions and cannot sequester more ferric ions which get available for the growth of an organism. Table 5 represents the mode of action of drugs against respective laboratory cultures.

Figure 4(a): Bacteriostatic activity of Rantac and Draminate each in combination with Desifer on the growth of S. pyogenes and M. smegmatis, respectively.  Click here to view figure

Figure 4(b): Bactericidal activity of Draminate in combination with Desifer on the growth of E. coli and S. typhi.  Click here to view figure

Table 5:  Mode of action of drugs against respective laboratory cultures

Determination of anticancer and cytotoxic activities of the selected drugs

Figure 5a: represents the cytotoxic activities of Draminate, Diclofenac, Rantac, and Sinarest alone and in combination with Desifer against the human embryonic kidney (HEK293) cell line. Click here to view figure

Figure 5b: represents anticancer activities of Draminate, Diclofenac, Rantac, and Sinarest alone and in combination with Desifer against human lung cancer cell line A549. Click here to view figure

Analysis of aqueous supernatants of Draminate, Diclofenac, Rantac, Sinarest, and Desifer.

Detection of active components in aqueous supernatant of drugs using thin layer chromatography:

Active components in the aqueous supernatant of drugs were determined using thin layer chromatography.

Figure 6a: shows the developed thin layer chromatogram loaded with aqueous supernatants of drugs. Click here to view figure

Active pharmaceutical ingredient (API) testing can be done using the most popular non-thermal methods for determining the compatibility of drugs and excipients infrared (IR), near-infrared (NIR), and Raman spectroscopy. Based on their physical and chemical characteristics, these approaches offer a distinctive fingerprint for the API and the excipients

Qualitative CAS assay for detection of active siderophores in the aqueous supernatant of Desifer

Figure 6b: shows the results of the qualitative CAS assay. Click here to view figure

Quantitative CAS assay for measuring the concentration of active siderophores aqueous supernatant of Desifer

Figure 6c: represents the standard graph DFO-B concentrations v/s absorbance at 630nm.  Click here to view figure

Discussion

To our knowledge, this was the first time that the antimicrobial activity of Draminate, Diclofenac, Rantac, and Sinarest has been assessed in combination with Desifer.

Tablet formulations of OTC drugs were analyzed to determine if these formulations had any antibacterial activities. Rantac, Draminate, and Diclofenac inhibited M. smegmatis, S. typhi, S. aureus, C. diphtheriae, S. pyogenes, and E. coli, but were ineffective against P. aeruginosa and K. pneumoniae.

This indicated that Rantac, Draminate, and Diclofenac had antibacterial activity. This result is as per the fact that all drugs used for treatment possess off-target effects as they share common targets and molecular pathways in cellular functioning.

A similar study had been conducted which supports the findings. In one such study, Draminate was found to have antibacterial activity against some NCTC bacterial cultures,^[30] in another study, dimenhydrinate (an active component of Draminate) was found to have antibacterial activity against S. aureus, E. coli, P. aeruginosa.^[31] Similarly, Diclofenac, at concentrations of 50-100 µg/ml, was also found to inhibit 397 bacterial strains when tested in-vitro.^[32] A study by Sun et al. found two sets of drug combinations that exhibited broad-spectrum antibacterial activity against a panel of ten common MDR clinical isolates. This included K. pneumoniae, P. aeruginosa, and E. coli, among others.^[33]

The Agar cup diffusion method was used to quantitate the antibacterial activity of the selected drugs in terms of MIC. Based on the results, dilutions were decided to study the inhibitory action of OTC drugs in combination with Desifer using the Agar cup diffusion and Micro broth dilution method. It was found that the OTC drugs had higher MICs when used individually, the MICs decreased to one-fourth of the individual MIC when used in combination with Desifer.

The inhibitory activity of Draminate, Sinarest, Rantac, and Diclofenac was enhanced in the presence of Desifer, this may be due to iron deficiency caused by the sequestration of Fe⁺³ ions. Pathogens have been found to colonize sites in the human body where they have easy access to iron or iron transport systems.^[34] This has been observed in pathogens like Mycobacterium species, extraintestinal pathogenic E. coli, H. pylori, and Burkholderia species, this shows the essential role of iron in their survival and virulence.^[35-38] Iron deficiency causes an increase in cell permeability, thus, the organisms have increased susceptibility to drugs. In addition, iron deficiency may also decrease the growth rate of bacteria. Therefore, the use of Desifer with drugs can be a good strategy to enhance the effectiveness of antibacterial drugs. Nick et al tested Deferoxamine for antimicrobial activity against various human pathogens like Plasmodium​,​ Pseudomonas, and​Staphylococcus spp.^[39]However, it had high MIC values, and therefore, they studied the activities of Deferoxamine in combination with other antibiotics. This was found to increase antimicrobial activity by 50-fold in some cases.^[40] However, in our study, micro broth dilution tests showed different results when Diclofenac was used with Desifer against S. aureus. It was found to inhibit S. aureus individually, but its inhibitory activity decreased in the presence of Desifer, suggesting that some organisms can survive iron deficiency.

