An Exploratory Study on the Antimicrobial Activity of Cetirizine Dihydrochloride

*Corresponding Author:

H. S. Maji
Department of Pharmaceutical Technology, JIS University, Kolkata-700 109, India
E-mail: hsmaji77@gmail.com

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Abstract

Antihistamines belong to H1 receptor antagonist class of drugs. Pharmacologically they are classified into two categories namely first generation, mostly sedating in nature and second generation, which are less sedating and quite selective in activity. Widespread uses of antihistamines were found during microbial infections of various types. The utmost deliberate effort in comprehending the actions of antihistamines within the extent of antimicrobials forms the basis of the investigation. The antimicrobial activity of antihistamines explored by previous researchers helped in determining the minimum inhibitory concentration of cetirizine against 51 strains of bacteria. The antiallergic drug cetirizine showed significant in vitro antimicrobial activity against 51 strains of bacteria belonging to 5 Gram-positive and 4 Gram-negative genera. The minimum inhibitory concentration of the drug determined both by agar dilution and broth dilution method ranged from 200-2000 μg/ml against most of the bacteria tested. Cetirizine was bactericidal in action in vitro on both Gram-positive and Gram-negative bacteria. In vivo experiment with this drug proved that it could offer significant protection to mice challenged with a virulent bacterium Salmonella typhimurium NCTC74. Thus, cetirizine has immense potential to be developed as an antibacterial agent. Therefore the present study would help in corroborating future prospects in disseminating efficacious use of cetirizine in mitigation of microbial infections.

Keywords

Non-antibiotic, antiallergic, antibacterial, bactericidal

The discovery of antimicrobial drugs, often a matter of chance and serendipity till the end of the 19th century, gradually become an exercise of detailed scientific knowledge and wisdom, which resulted in “drug explosion”. Among the antimicrobial agents, largest share belongs to the antibiotics. Enormous use of antibiotics has led not only to emergence of drug resistant bacteria, but also to increasing infections with opportunistic microorganisms. Antimicrobial drugs are the greatest contribution of the 20th century to therapeutics. Their advent changed the outlook of the physician about the power of drugs on diseases. As a class, they are one of the most frequently used as well as misused drugs. So, pharmaceutical industries and research organizations are constantly making an effort to synthesize new antibiotics to combat drug resistance. Extensive studies of various workers detect antimicrobial action in different types of drugs belonging to different pharmacological classes, such as antihistamines like bromodiphenhydramine and diphenhydramine [1], methdilazine [2], promethazine [3], trimeprazine [4], terfenadine [5], tranquilizers like promazine [6], antihypertensives like propranolol [7], methyl dihydroxyphenylalanine (methyl DOPA) [8], dobutamine [9], amlodipine [10], oxyfedrine [11], lacidipine [12], antispasmodics like dicyclomine [13,14], antipsychotics like chlorpromazine [15], fluphenazine [16], thioridazine [17], prochlorperazine [18], flupenthixol [19], antiinflammatory agents like diclofenac [20-24], flurbiprofen [25] and sympathomimetic drug dopamine hydrochloride [26]. Such drugs, having antimicrobial activity in addition to their predesignated pharmacological activity, have been grouped together under the banner of “non-antibiotics” [27].

The present study was performed with cetirizine, an antiallergic drug, to observe its antimicrobial activity by in vitro and in vivo experiments. The antimicrobial activity of this non-antibiotic could be useful in the fight against antimicrobial resistance.

Materials and Methods

Liquid media used in this study were peptone water (PW) containing 1.0% peptone (Oxoid) plus 0.5% AnalaR NaCl, nutrient broth (NB, Oxoid) and Muller Hinton Broth (MHB, Oxoid). Solid media were nutrient agar (NA), prepared by solidifying NB with 2.0% agar (NA, Oxoid), and Muller-Hinton agar (MHA, Oxoid), pH 7.2-7.4.

