This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Brazilian Journal of Microbiology (2011) 42: 980-991
`ISSN 1517-8382
`
`IN VITRO ANTIBACTERIAL ACTIVITY OF SOME ANTIHISTAMINICS BELONGING TO DIFFERENT GROUPS
`
`AGAINST MULTI-DRUG RESISTANT CLINICAL ISOLATES
`
`Moustafa A. El-Nakeeb, Hamida M. Abou-Shleib, Amal M. Khalil, Hoda G. Omar, Omar M.El-Halfawy*
`
`Pharmaceutical Microbiology Department, Faculty of Pharmacy, Alexandria University, Egypt.
`
`Submitted: March 14, 2010; Approved: March 14, 2011.
`
`ABSTRACT
`
`Antihistaminics are widely used for various indications during microbial
`
`infection. Hence,
`
`this paper
`
`investigates the antimicrobial activities of 10 antihistaminics belonging to both old and new generations
`
`using multiresistant Gram-positive and Gram-negative clinical
`
`isolates. The bacteriostatic activity of
`
`antihistaminics was investigated by determining their MIC both by broth and agar dilution techniques
`
`against 29 bacterial strains. Azelastine, cyproheptadine, mequitazine and promethazine were the most active
`
`among the tested drugs. Diphenhydramine and cetirizine possessed weaker activity whereas doxylamine,
`
`fexofenadine and loratadine were inactive even at the highest tested concentration (1 mg/ml). The MIC of
`
`meclozine could not be determined as it precipitated with the used culture media. The MBC values of
`
`antihistaminics were almost
`
`identical
`
`to the corresponding MIC values. The bactericidal activity of
`
`antihistaminics was also studied by the viable count technique in sterile saline solution. Evident killing
`
`effects were exerted by mequitazine, meclozine, azelastine and cyproheptadine. Moreover, the dynamics of
`
`bactericidal activity of azelastine were studied by the viable count technique in nutrient broth. This activity
`
`was found to be concentration-dependant. This effect was reduced on increasing the inoculumsize while it
`
`was increased on raising the pH. The post-antimicrobial effect of 100 pg/ml azelastine was also determined
`
`and reached up to 3.36 h.
`
`Key words: Antihistaminics; bactericidal activity; bacteriostatic activity; Gram-negative isolates; Gram-
`
`positive isolates
`
`INTRODUCTION
`
`phenothiazines where all the classes share a certain common
`
`structural feature (33). However, pharmacologically, they are
`
`Antihistaminics
`
`are
`
`histamine Hy,-antagonists
`
`-also
`
`classified into first-generation, whose members are sedating,
`
`
`
`known as H,-receptor antagonists and H,-antihistaminics (34). are_relativelyand second-generation, whose members
`
`
`
`
`
`Chemically, they are classified into several classes including
`
`nonsedating and more selective; such classification is now
`
`ethanolamines, ethylenediamines, piperazines, piperidines and more commonlyused (32).
`
`*Corresponding Author. Mailing address: Pharmaceutical Microbiology Department, Faculty of Pharmacy, Alexandria University, El-Khartoom Square,
`
`Azarita, P.O. 21521, Alexandria, Egypt.; Tel: 002-010-8145125.; E-mail: omar. alhitawy @ alexpharmacy.edu.es
`
`980
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`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`Antihistaminics are available on the market
`
`in many
`
`almost received no attention from the microbiological point of
`
`pharmaceutical dosage forms for a variety of uses which
`
`view; for that reason, this paper deals with the microbiological
`
`mainly include the managementof allergic conditions and the
`
`testing of possible activities of 10 antihistaminics belonging to
`
`symptomatic treatment of cough and cold when used in
`
`compound
`
`preparations
`
`(34). Other
`
`uses
`
`of
`
`some
`
`antihistaminics include their use as antiemetic, anti-motion
`
`both old and new generations using antibiotic multiresistant
`clinical isolates.
`
`sickness, antiparkinsonism, sleep aids and appetizers (33, 34).
`
`MATERIALS AND METHODS
`
`The use of antihistaminics in the drug regimen for patients
`
`who acquire microbial infection is inevitable and that gave rise
`
`Microorganisms
`
`to
`
`the
`
`need
`
`to
`
`assess
`
`the
`
`antimicrobial
`
`activity
`
`of
`
`A total of twenty five bacterial isolates was used in this
`
`antihistaminics. Few studies were previously carried out to
`
`study belonging to two Gram-positive and four Gram-negative
`
`demonstrate
`
`the
`
`antimicrobial
`
`activity of
`
`a number of
`
`genera. They were human isolates
`
`identified by classical
`
`antihistaminics which belonged mainly to the first generation
`
`microscopical
`
`and biochemical
`
`procedures
`
`a9,
`
`23).
