`
`Brazilian Journal of Microbiology (201 1)42: 980—991
`ISSN1517—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, 201 l.
`
`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 inoculum size while it
`
`was increased on raising the pH. The post-antimicrobial effect of 100 rig/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
`
`Ill-antagonists
`
`-also
`
`classified into first-generation, whose members are sedating,
`
`known as Hl-receptor antagonists and Hl-antihistaminics (34).
`
`and
`
`second-generation, whose members
`
`are
`
`relatively
`
`Chemically, they are classified into several classes including
`
`nonsedating and more selective; such classification is now
`
`ethanolamines, ethylenediamines, piperazines, piperidines and more commonly used (32).
`
`*Corresponding Author. Mailing address: Pharmaceutical Microbiology Depaitment, Faculty of Pharmacy, Alexandria University, El—Khartoom Square,
`
`Azarita, PO. 21521, Alexandria, Egypt.; Tel: 002—010—8145125.; E—mail: omaralhlfaw r@alex harmac 7.edu.e ‘
`
`980
`
`UCB Biopharma SPRL (IPR2019—00400)
`Exhibit 2030 Page 1
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 1
`
`
`
`El-Nakeeb, M.A. el al.
`
`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 management of 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
`
`sickness, antiparkinsonism, sleep aids and appetizers (33, 34).
`
`The use of antihistaminics in the drug regimen for patients
`
`both old and new generations using antibiotic multiresistant
`clinical isolates.
`
`MATERIALS AND METHODS
`
`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
`
`(19,
`
`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 el al.
`
`(20) found that diphenhydramine and
`
`(NCTC 10418), Klebsiella pneumoniae (ATCC 35657) and
`
`bromodiphenhydramine inhibited several Gram-positive and
`
`Pseudomonas
`
`acruginosa
`
`(ATCC 9027).
`
`They were
`
`Gram-negative strains at concentrations ranging from 0.05 to
`
`maintained at 4GC as slant cultures of sterile nutrient agar for a
`
`maximum of one month (35). Long term preservation was
`
`0.2 mg/ml. On the other hand, Semenitz (30) reported much
`
`higher MIC values for diphenhydramine that ranged from 1.8
`
`performed by freezing in 15% glycerol broth (26).
`
`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
`
`(A26),
`
`Cetirizine
`
`dihydrochloride
`
`(Cat),
`
`1.6 ug/ml against a S. humus 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
`
`(Fa),
`
`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 preserved at 40C.
`
`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 members of antihistaminics while the new ones particularly
`
`(azclastinc,
`
`cctirizine,
`
`cyprohcptadine,
`
`diphenhydramine,
`
`those belonging to the second generation of antihistaminics
`
`doxylamine, meclozine and promethazine) were dissolved in
`
`981
`
`UCB Biopharma SPRL (IPR2019—00400)
`Exhibit 2030 Page 2
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 2
`
`
`
`El-Nakeeb, MA. 61 al.
`
`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 pg/ml
`azelastine were deterlnined 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
`
`Determination of minimum inhibitory concentration (MIC)
`of antihistaminics
`
`aseptically withdrawn from each flask at 0, 3, 6 and 24 hours
`for the Viable count determination.
`
`Kingdom)
`
`at 37°C and 35
`
`strokes/min. Samples were
`
`The MIC of each antihistaminic against Various strains
`
`employed in this
`
`study was determined by the broth
`
`Study of the effect of some factors 011 the bactericidal
`
`macrodilution technique and the agar dilution technique as well
`
`activity of azelastine
`
`(3).
`
`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 of each antihistaminic against the tested strains
`
`azelastine concentrations ranging from 0 to 200 pg/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
`
`inoculum size used was ~ 106 cells/ml.
`
`visible growth in the MIC experiment into test tubes containing
`
`The effect of inoculum size was also studied; three inocula
`
`5 ml antihistaminic—free sterile nutrient broth (Oxoid).