Total 51 bacterial strains belonging to 5 Gram-positive and 4 Gram-negative genera, comprising of 13 Grampositive and 38 Gram-negative strains were tested (Table 1, Figure 1). Many of the strains were of human origin, identified [28-30] and preserved in freeze dried state. Many of the standard strains like Staphylococcus aureus (ATCC 29157), Bacillus subtilis (ATCC 6633), Micrococcus lutea (ATCC 9341), Vibrio cholerae (ATCC 14033), Escherichia coli (ATCC 25922) and even multidrug resistant (MDR) strains like E. coli (R239), E. coli (R 224), Vibrio cholerae (DN 8), S. aureus (ML 145) and S. aureus (ML 335) were included in the study. All microorganisms were maintained at 4° at slant culture for a maximum of one month and as freeze dried culture for long term preservation [31].

Table 1: Preliminary screening of cetirizine as an antimicrobial agent by agar dilution method

Preparation of cetirizine stock solution

Cetirizine used in this study was obtained as pure dry powder of pharmaceutical grade. Specified amount of the drug was accurately weighed and transferred into a suitable sterile volumetric flask and dissolved in sterile distilled water. The flask is covered properly to protect it from light.

Determination of minimum inhibitory concentration (MIC) of cetirizine

MIC of cetirizine was accurately determined with respect to different test bacteria following the standard guideline of agar dilution techniques as described by the clinical laboratories and standard institutes (CLSI 2006). For this purpose, cetirizine was dissolved in sterile distilled water and added to molten Muller Hinton Agar (MHA) at concentration of 0 (control), 200, 400, 1000, 1400 and 2000 μg/ml. Gram-positive bacteria were grown in NB and Gram-negative bacteria in PW for 18 h and bacteria were harvested during the secondary growth phase. A suspension of organism was prepared in 5 ml sterile distilled water. The turbidity of the suspension was adjusted to a 0.5 McFarland standard with a UV/Vis spectrophotometer (Chemito UV 2600 double beam spectrophotometer) at 625 nm, which corresponded to 2.4×10⁸ CFU/ml. the inocula were prepared by further diluting the suspension 1:100 with sterile distilled water in such a manner that a 2 mm (internal diameter) loopful of culture contain 105 CFU. These were spot inoculated on the MHA plates containing increasing concentrations of the drug including the control. The plates were incubated at 37° and examined for the appearance of growth after 24 h (extended up to 72 h, if necessary). The MIC was determined as the concentration of the drug that resulted in no visible growth.

Determination of effect of cetirizine on S. aureus ML 281 and Salmonella typhi 62

To determine the bacteriostatic or bactericidal action of the antihistaminic compound, strains that are sensitive to cetirizine was selected and each of them was grown in 4 ml NB for 18 h. From that 18 h old broth culture, 2 ml was taken and added into another 4 ml of fresh NB. This was incubated at 37° for 2 h so that the bacterial culture could attain logarithmic growth phase. The number of viable cells in the culture was determined by the CFU count technique as described by Miles and Mishra (1938) [34]. At this stage cetirizine was added at a concentration higher than the respective MIC values against the selected sensitive stains. CFU counts from the culture were individually taken after 2 h, 6h and finally after 18 h [32].

In vivo antimicrobial activity

Male albino mice of Swiss strain weighing 18-20 g were taken for in vivo study. Animals were maintained at standard conditions at 21±1° and 50-60% relative humidity with a 14 h photo period. Water and dry pellet diet were given ad libitum. The virulence of the test strain S. typhimurium NCTC74 was exalted by repeated mouse passages and the median lethal dose (MLD or LD₅₀) of the passaged strains was determined. From this the 50×MLD of the strains corresponding to 0.95×10⁹ CFU/mouse suspended in 0.5 ml NB served as the challenge dose [33] for all groups of animals. Reproducibility of the challenged dose was ensured by standardization of its optical density in a colorimeter at 640 nm and determination of the CFU count in NA [34].