`
`In
`
`especially the ethanolamine and phenothiazine antihistaminics;
`
`addition,
`
`the
`
`following
`
`standard
`
`strains were
`
`used:
`
`however, the published results are rather controversial.
`
`Staphylococcus aureus
`
`(ATCC 6538P),
`
`Escherichia coli
`
`Dastidar et al.
`
`(20) found that diphenhydramine and
`
`(NCTC 10418), Klebsiella pneumoniae (ATCC 35657) and
`
`bromodiphenhydramine inhibited several Gram-positive and
`(ATCC=9027).
`Pseudomonas
`aeruginosa
`They were
`maintained at 4°C as slant cultures ofsterile nutrient agar for a
`
`Gram-negative strains at concentrations ranging from 0.05 to
`
`0.2 mg/ml. On the other hand, Semenitz (30) reported much
`
`maximum of one month (35). Long term preservation was
`
`performedby freezing in 15% glycerol broth (26).
`
`higher MIC values for diphenhydramine that ranged from 1.8
`
`to 15 mg/ml. However, certain members of phenothiazine
`
`antihistaminics were shown to have MIC that ranged mostly
`
`Antihistaminics
`
`between 10 and 200 ug/ml against several Gram-positive and
`
`Gram-negative bacterial
`
`strains
`
`(16,
`
`18,
`
`21,
`
`27,
`
`29).
`
`The antihistaminics used in this study were obtained as
`Azelastine
`
`of pharmaceutical grade:
`
`pure dry powders
`
`Nonetheless, Shibl et al. (31) revealed MIC values as low as
`
`hydrochloride
`
`(Aze),
`
`Cetirizine
`
`dihydrochloride
`
`(Cet),
`
`1.6 ug/ml against a S. aureus strain. In addition to the varied
`
`Cyproheptadine
`
`hydrochloride
`
`(Cyp),
`
`Diphenhydramine
`
`MIC ranges,
`
`the
`
`spectrum of antibacterial
`
`activity was
`
`somehow variable in the previous studies where the tested
`
`hydrochloride
`Fexofenadine
`
`(Dip),
`
`Doxylamine
`
`hydrochloride
`
`(Fex),
`
`succinate
`Loratadine
`
`(Dox),
`
`(Lor),
`
`phenothiazines were generally potent against the Gram-positive
`
`Meclozine hydrochloride
`
`(Mec), Mequitazine
`
`(Meq)
`
`and
`
`microorganisms; however
`
`their effect against
`
`the Gram-
`
`Promethazine (Pro). They were preservedat 4°C.
`
`negative ones was either comparable to that against the Gram-
`
`positive ones or inferior to it. Moreover, some phenothiazine
`
`Preparation of antihistaminic stock solutions
`
`antihistaminics showed certain anti-tuberculosis activity (13,
`
`Specified amounts of
`
`the tested antihistaminics were
`
`17, 36).
`
`accurately weighed and transferred separately into suitable
`
`As previous studies were almost restricted to some of the
`
`sterile volumetric flasks. Water soluble antihistaminic powders
`
`old membersof antihistaminics while the new onesparticularly
`
`(azelastine,
`
`cetirizine,
`
`cyproheptadine,
`
`diphenhydramine,
`
`those belonging to the second generation of antihistaminics
`
`doxylamine, meclozine and promethazine) were dissolved in
`
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`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`sterile distilled water. Fexofenadine was dissolved in the
`
`minimal amount of methanol then diluted with sterile distilled
`
`Determination of the dynamics of bactericidal activity of
`azelastine
`
`water. Loratadine and mequitazine were dissolved in minimum
`
`amounts of 95% ethanol and dimethylsulphoxide (DMSO)
`
`Dynamics of bactericidal activity of 50 and 100 ug/ml
`azelastine were determined in nutrient broth. The inoculum
`
`respectively then diluted with sterile distilled water to form a
`
`size used was ~10° cells/ml. The systems were incubated in the
`
`colloidal dispersion.
`
`shaking orbital incubator (A. Gallenkamp & Co. Ltd, United
`
`Kingdom)
`
`at 37°C and 35.
`
`strokes/min. Samples were
`
`Determination of minimum inhibitery concentration (MIC)
`of antihistaminics
`
`aseptically withdrawn from each flask at 0, 3, 6 and 24 hours
`for the viable count determination.
`
`The MIC of each antihistaminic against various strains
`
`employed in this
`
`study was determined by the broth
`
`Study of the effect of some factors on the bactericidal
`
`macrodilution technique and the agar dilution technique as well
`
`activity of azelastine
`
`@G).