`
`of about 103, 105 and 107 cells/n11 were used. Similarly,
`
`the
`
`effect of 4 different pH values (5, 6, 7 and 8) was studied using
`
`Determination of the bactericidal activity of antihistaminics
`
`the corresponding sterile phosphate buffers (14) at ~ 106
`
`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 ~ 106 cells/ml in the sterile
`
`Determination of the post-antimicrobial effect
`
`(PAE) of
`
`saline solution. Proper controls lacking the antihistaminics
`Were included in each test.
`
`azelastine turbidimetrically (22)
`
`An overnight broth culture of each of the selected isolates
`
`Samples were aseptically withdrawn at 0, 6 and 24 h and
`
`was 10'3 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
`
`982
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 3
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 3
`
`
`
`E1-Nakeeb, M.A. el al.
`
`Antibacterial activity of some antihistaminics
`
`nm, until an absorbance of ~0.25 was reached (equivalent to
`
`study was investigated through MIC determination against all
`
`~107 cells/ml). Treatment was carried out with 100 ug/ml
`azelastine. A control untreated flask was included in the
`
`experiment.
`
`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 1 h, at the end of which
`
`isolates
`
`showed similar
`
`responses
`
`to the action of
`
`the
`
`drug activity was stopped by performing a 10'3 dilution to the
`
`antihistaminics. The Ps. acruginosa 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 OD. was reached and the PAE was
`
`against both Gram-positive and Gram-negative bacteria. The
`
`obtained MIC range of promethazine was similar to the results
`
`calculated as described by Dominguez et 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
`
`a]. (16) as well as Molnar el al.(29) concerning the MIC of
`
`promethazine. However, Shibl et al. (31) determined the MIC
`
`of promethazine against only 1 strain (S. aureus NCTC 6571
`
`standard strain) and found it to be 6.2 ug/ml, far lower than that
`
`obtained in the present work (Table I). 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
`
`l), 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 Klebsiellu spp. strains.
`
`
`Table 1. MIC ranges of antihistaminics
`
`Meq
`Pro
`Cyp
`Aze
`Othersl
`Others2
`
` .
`
`.
`Orgamsm (number of stralns)
`
`MIC, [lg/ml
`
`S. aureus (5)
`
`S. epidennidis (2)
`
`E. faecium (2)
`E. coli (6)
`
`62.5-125
`
`125-250
`
`62.5-125
`
`125-250
`
`500-1000
`
`125
`
`62.5
`125-250
`
`62.5
`
`62.5-125
`250-500
`
`125
`
`62.5
`125-250
`
`125
`
`125
`1000
`
`1000
`
`1000
`500- >1000
`
`>1000
`
`>1000
`
`>1000
`>1000
`
`>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. mirabilz‘s (3)
`
`PS. aeruginosa (6)
`500
`125-1000
`>1000
`>1000
`1000—>1000 >1000
`Otherslz Dip and Cet
`Otherszz Dox, Fex, Lor and Mec
`
`983
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 4
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 4
`
`
`
`El-Nakeeb, M.A. el al.
`
`Antibacterial activity of some antihistaminics
`
`Other antihistaminics shown also in Table 1 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
`
`cetilizine, 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 this test,
`
`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
`
`aurcus, 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 rig/ml)
`were lower than those selected for the other antihistaminics
`
`1. The other
`
`studied antihistaminics namely meclozine,
`
`(100 and 200 ug/ml) because of their 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 shows the results of
`
`phosphates and proteins upon the addition to either nutrient
`
`the 100 ug/ml concentration of all tested antihistaminics to
`
`agar or nutrient broth.
`
`compare between their relative activities.
`
`Table 2. Bactericidal effect of 100 rig/ml antihistaminics against selected isolates in 0.9% saline solution by viable count technique at 37°C.
`
`Antihistaminic
`Time, h
`Organism":
`
`sa103
`sa104
`se101
`Ef101
`E0103
`E0105
`Kl102
`PS102
`
`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
`-0.1 17
`-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
`6
`-0.376
`-0.209
`0.058
`-0.327
`0.165
`0.282
`-0.008
`-0.330
`Lor
`
`-0.39224 -0.881 -1.082 -0.444 -0.394 -0.032 -0.524 -0.017
`
`
`
`
`
`
`
`*Organisms. Sam and Sam: S. aureus isolates, Selm: S. epidermidis, Eflm: E. faecium isolate, Ecm and Ecms: E. coli isolates,
`Klmz: Kl. Pneumonia isolate, Psmz: PS. aeruginosa isolate
`
`984
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 5
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 5
`
`
`
`El-Nakeeb, M.A. el (11.