To determine the toxicity of cetirizine, 40 mice were taken, 20 of which were injected with 60 μg of drug while the remaining 20 received 100 μg of cetirizine. They were kept under observation for up to 100 h. The protective capacity of cetirizine was judged as follows: two groups of mice, 20 animals per group were kept in separate cages. Group I was intraperitoneally administered 60 μg cetirizine per mouse and group II was given 100 μg of the drug per mouse. After 3 h each group was challenged with 50 MLD of S. typhimurium NCTC 74. A control group of 40 mice was also injected similarly with the same bacterial strain and 0.1 ml sterile saline instead of cetirizine. The protective capacity of the drug was determined by recording the mortality of mice in different groups up to 100 h of treatment and statistically by chi square test.

In another study, 4 groups of 5 mice were used. Group 1 and 3 were injected 100 μg of cetirizine intraperitoneally and each mice of group of 2 and 4 received 0.5 ml of sterile saline instead of drug. After 3 h of treatment, all groups were given 50 MLD challenges of S. typhimurium NCTC74. After 2 h, all mice of group 1 and 2 were sacrificed and their heart blood was collected, livers and spleens were separated aseptically and homogenized in tissue homogenizers. CFU count of individual organs was determined separately. The same procedure was applied to groups 3 and 4, 18 h after challenge. The data obtained were statistically analyzed by student t-test. All the animal experiments were carried out following Institutional Animal Ethical Committee guidelines (955/A/06/CPSEA2006).

Results and Discussion

A primary screening of cetirizine against 7 bacteria belonging to Gram-negative and 4 Gram-positive genera shows satisfactory antimicrobial activity against most of the test bacteria (Table 1). In an elaborate in vitro study, the drug was tested against 42 different strains of bacteria belonging to both Grampositive and Gram-negative genera. Six S. aureus three Bacillus sp., one Enterococcus, Three E. coli, four V. cholera, three Shigells and twenty two Salmonella sp. were used in the elaborate study. Many of them were inhibited at 200-2000 μg/ml concentration; few were also susceptible below 200 μg/ml concentration. The order of sensitivity towards cetirizine was Bacillus sp., Vibiro cholera, S. aureus, Escherichia coli, Shigella sp. But few strains of S. aureus, E. coli, Shigella sp. and Salmonella sp. were not inhibited at test concentration.

The MIC of cetirizine against S. typhi 62 and S. aureus ML281 was 1000 μg/ml; in logarithmic growth phase their CFU count was 5×10⁶ CFU/ml for both of them. At 0 h, 2×MIC of cetirizine of the test organisms was added to each of the culture tubes. Subsequently, when the CFU counts were determined after 2, 6, and 18 h, it was noticed that there was a gradual decrease in the number of viable cells up to 6 h for both bacteria. The decrease in CFU count were 5×10¹⁰, 4×10¹⁰, 2×10⁷ in the case of S aureus ML281 whereas 5×10¹⁰, 1×10⁸, 1×10⁶ in the case of S. typhi 62, respectively. However there were no viable cells found after 18 h, proving the bactericidal property of drug (Figure 2).

Table 2 shows that in control group 49 out of 60 animals died within 100 h of challenge. No mortality was recorded in those groups that received highest concentration of drug (100 μg/ml). As can be seen in Table 3, by comparing the CFU count in heart blood, liver and spleen at 2 h and 18 h, it is evident that there is no significant increase in viable count in drug treated group even after 18 h, thus clearly indicating the bacteriostatic nature of cetirizine (Figure 3).

Table 2: Determination of in vivo protection by cetirizine

Table 3: Efficacy of cetirizine in reducing bacterial counts in challenged mice

The search for antimicrobials has now been extended to a class of compounds named non-antibiotics which are employed for the therapy of noninfectious pathologies and which demonstrate significant antimicrobial activity against some of the most pathogenic infectious agents such as vancomycin resistant or methicillin resistant S. aureus [35] or MDR Mycobacterium tuberculosis [36-38].