`
`Determination of minimum bactericidal concentration
`
`(MBC) of antihistaminics
`
`The effect of different factors on the bactericidal activity
`
`of azelastine was determined in nutrient broth by surface viable
`
`count technique. All systems were incubated in the shaking
`orbital incubator at 37°C and 35 strokes/min. The effect of
`
`The MBC ofeach antihistaminic against the tested strains
`
`azelastine concentrations ranging from 0 to 200 ug/ml was
`
`was determined by the broth macrodilution technique (12) by
`
`determined after withdrawing samples at 0 and 6 hours. The
`
`subculturing 0.1 ml portions of each test tube showing no
`
`inoculumsize used was ~ 10° cells/ml.
`
`visible growth in the MIC experimentinto test tubes containing
`
`5 ml antihistaminic-free sterile nutrient broth (Oxoid).
`
`Determination of the bactericidal activity of antihistaminics
`
`The effect of inoculum size wasalso studied; three inocula
`of about 10°, 10° and 10” cells/ml were used. Similarly,
`the
`
`effect of 4 different pH values (5, 6, 7 and 8) was studied using
`the corresponding sterile phosphate buffers (14) at ~ 10°
`
`using the viable count technique
`
`cells/ml inoculum size. The pH of each system was checked
`
`The bactericidal activity of antihistaminics
`
`in final
`
`using pH meter (pH 211 Microprocessor pH meter, HANNA,
`
`concentrations ranging from 50 to 200 ug/ml was determined
`in sterile saline solution. Stock solutions of the antihistaminics
`
`Romania) after addition of azelastine and adjusted if necessary.
`
`Proper controls lacking azelastine were included for each
`
`at 10X the required concentrations were 10-fold diluted into
`
`inoculum size and pH. After
`
`incubation,
`
`samples were
`
`the prepared bacterial suspensions and mixed at the zero time
`then incubated at 37°C for 24 hours. The final inoculum for
`
`aseptically withdrawn at 0, 3, 6 and 24 hours for the previously
`
`described viable count technique.
`
`each of the tested isolates was ~ 10° cells/ml in the sterile
`
`Determination of the post-antimicrobial effect
`
`(PAE) of
`
`saline solution. Proper controls lacking the antihistaminics
`were included in eachtest.
`
`Samples were aseptically withdrawn at 0, 6 and 24 h and
`
`azelastine turbidimetrically (22)
`
`An overnight broth culture of each of the selected isolates
`was 10° diluted in prewarmed sterile nutrient broth and
`
`10-fold serially diluted with sterile saline solution. The number
`
`incubated in a water-bath (GFL, Germany) at 37°C with
`
`of survivors was determined by the surface viable count
`
`agitation (50 rpm). The absorbance of
`
`the culture was
`
`technique. The plates were incubated at 37°C for 24 h.
`
`monitored with aspectrophotometer using a wavelength of 600
`
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`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`nm, until an absorbance of ~0.25 was reached (equivalent to
`~10’ cells/ml). Treatment was carried out with 100 pg/ml
`azelastine. A control untreated flask was included in the
`
`experiment.
`
`study was investigated through MIC determination against all
`
`the
`
`tested organisms both by broth and agar dilution
`
`techniques. Both methods yielded similar results shown in
`Table 1. Both the standard strains and the multiresistant clinical
`
`The bacteria—drug contact lasted | h, at the end of which
`drug activity was stopped by performing a 10% dilution to the
`
`isolates
`
`showed similar
`
`responses
`
`to the action of
`
`the
`
`antihistaminics. The Ps. aeruginosa strains were insensitive to
`
`cultures in drug-free prewarmed nutrient broth. The control
`
`the tested antihistaminics at the studied concentration range
`
`culture was also subjected to the same dilution and growth
`
`except the phenothiazine ones, mequitazine and promethazine.
`
`turbidity was determined under identical conditions without
`
`The tested phenothiazines and cyproheptadine were the most
`
`antihistaminic exposure. All the cultures were further incubated
`
`effective among the studied antihistaminics and were active
`
`at 37°C with agitation and the absorbance was measured hourly
`at 600 nm until > 0.1 O.D. was reached and the PAE was
`
`against both Gram-positive and Gram-negative bacteria. The
`
`obtained MIC range of promethazine wassimilar to the results
`
`calculated as described by Dominguezet al (22).
`
`obtained by Kristiansen and Moratensen (27), Chakrabarty et
`
`RESULTS AND DISCUSSION
`
`In the present work, a total of 29 bacterial strains and
`clinical isolates obtained from different sources were used. The
`
`identified clinical isolates showed multiresistance to different
`
`extents upon testing their
`
`susceptibility to 25 different
`
`antibiotics by the disc diffusion technique (4). Multiresistance
`was considered on the basis that the studied clinical isolates
`
`al. (16) as well as Molnar et al.(29) concerning the MIC of
`
`promethazine. However, Shibl ef al. (31) determined the MIC
`
`of promethazine against only | strain (S. aureus NCTC 6571
`
`standard strain) and foundit to be 6.2 ug/ml, far lower than that
`
`obtained in the present work (Table 1). In that case, the authors
`
`only performed broth dilution technique using a different
`medium.