The growth curve analysis was done to identify the bactericidal action of drugs. In combination treatment with Desifer, Sinarest, and Diclofenac showed bacteriostatic activity. The​drugs Draminate, Sinarest, and Diclofenac in combination with Desifer showed good inhibitory activity against ​M. smegmatis, suggesting the future use of these Desifer-drug conjugates in the treatment of MDR ​M. tuberculosis infection. Studies have been conducted using artemisinin in combination with Deferasirox against ​ Mycobacterium tuberculosis strains, this combination was found effective (Artemisinin alone was found to be ineffective).^[41]​As liquid media is used in the micro broth dilution method, Desifer has better access to ferric ions, leading to increased iron sequestration. Hence, enhanced inhibitory activity was observed using this method compared to the agar cup diffusion method.

These results suggest the use of combination therapy with OTC drugs and Desifer as an alternative therapy. However, the safety of this treatment strategy must be evaluated for its safety, cytotoxicity was checked using HEK293 cell lines. Diclofenac and Desifer exhibited low cytotoxicity, and hence, can be good candidates for repurposing. Some studies have reported diclofenac-induced cytotoxicity in leukocytes due to enzyme-mediated transformation^[42], and cytotoxicity in Fibroblast 3T3-L1 preadipocytes^[43]. Desifer can also cause cytotoxicity by iron depletion which encourages BclxL downregulation and proximal tubular cell death.^[44] However, further investigation is warranted to understand the causes of cytotoxicity.

The OTC drugs like Rantac and Draminate are FDA approved and are consumed commonly by the population taking their safety as guaranteed. However, some studies have shown their toxicity to normal human cell lines. Side effects of OTC medications like antacids, cough, and cold formulation have been investigated where case studies have reported significant morbidity and even mortality in both acute overdoses and when administered in the right dosage but for prolonged periods.^[45]  Ranitidine-induced anaphylaxis has also been observed.^[46]

When checked for anticancer activity against the Human lung cancer cell line A549, drugs Desifer, Draminate, Rantac, and Sinarest individually and in combination with Desifer showed anticancer activity. Although Diclofenac did not show anticancer activity individually, in combination with Desifer, it had anticancer activity, thus, it could be used in combination chemotherapy. Most used drugs exhibit anticancer activity. Rantac has been a part of the Coordinated Undermining of Survival Paths 9 (CusP9) regime for glioblastoma, for which limited therapies are available.^[43]Deferasirox demonstrated similar activity in inhibiting the proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines.^[44] Draminate, Rantac, and Sinarest alone and in combination with Desifer were cytotoxic (fig.5a) as well as had anticancer activity(fig.5b). Cytoxicity may not limit their use, as distinguished anticancer agents usually possess severe side effects at their effective concentrations. However, there are ways to manage the toxicities of anticancer drugs.^[45]

TLC analysis was conducted to find several components in the drug supernatants. Desifer and Rantac showed the presence of one component on TLC analysis, Draminate, Sinarest, and Diclofenac showed the presence of two components each. Chemical testing of Desifer supernatant with CAS dye confirmed the presence of its active ingredient, Deferasirox, furthermore, on TLC analysis, Desifer supernatant showed only one component. Thus, the antibacterial activity of Desifer can be attributed to Deferasirox.

The study has several limitations. All the above experiments were done assuming that the active ingredients of all drugs used were completely water-soluble. Quantitative estimation of deferasirox in Desifer supernatant was done using CAS reagent with the use of DFO-B (injectable) as a standard. It was found that16.7 mg/ml of DFO-B corresponds to 5mg/ml of deferasirox. According to the Merck index, Dimenhydrinate, the active component of Draminate, has a solubility of 3mg/ml in water. However, no analytical methods were used to confirm the concentrations of active ingredients of Draminate, Diclofenac, Rantac, and Sinarest in their aqueous supernatants. 

Conclusion

Desifer and Diclofenac, Dimenhydrinate, Sinarest, Rantac supernatants individually showed antibacterial activity against laboratory cultures. This inhibitory action may indicate their potential activity against pathogenic strains. The enhanced antibacterial activity of drugs in combination treatment can be attributed to iron deprivation, chelation of essential non-iron metals, and increased membrane permeability due to the action of Desifer. A rifampin and isoniazid-resistant strain of​ M. smegmatis was used as a model for pathogenic MDR ​M. tuberculosis​. Commonly used drugs like Draminate, Sinarest, and Diclofenac showed good inhibitory activity against ​M. smegmatis in the presence of Desifer, suggesting the future use of these Desifer-drug conjugates for MDR​M. tuberculosis​infections. However, the safety and efficacy of such a treatment must be analyzed.

In the wake of emerging drug resistance among pathogenic bacteria, such studies would have wide applications in chemotherapy.

Acknowledgment

We would like to thank the Department of Microbiology, St. Xavier’s College, Mumbai, for providing us with the laboratory facilities to conduct this work. 

Conflict of interest

The author(s) declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article. 

Funding Source

The author(s) received no financial support for the research, authorship, and/or publication of this article. 

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