`
`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
`
`aemginosa (Table 2). However, its effect against the Gram-
`
`stated that increasing the hydrophobicity increases the surface
`
`positive
`
`isolates was more pronounced. Azelastine
`
`and
`
`activity
`
`(6).
`
`Therefore,
`
`based
`
`on
`
`this
`
`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 CMC is relatively 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
`
`solution, it can be concluded that active growth of the tested
`
`and azelastine (15, 24)
`
`increases their surface activity and
`
`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 bulky lipophilic
`
`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, ll). 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 salne
`
`(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 el al. (31)
`
`counter ion effect (succinate) which may lead to altered surface
`
`who demonstrated that certain phenothiazine antihistaminics
`
`activity and change in the aggregation character from micelle
`
`985
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 6
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 6
`
`
`
`El-Nakeeb, M.A. el al.
`
`Antibacterial activity of some antihistaminics
`
`formation to a stacking process: continuous self-association
`
`antihistaminic or any member of its class were available, it has
`
`with no CMC (6, 8).
`
`been of interest 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 l-antagonists and belonging
`
`also determined by the viable count
`
`technique. Since it
`
`to both first and second generations, for bacteriostatic and
`
`possessed marked killing 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 and its 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.
`
`3C00KO3U!0
`
`\l U1
`
`\l
`
`F".3OU!
`LII
`LII
`
`l
`
`
`
`
`
`
`
`.l”
`
`
`
`
`
`Logsurvivors,CFU/ml
`
`.=~a>1>1909c0.w3U!OU]3UiOU]
`
`LII
`
`LII
`
`
`
`
`
`Logsurvivors,CFU/ml
`
`
`
`
`
`
`
`
`
`
`12
`
`18
`
`24
`
`986
`
`UCB Biopharma SPRL (IPR2019—00400)
`Exhibit 2030 Page 7
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 7
`
`
`
`El-Nakeeb, MA. 61 al.
`
`Antibacterial activity of some antihistaminics
`
`00 U
`
`
`
`9°C
`
`\l UI
`
`
`
`Logsurvivors,CFU/ml .'p tn
`
`savageOUIOUIO
`
`12
`
`Time, h
`
`18
`
`24
`
`Figure 1. Dynamics of the antibacterial activity of Aze 50 and 100 ug/ml against
`
`S. aureus Sam (A), S. uureus Salm (B) and E. faec‘ium Efml (C) isolates at 37°C
`
`0 Control; A AzeSO; I AzelOO
`
`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 pig/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 ([0, 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
`
`shown in 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 Ecm; isolate at slightly acidic or
`neutral
`the bactericidal
`
`activity dramatically
`
`pH. However.
`
`studied against one S. aureus and another E. coli isolates (Fig. 4).
`
`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 E. coli 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. el al.
`
`Antibacterial activity of some antihistaminics
`
`
`OK\Iso 1
`
`Logsurvivors,CFU/ml U]
`
`
`3
`
`’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’l”””””””””””””””””””””””””l”””””””””””””””””””””””””T’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’’l”””””””””””””””””””””””””l
`
`25
`
`50
`
`75
`
`100
`
`125
`
`150
`
`Concentration, ug/ml
`
` 0
`
`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:
`
`
`
`
`ASaHMZ 6.53; .Ef101: 6.71,
`
`EC103: 6.97 and AP5102: 6.52
`
`Table 3. Effect of inoculum size on the dynamics of antibacteiial activity of Aze 50 & 100 uglml against S.
`
`aureus Sam and E.