`
`Azelastine, a new generation phthalazinone derivative,
`
`were resistant to antibiotics belonging to at least 3 classes and
`
`demonstrated significant bacteriostatic activity which was more
`
`up to all tested antibiotics. Whereas the standard strains used
`
`pronounced against the tested Gram-positive organisms (Table
`
`were selected so that
`antibiotics.
`
`they were sensitive to the tested
`
`1), and hence it was used in further studies for reasons
`
`discussed later. It showed moderate activity against the tested
`
`The bacteriostatic activity of the antihistaminics under
`
`E. coli and Klebsiella spp. strains.
`
` .
`
`.
`Organism (numberofstrains)
`
`
`Table 1. MIC ranges of antihistaminics
`
`Meq
`Pro
`Cyp
`Aze
`Others'
`Others”
`MIC, pg/ml
`62.5-125
`125-250
`
`S. aureus (5)
`
`62.5-125
`
`125-250
`
`500-1000
`
`>1000
`
`S. epidermidis (2)
`E. faecium(2)
`E. coli (6)
`
`125
`62.5
`125-250
`
`62.5
`62.5-125
`250-500
`
`125
`62.5
`125-250
`
`125
`125
`1000
`
`>1000
`1000
`>1000
`1000
`500- >1000 >f000
`
`>1000
` 500->1000
`125-1000
`62.5-250
`125-250
`62.5-125
`Klebsiella spp. (5)
`1000->1000 >1000
`>1000
`250-1000
`500-1000
`250-1000
`Pr. mirabilis (3)
`Ps. aeruginosa (6) 1000->1000 >1000 500 125-1000 >1000 >1000
`
`Others': Dip and Cet
`Others*: Dox, Fex, Lor and Mec
`
`
`
`
`
`
`
`
`
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`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`Other antihistaminics shown also in Table | possessed a
`
`The bactericidal activity of the antihistaminics under study
`
`slight bacteriostatic activity, under the conditions of the test,
`
`was evaluated by the dilution methods (data not shown). The
`
`against both tested Gram-positive and Gram-negative bacteria.
`
`MBC values of the antihistaminics were mostly identical
`
`to
`
`These histamine
`
`antagonists were diphenhydramine
`
`and
`
`their MIC values. The bactericidal activity of the tested
`
`cetirizine, however,
`
`they lacked activity against
`
`the Ps.
`
`antihistaminics was also investigated by the viable count
`
`aeruginosa
`
`and Pr. mirabilis
`
`isolates
`
`in
`
`the
`
`tested
`
`technique against 4 Gram-positive and 4 Gram-negative
`
`concentration range. This agrees in part with the findings of
`
`clinical isolates exposed for 6 and 24 hr (Table 2). In thistest,
`
`Semenitz (30) and Dastidar et al. (20) regarding the activity of
`
`sterile saline solution which did not precipitate any of the
`
`diphenhydramine. Moreover,
`
`Semenitz (30) reported 2 to 8
`
`tested antihistaminics was used. This system was also used to
`
`folds higher MIC values than those obtained in the present
`
`simulate some pharmaceutical dosage forms such as eye drops
`
`study (Table 1). On the contrary, Dastidar et al. (20) mentioned
`
`or nasal sprays or drops where the vehicle is the physiological
`
`200 ug/ml as MIC of diphenhydramine against several S.
`
`isotonic solution. In case of azelastine, cyproheptadine and
`
`aureus, E. coli, Kl. pneumoniae and Pr. mirabilis isolates. This
`
`value is approximately half to quarter the level of MIC in Table
`
`mequitazine the two concentrations used (50 and 100 Lg/ml)
`were lower than those selected for the other antihistaminics
`
`1. The other
`
`studied antihistaminics namely meclozine,
`
`(100 and 200 pg/ml) becauseoftheir relative high antibacterial
`
`loratadine, fexofenadine and doxylamine did not exhibit any
`
`activity.
`
`In general,
`
`increasing the concentration of
`
`the
`
`bacteriostatic effects in the studied range of concentrations
`
`antihistaminics resulted in higher killing effects whenever an
`
`(Table 1). It is noteworthy to mention that the lack of activity
`
`antihistaminic demonstrated antibacterial activity against a
`
`of meclozine might be attributed to its precipitation with the
`
`tested clinical isolate. However, Table 2 showsthe results of
`
`phosphates and proteins upon the addition to either nutrient
`
`the 100 g/ml concentration of all tested antihistaminics to
`
`agar or nutrient broth.