Cetirizine dihydrochloride is an antagonist of histamine, mostly against H1 receptor. It inhibits effect of histamine in H1 receptor of smooth muscle. It also blocks capillary permeability to prevent edema, but as a second generation ethanolamine, it does not cause sedation. Cetirizine HCl is a white powder having a molecular weight of 461.8. Chemically it is (±)-2- [2- [4- [(4-chloropenyl)phenylmethyl]ethoxy] acetic acid dihydrochloride (Figure 4). This drug is sensitive to light and freely soluble in water, partially insoluble in acetone and methyl chloride [39,40].

Cetirizine being a H1 receptor antagonist is used in conditions like upper respiratory allergies, pollinosis, urticarial/atopic dermatitis; also used as adjuvant in seasonal asthma. It is a metabolite of hydroxyzine with marked affinity for peripheral H1 receptor; penetrates brain poorly but subjective drowsiness has been experienced at higher doses. It is not metabolized, does not prolong cardiac action potential or produce arrhythmias when given with erythromycin, ketoconazole. Cetirizine also inhibits release of histamine and cytotoxic mediators from platelets as well as eosinophil chemotaxis during the secondary phase of allergic response. Thus it may benefit allergic disorder by other actions as well. It attains high and longer lasting concentration in skin which may be responsible for superior efficacy in urticarial/atopic dermatitis, as well as once daily dosing although the elimination half-life (t1/2) is 7-10 h [41,42]. In addition to these pharmacological actions, cetirizine has significant activity on several Gram-positive and Gram-negative bacteria in vitro and S. typhimurium in vivo. The study reaffirms the antimicrobial activity of this class of drugs.

Most of the bacteria tested were inhibited within 1000 μg/ml concentration of the drug whereas few Gram-positive and Gram-negative bacteria were killed by the drug at much lower concentration (200-400 μg/ml) of the drug. In an in vitro study, cetirizine is proved as a bactericidal agent, which was performed in a Gram-positive bacteria S. aureus ML281 and a Gram-negative bacteria S. typhi 62. So from the in vitro study it can be concluded that cetirizine produced antimicrobial activity at around 1000 μg/ml concentration but in the in vivo study cetirizine gave significant protection to the challenged mice (with S. typhimurium NCTC74) at 100 μg/ml concentration. In another in vivo study where the viable count of organ homogenates and heart blood were compared to control and drug treated challenged mice, the result were highly significant (P<0.5). Examinations among various classes of pharmacological agents have revealed that in general the tricyclic phenothiazines possess discernible antimicrobial action [43]. Extensive reviews of literature have revealed that antimicrobial properties of several phenothiazines and other antimicrobial agents are due to the presence of aromatic rings [44-46].

Antihistaminic drug cetirizine contains aromatic ring and piperazine ring with halogens. The promising antimicrobial activity of this drug may be attributed to these structural components. Thus, cetirizine stands a chance of being developed as an antimicrobial agent to combat microbial resistance and bacterial infection associated with allergic reactions.

The main limiting factor of non-antibiotics to display their antimicrobial characteristics in mammalian system is that the maximum serum level remains (approximately 1 mg per liter) lower than the concentration required for inhibiting microbial growth. However this level might be sufficient to modify microbial metabolism and act synergistically with certain antibiotics [47]. On the other hands the currently published information describes in vitro and in vivo efficacy in animals. There is very limited clinical information that indicates clinically relevant activity of non-antibiotics in human. In addition, there is a need to take thermodynamics into account in vivo. On the basis of this information new approaches to the infection can be easily designed.

Acknowledgements

The authors wish to thank Ms. Dusap Pharmaceuticals Limited, Kolkata for providing the sample to cetirizine dihydrochloride to carry out the research work. The authors are also thankful to Gupta College of Technological Sciences, India for providing necessary infrastructural facility to carry out the entire research work.

Conflicts of interest

There are no conflicts of interest.

Financial support and sponsorship

Nil.

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