`
`
`coli ECI03 isolates by viable count technique at 37"C
`
`Inoculum Size (cells/ml)
`
`Organism
`Time, h
`-103
`~105
`~107
`pig/ml
`
`Log Plating Efficiency
`-0.501
`4.710
`
`Aze Cone.
`
`50
`
`3
`6
`
`-0.125
`-0.500
`
`-0.495
`-0.806
`
`s
`
`a 3
`‘0
`
`-0.986
`-0.885
`—2.624
`24
`-0.732
`-0.692
`4.246
`3
`100
`6
`4.830
`4.820
`4.533
`
`24
`-3.584
`4.381
`4.361
`
`50
`
`EC103
`
`3
`
`6
`24
`3
`
`-0.371
`
`-0.911
`-0.100
`-0.688
`
`-0.068
`
`-0.051
`-0.033
`-0.402
`
`-0.051
`
`0.067
`-0.060
`-0.153
`
`-0.514
`4.302
`4.251
`6
`100
`24 -0.557 -0.913 -0.492
`
`
`
`
`
`
`
`988
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 9
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 9
`
`
`
`El-Nakeeb, MA. 61 al.
`
`Antibacterial activity of some antihistaminics
`
`8.5
`8
`7.5
`7
`6.5
`6
`5.5
`5
`4.5
`
`
`
`
`LogPlatingEfficiency
`
`
`
`
`
`-1.0 ‘
`
`:5c
`
`
`
`LogPlatingEfficiencya:sGO
`
`I
`
`Figure 3. Effect of pH on the antibacterial activity of Aze 100 ug/ml against S. aureus Sam (A) and E. coli E0103 (B) isolates by
`
`Viable count technique at 370C
`
`03h;A6h;l24h
`
`989
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 10
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2030 Page 10
`
`
`
`El-Nakeeb, M.A. el al.
`
`Antibacterial activity of some antihistaminics
`
`0.7
`
`0.6
`
`0.5
`
`
`
`
`
`
`
`
`
` Opticgldensity be
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`; .5
`
`F N
`
`
`
`0.1
`
`1
`
`2
`
`3
`
`Time,h 4
`
`5
`
`6
`
`Figure 4. Turbidimetric growth curves showing PAE of Aze against S. uureus Sam and E. coli Ecmg clinical isolates
`
`
`
`
`l Sam control;
`
`Sam treated With Aze; o Ecm control; 0 Ec103 treated with Aze
`
`U1
`
`REFERENCES
`
`(1975). Toxicity of
`Abernathy, C.O.; Lukacs, L; Zimmerman, H].
`tricyclic antidepressants to isolated rat hepatocytes. Bier-hem Pharmacnl
`24(3). 347—50.
`
`(1980). The
`Ahmed. M.; Hadgraft. 1.; Burton. J.S.; Kellaway. 1W.
`interaction of mequitazine with phospholipid model membranes. Chem
`Phys Lipids 27(3), 251—62.
`1M.
`(2001).
`of minimum inhibitory
`Determination
`Andrews,
`concentrations. JAntimicrob Chemorher 48 Suppl 1, 5-16.
`Andrews, J.M.
`(2007). BSAC standardi7ed disc susceptibility testing
`method (version 6). J Antimicrob Chemorher 60( 1), 20—41.
`
`(1974). Aggregation of antihistamines in
`Attwood, D.; Udeala, OK.
`aqueous
`solution:
`micellar properties of
`some diphenylmethane
`derivatives. J Pharm Pharmacol 26(11). 854—60.
`
`(1975). The surface activity of some
`Attwood. D.; Udeala, OK.
`antihistamines at the air—solution interface. J Pharm Pharmacnl 27(10).
`754—58.
`
`Attwood, D.; Udeala, O.K. (1975). The interaction of antihistamines with
`
`lecithin monolayers. J Pharm Pharmacol 27(1 1), 806—10.
`Attwood, D.; Udeala, OK.
`(1976). Aggregation of antihistamines in
`aqueous solution: effect of counterions on self—association of pyridine
`derivatives. J Pharm Sci 65(7), 1053—7.
`Attwood, D.; Natarajan, R.
`(1981). Effect of pH on the micellar
`
`properties of