`
`compare betweentheir relative activities.
`
`Table 2. Bactericidal effect of 100 pg/ml antihistaminics against selected isolates in 0.9% saline solution by viable count technique at 37°C.
`
`Time, h
`
`Antihistaminic
`
`Organism*
`
`Sayo3
`Sayo4
`Seior
`Efig.
`Ecig3
`Ecos
`Kliop
`Psi02
`
`Log Plating Efficiency
`Meq
`6
`-3.579
`-3.146
`-3.844
`-4.145
`-4.179
`-1.948
`-2.293
`-3.672
`
`24
`-2.838
`-4.040
`-3.777
`-3.476
`-3.426
`-2.558
`-3.845
`-2.796
`Cyp
`6
`-1.389
`- 1.032
`-0.013
`-1.192
`-0.062
`-0.320
`0.029
`-0.393
`
`24
`- 1.663
`-2.087
`-0.456
`-2.176
`0.068
`- 1.102
`0.014
`-0.199
`Aze
`6
`-2.580
`-1.690
`-0.289
`-3.146
`-3.702
`-0.550
`-0.110
`-0.371
`
`24
`-2.838
`-2.699
`-1.875
`-3.476
`-5.264
`-1.373
`-0.474
`-0.489
`Mec
`6
`0.363
`-1.243
`-1.146
`-1.192
`-0.599
`-1.333
`-0.069
`-1.092
`
`24
`-2.838
`-4.040
`-2.477
`-2.000
`0.078
`-2.868
`-0.469
`-0.799
`
`Dip
`6
`-0.103
`-0.544
`0.196
`-0.146
`-0.096
`-0.032
`0.083
`-0.009
`
`24
`-1.839
`-0.087
`-0.234
`-1.097
`0.014
`-1.248
`-0.026
`-0.246
`Cet
`6
`-0.349
`-0.669
`-0.088
`-0.867
`-0.660
`-0.897
`-0.008
`-0.029
`
`24
`- 1.838
`-3.040
`-0.499
`-1.097
`- 1.094
`- 1.558
`-O.117
`-0.938
`Dox
`6
`0.101
`0.385
`-0.414
`0.000
`0.131
`0.249
`0.071
`-0.417
`
`24
`0.349
`0.357
`-1.632
`-0.310
`0.043
`-0.409
`0.012
`-0.074
`Fex
`6
`0.312
`0.535
`0.234
`-0.105
`0.062
`0.103
`-0.026
`-0.827
`
`24
`0.426
`-0.219
`-0.135
`-0.316
`0.057
`-1.134
`0.024
`-1.090
`Lor
`6
`-0.376
`-0.209
`0.058
`-0.327
`0.165
`0.282
`-0.008
`-0.330
`24 -0.881 -0.392 -1.082 -0.444 -0.394 -0.032 -0.524 -0.017
`
`
`
`
`
`
`
`
`*Organisms. Sajo3 and Sajo4: S. aureus isolates, Seio: S. epidermidis, Efip: E. faecium isolate, Eci9; and Ecios: E. coli isolates,
`Kliw:: Kl. Pneumonia isolate, Psig2: Ps. aeruginosa isolate
`
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`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`Again, mequitazine demonstrated significant bactericidal
`
`could be adsorbed onto the surface of the bacterial cells which
`
`effects against all
`
`the tested clinical
`
`isolates including Ps.
`
`might facilitate their effect on their membranes. It has been
`
`aeruginosa (Table 2). However, its effect against the Gram-
`
`stated that increasing the hydrophobicity increases the surface
`
`positive on_thisisolates was more pronounced. Azelastine and activity (6). Therefore, based assumption,
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`cyproheptadine also exhibited remarkable killing activities
`
`antihistaminics with the most powerful antibacterial activity
`
`mainly against the Gram-positive isolates (Table 2). Cetirizine
`
`should be the most surface-active and be highly hydrophobic.
`
`and diphenhydramine also produced moderate bactericidal
`
`The
`
`phenothiazine
`
`derivatives
`
`for
`
`example,
`
`possess
`
`effects, but in relatively higher concentration (data not shown).
`
`considerable surface activity where their CMCisrelatively low
`
`In the above mentioned bactericidal activity tested in
`
`saline solution, meclozine showed remarkable bactericidal
`
`characters gained partly by the
`
`and aggregation number is high owing to their hydrophobic
`in the
`
`sulphur
`
`atom
`
`effects against all the tested clinical isolates being more active
`
`phenothiazine
`
`ring
`
`(2).
`
`Thus,
`
`both
`
`promethazine
`
`and
`
`against the Gram-positive ones (Table 2). This was contrary to
`
`mequitazine
`
`exhibit
`
`recognizable
`
`antibacterial
`
`effects.
`
`the results of the MIC experiment in which precipitation of the
`
`Similarly, cyproheptadine, having considerable surface activity
`
`drug occurred with the
`
`test medium. From the
`
`above
`
`(10), manifests marked antibacterial effects. Likewise,
`
`the
`
`bactericidal activity of antihistaminics estimated in saline
`
`chlorine atom substituent in the aromatic rings of meclozine
`
`increases their surface activity and
`
`solution, it can be concluded that active growth of the tested
`
`and azelastine (15, 24)
`
`organisms does not
`
`seem to be essential
`
`for whatever
`
`reduces their CMC values (10). On the other hand, moderate
`
`antibacterial activity that the drugs might have.
`
`antibacterial activity seems to be related to intermediate surface
`
`The variation in the magnitude of antibacterial effects
`
`characters. Diphenhydramine, having moderate surface activity
`
`among different antihistaminics is however difficult to explain
`
`manifested as intermediate CMC values (5), demonstrated
`
`since screening the literature revealed that no extensive studies
`
`certain antibacterial effects. Similarly, cetirizine have reduced
`
`were published on the antibacterial activity of the different
`
`surface properties, although possessing a chlorine substituent
`
`classes of
`
`antihistaminics. Thus,
`
`reviewing
`
`a_ possible
`
`which should increase its hydrophobic character,
`
`as
`
`the
`
`explanation of such varied antimicrobial activity will be
`
`sustituent’s effect
`
`is counteracted by the highly hydrophilic
`
`attempted. Such explanation may relate the mechanism of
`antibacterial
`action of antihistaminics
`to their
`chemical
`
`structure by analogy with other therapeutic classes having
`similar structural features. Since the main structural feature of
`
`carboxylic group substituent. The rest of antihistaminics
`
`demonstrated poor antimicrobial activity which matched with
`their
`low surface characters where both loratadine and
`
`fexofenadine
`
`show considerable
`
`hydrophilic
`
`characters
`
`antihistaminics is a tertiary amino group and a bulkylipophilic
`
`imparted by ester group attached to the nitrogen atom in the
`
`aromatic moiety, they possess certain surfactant-like characters
`
`former and a hydroxyl group substituent in the latter which
`
`(5, 11). Owing to surface activity of amphipathic compounds, it
`
`would
`
`significantly
`
`reduce
`
`their
`
`surface
`
`activity
`
`and
`
`has been reported that
`
`they might cause alteration in the
`
`consequently the possible effect upon the membranes (10). On
`
`function and permeability of biological membranes in general
`
`the other hand, doxylamine although belonging to the same
`
`(25, 28). The extent of adsorption onto the membranes due to
`
`chemical class as diphenhydramine, it possessed much weaker
`
`surface activity has been correlated with their damaging effects
`
`antibacterial effect. This might be attributed to the different
`
`(1, 10). These postulates were investigated by Shibl et al. (31)
`
`counter ion effect (succinate) which maylead to altered surface
`
`who demonstrated that certain phenothiazine antihistaminics
`
`activity and change in the aggregation character from micelle
`
`985
`
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`Exhibit 2030 Page 6
`
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`
`

`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`formation to a stacking process: continuous self-association
`
`antihistaminic or any memberofits class were available, it has
`
`with no CMC (6, 8).
`
`been ofinterest to investigate its antimicrobial activity.
`
`After screening antihistaminics, representing most of the
`
`The dynamics of bactericidal activity of azelastine was
`
`chemical classes of the histamine H,-antagonists and belonging
`
`also determined by the viable count
`
`technique. Since it
`
`to both first and second generations, for bacteriostatic and
`
`possessed markedkilling effects, 2 concentrations of azelastine
`
`bactericidal activities,
`
`further
`
`studies were performed on
`
`were tested against 3 Gram-positive and 2 Gram-negative
`
`azelastine in order to investigate its observed antibacterial
`
`clinical isolates. Azelastine continued to demonstrate powerful
`
`activity. This antihistaminic is relatively new, belonging to the
`
`bactericidal effects against the tested clinical isolates especially
`
`phthalazinone class of antihistaminics and showed promising
`
`against the Gram-positive ones (Fig. 1) whereas the higher
`
`antimicrobial activity. Moreover,it is clinically used locally as
`
`tested concentration was required to exert similar effects
`
`nasal sprays and eye drops andits therapeutic dose lies within
`
`against
`
`the tested E. coli
`
`isolate (not shown). However,
`
`it
`
`the range of its demonstrated antibacterial activity. And since
`
`lacked the activity against Ps. aeruginosa isolate as shown also
`
`no studies
`
`regarding the microbiological
`
`aspect of
`
`this
`
`in the previous experiments.
`
`
`
`
`
`LL
`
`~ aA
`
`n aA>:°
`
`
`Logsurvivors,CFU/ml ~
`
`
`CFU/ml Ui ut
`a2NN2KeEPohotheothOolt
`Logsurvivors,
`
`
`
`
`
`
`
`
`1Ll
`
`
`
`12
`
`18
`
`24
`
`986
`
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`Exhibit 2030 Page 7
`
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`
`

`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`oOoo owa
`
`~ La
`
`
`
`
`
`Logsurvivors,CFU/ml
`
`=e
`
` BOWWeONwaoOUMoOUMOS
`
`
`
`tad a
`
`12
`Time, h
`
`18
`
`24
`
`Figure 1. Dynamics of the antibacterial activity of Aze 50 and 100 ug/ml against
`
`S. aureus Sayp3 (A), S. aureus Sajo4 (B) and E. faecium Efp, (C) isolates at 37°C
`
`e Control; A Aze50; m Azel00
`
`The effect of some factors was studied on the bactericidal
`
`number of monomer molecules forming the micelle). At pH values
`
`activity of azelastine. First, the activity of azelastine was tested in
`
`approaching the pKa, an increase in the aggregation number
`
`different concentrations ranging between 0 and 200 pg/ml. The
`
`occurs due to the increased percentage of the non-ionized more
`
`outcome of
`
`this experiment
`
`showed that
`
`the antihistaminic
`
`hydrophobic base. Thus,
`
`it has been reported that
`
`the non-
`
`bactericidal activity was in general concentration-dependant(Fig.
`
`protonated drug present at high pH values demonstrated a dramatic
`
`2). Table 3 showed that
`
`the bactericidal activity of azelastine
`
`increase in the surface activity (37). It was also reported that as the
`
`increased by decreasing the inoculum sizes of the tested organisms
`
`hydrophobicity
`
`of
`
`compounds
`
`increased,
`
`their
`
`effect
`
`on
`
`in most cases. The inoculum size effect seems to be dependant on
`the ratio between antihistaminic molecules and the number of
`
`membranes increased (10, 25). This might be the reason beyond
`
`the increased antibacterial effects of azelastine against the tested
`
`cells. In case of low inoculum,the drug could saturate more of the
`
`isolates at high pH values especially at pH 8 (Fig. 3). This also
`
`available sites of adsorption and hence be more active. The effect
`
`agreed with Attwood and Udeala (7) who reported that the surface
`
`of pH of the medium on the bactericidal activity of azelastine is
`
`pressure increase was larger in the presence of phosphate buffer at
`
`shownin Fig. 3. Fig. 3A reveals that raising the pH of the medium
`
`pH 6-8, nonetheless, they were not sure whether this effect was
`
`steadily increased the bactericidal effect of azelastine against the
`
`caused by the buffer components or was a pH effect.
`
`S. aureus isolate under study. On the other hand, azelastine almost
`
`Finally,
`
`the post-antimicrobial effect of azelastine was
`
`had no effect against E. coli Ec; isolate at slightly acidic or
`
`studied against one S. aureus and another E. coli isolates (Fig. 4).
`
`neutral pH. However,
`
`the bactericidal
`
`activity dramatically
`
`The post-exposure effect was sustained for 3.36 hr against the
`
`increased at pH 8 both at 3 and 6 hr (Fig. 3B). The pH of the
`
`tested S. aureus isolate compared to only about half an hour for
`
`medium affects the surface activity and aggregation properties of
`
`the &. colt isolate. This effect might be beneficial in reducing the
`
`the antihistaminics (9, 37). At
`
`low pH values, protonation of
`
`dose and prolonging the time interval of administration thus
`
`tertiary amine groups occurs leading to elevated critical micelle
`
`decreasing any possible adverse effects.
`
`concentrations (CMC) as well as lower aggregation number(n, the
`
`987
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2030 Page 8
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 8
`
`

`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`
`
`
`Logsurvivors,CFU/ml mn
`
`Bbeyponeeeey[ornnnnnnannnnanl
`
`AnI
`
`0
`
`25
`
`50
`
`125
`100
`75
`Concentration, pg/ml
`
`Figure 2. Effect of Aze concentrations on the growth of 5 clinical isolates at 6 hours using the viable count technique.
`
`The following initial inocula (Log CFU/ml) were used in nutrient broth at 37°C:
`
`
`
`ASaj04: 6.53; @Efy): 6.71; Ec y:: 6.97 and APS199: 6.52
`
`Table 3. Effect of inoculum size on the dynamics of antibacterial activity of Aze 50 & 100 ug/ml against S. aureus Say, and E.
`
`coli Ecjo3 isolates by viable count technique at 37°C
`
`Aze Conc.
`
`Inoculum Size (cells/ml)
`
`Organism
`Time, h
`~10°
`~10°
`~10"
`pg/ml
`
`Log Plating Efficiency
`3
`-0.125
`-0.501
`-0.495
`6
`-0.500
`-1.710
`-0.806
`24
`-2.624
`-0.885
`-0.986
`3
`-1.246
`-0.692
`-0.732
`6
`-1.830
`-1.820
`-1.533
`
`5
`
`103
`
`50
`
`100
`
`24
`-3.584
`-1.381
`-1.361
`
`3
`-0.371
`-0.068
`-0.051
`
`50
`
`Ecio3
`
`6
`24
`3
`
`-0.911
`-0.100
`-0.688
`
`-0.051
`-0.033
`-0.402
`
`0.067
`-0.060
`-0.153
`
`100
`6
`“1.251
`-1.302
`-0.514
`-0.913 -0.49224 -0.557
`
`
`
`
`
`
`
`988
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2030 Page 9
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 9
`
`

`

`E]-Nakeeb, M.A.et al.
`
`Antibacterial activity of some antihistaminics
`
`45
`
`5
`
`5.5
`
`6
`
`6.5
`
`7
`
`75
`
`8
`
`8.5
`
`2.
`
`
`
`LogPlatingEfficiency
`
`6 4
`
`.5
` 0.0
`
`1.0 7
`
`sJ
`
`
`
`LogPlatingEfficiencyb&rmSo
`
`1
`
`Figure 3. Effect of pH on the antibacterial activity of Aze 100 pg/ml against S. aureus Sajo3 (A) and EF. coli Ec;93 (B) isolates by
`viable count technique at 37°C
`
`e3h A6h,824h
`
`989
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2030 Page 10
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 10
`
`

`

`El-Nakeeb, M.A.etal.
`
`Antibacterial activity of some antihistaminics
`
`0.7
`
`= a
`
`
`
`
`
`
`
`
`
`= tn
`
`=
`
`= N
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`|
`
`
`
` Opticaldensity te
`
`1
`
`2
`
`3
`
`Time, h 4
`
`5
`
`6
`
`Figure 4. Turbidimetric growth curves showing PAE of Aze against S. aureus Say); and E. coli Ec,,3 clinical isolates
`
`
`
`
`m Sa)93 control;
`
`Sajo3 treated with Aze; @ Ecj93 control; o Ecjg3 treated with Aze
`
`REFERENCES
`
`10.
`
`Attwood, D.; Florence, A.T. (1983). Surfactant systems: Their chemistry,
`
`(1975). Toxicity of
`Abernathy, C.0.; Lukacs, L.; Zimmerman, H.J.
`tricyclic antidepressants to isolated rat hepatocytes. Biochem Pharmacol
`24(3), 347-50.
`
`(1980). The
`Ahmed, M.; Hadgraft, J.; Burton, J.S.; Kellaway, LW.
`interaction of mequitazine with phospholipid model membranes. Chem
`Phys Lipids 27(3), 251-62.
`of minimum inhibitory
`Andrews,
`J.M.
`(2001). Determination
`concentrations. J Antimicrob Chemother 48 Suppl |, 5-16.
`Andrews, J.M.
`(2007). BSAC standardized disc susceptibility testing
`method (version 6). J Antimicrob Chemother 60(1), 20-41.
`
`(1974). Aggregation of antihistamines in
`Attwood, D.; Udeala, O.K.
`aqueous
`solution: micellar properties of
`some diphenylmethane
`derivatives. J Pharm Pharmacol 26(11), 854-60.
`
`tr
`
`13.
`
`14.
`
`pharmacy and biology. Chapman and Hall Ltd.. London, p. 124-228,
`388-468, 607-610.
`
`(1998). Physicochemical principles of
`Attwood, D.; Florence, A.T.
`pharmacy. Macmillan, London, p. 199-250.
`Barry, A.L.; Craig, W.A.; Nadler, H.; Reller, L.B.; Sanders, C.C.;
`Swenson, J.M. (1999). Methods for Determining Bactericidal Activity of
`Antimicrobial Agents; Approved Guideline. NCCLS document M26-A,
`Vol. 19 (18). NCCLS, Pennsylvania, USA.
`
`Bettencourt, M.V.; Bosne-David, S.; Amaral, L. (2000). Comparative in
`vitro
`activity
`of
`phenothiazines
`against multidrug-resistant
`Mycobacterium tuberculosis. Int J Antimicrob Agents 16(1), 69-71.
`British-Pharmacopoeia-Commission, ed. (2007). App