`fq molecules
`
`fib)
`
`Article
`Interaction between Different Pharmaceutical
`Excipients in Liquid Dosage Forms—Assessmentof
`Cytotoxicity and Antimicrobial Activity
`
`Daniel Nemes !'.., Renaté Kovacs 2, Fruzsina Nagy 2 Mirtill Mezé 1, Nikolett Poczok !,
`Zoltan Ujhelyi 1, Agota Peté 1, Palma Fehér 1, Ferenc Fenyvesi ', Judit Varadi',
`Miklés Vecsernyés ! and Ildiké Bacskay 1*
`
`1 Departmentof Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Debrecen 4032,
`Hungary; nemes.daniel@pharm.unideb-hu (D.N.); mmirtill95@gmail.com (M.M_.);
`poczok.niki@freemail.hu (N.P); ujhelyi.zoltan@pharm.unideb.hu (Z.U.); agota713@gmail.com (AP);
`feher.palma@pharm.unideb.hu (P.F.); fenyvesi.ferenc@pharm.unideb.hu (F.F.);
`varadijudit@pharm.unideb.hu (J.V.); vecsernyes.miklos@pharm.unideb.hu (M.V.)
`Departmentof Medical Microbiology, Faculty of Medicine, University of Debrecen, Debrecen 4032, Hungary;
`kovacs.renato@med.unideb.hu (R.K.); nagyfru@freemail.hu (F.N.)
`Correspondence: bacskay.ildiko@pharm.unideb.hu
`
`Received: 25 June 2018; Accepted: 19 July 2018; Published: 23 July 2018
`
`check for
`updates
`
`the safety of parabens as pharmaceutical preservatives is debated.
`Abstract: Nowadays,
`Recent studies investigated their
`interference with the oestrogen receptors, nevertheless
`their carcinogenic activity was also proved.
`That was the reason why the re-evaluation
`of the biocompatibility and antimicrobial activity of parabens is required using modern
`investigation methods. We aimed to test the cytotoxic, antifungal and antibacterial effect of
`parabens on Caco-2 cells, C. albicans, C. parapsilosis, C. glabrata, E. coli, P. aeruginosa and S. aureus.
`Two complex systems (glycerol—Polysorbate 20; ethanol—Capryol PGMC™)were formulated to
`study—with the MTT-assay and microdilution method, respectively—how other excipients may
`modify the biocompatibility and antimicrobial effect of parabens. In the case of cytotoxicity, the
`toxicity of these two systems was highly influenced by co-solvents and surfactants. The fungi
`and bacteria had significantly different resistance in the formulations and in some cases the
`excipients could highly modify the effectiveness of parabens both in an agonistic and in a
`counteractive way. These results indicate that with appropriate selection, non-preservative excipients
`can contribute to the antimicrobial safety of the products, thus they may decrease the required
`preservative concentration.
`
`Keywords: excipient interaction; surfactant;
`Caco-?2 cells
`
`
`liquid dosage forms; cytotoxicity; preservative;
`
`1. Introduction
`
`Although tablets and capsules are the most popular types of pharmaceutical dosage forms,
`different oral liquid formulations (syrups, herbal extracts, suspensions, emulsions, etc.) still have
`specific therapeutic indications, mainly in paediatrics. Flavouringis a crucialpart of these formulations
`because patient compliance is highly dependent on the taste of the product. Usually they contain
`high amount of sweet carbohydrates (glycose, fructose, maltitol, xylitol, sorbitol, etc.), which can be
`metabolized by different microorganisms, thus the product can be easily contaminated [1]. It must
`be noted, that these liquid preparations are opened and closed multiple times during their life-time
`and each application increases the possibility of contamination. In order to avoid it, an appropriate
`
`Molecules 2018, 23, 1827; doi:10.3390/molecules23071827
`
`www.mdpi.com /journal/molecules
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`Molecules 2018, 23, 1827
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`amountof preservatives must be used, which can kill or inhibit the growth of bacteria, fungi and
`other unicellular. The exact mechanism ofaction of preservatives is unclear in somecases, but as the
`cell membraneis the only commonsubcellular componentin these microbes, they mostly distort the
`structure of the membraneresulting in several consequences[2]. Their cytotoxicity is mostly based on
`these effects as well [3].
`One of the most widely used group of pharmaceutical preservatives is the parabens. They are
`derivatives of 4-hydroxybenzoic acid in the form of its carboxylic esters. The most commonly used
`parabens(Figure 1) are methyl paraben (MP)(E218), ethyl paraben (EP) (E214), propyl paraben (PP)
`(E216), butyl paraben (BP), heptyl paraben andtheir respective sodium salts. The longerthe alkyl
`chain, the lowerthe solubility in water is. Hence, some co-solvent such as ethanolis usually required
`to increase their solubility and it mustalso be noted, that the sodium salts are less frequentin different
`formulations. Generally, they are considered as synthetic compounds,but in the recent years many
`natural sources were found [4-6]. They are preferred in the pharmaceutical and cosmetic industries,
`because of their odourless and tasteless characteristics, great chemical stability over a wide range of
`pH values and a broad spectrum of antimicrobialactivity [7].
`
`Q
`yoCH,
`Ry
`U
`ee
`OH
`
`oO
`OCH,
`Noe
`UO
`ye
`OH
`
`©
`o
`VOLcH, yOHs
`vx
`aSs
`
`OH
`
`OH
`
`Figure 1. Chemical structure of the most commonly used parabens in growing alkyl chain length:
`methyl paraben, ethyl paraben, propyl paraben, butyl paraben.
`
`In the case of
`The esters of 4-hydroxybenzoic acid also have certain well-known risks.
`topical application, contact dermatitis is a well-known problem [8,2] however, the latest results
`are controversial, describing a low occurrenceof allergic reactions caused by parabens[10,11] or a
`severe influence on sensitization [12]. Recent studies have indicated the carcinogeniceffect of parabens,
`as they interfere with oestrogen receptors [13,14]. Furthermore, in vivo evidence suggests that urine
`paraben levels can be associated with menstrual cycle problems [15]. They are able to penetrate
`throughthe skin from cosmetic products [16,17]. Their direct cytotoxic behaviour has been reported
`on corneal epithelial cells [18], on dermal fibroblasts [1%] and onliver cells [20]. Paraben exposureis
`not only restricted to the users of cosmetics [21], as they can pass throughthe placenta [22] and can
`be measured in the milk of lactating mothers [23]. These results suggest a decline in the use of these
`4-hydroxybenzoic acid derivatives in oral and topical formulations during the next few years.
`An oral, liquid pharmaceutical preparation contains many excipients, which is the reason why
`cytotoxicity tests of each chemical by itself is not enough to gain a comprehensive view of the
`biocompatibility profile of the product. There are only few studies on how the biocompatibility of an
`excipient is influenced if other components are present in the test systems. However, in order to get
`authorized by governmental authorities, the whole product cannot be toxic, but positive interactions
`might decrease the appropriate concentration of additivesi.e., the quantity of preservatives may also
`be reduced. However, serious cytotoxicity values may be measured,if the excipients can potentiate
`their harmfuleffects [24]. As the cytotoxic effects of surface-active agents are well-known[25], they
`might have synergetic antimicrobial activity with preservatives. Different co-solvent mixtures can
`
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`Molecules 2018, 23, 1827
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`have different biocompatibility profiles and might modify the toxicity of preservatives, increasing their
`effect on the cell membranes by creating a better chemical environmentatthesite of action [24].
`In this study, our objective was to investigate the cytotoxicity and antimicrobial properties on
`Caco-2 cells and on various pathogenic microorganismsof different 4-hydroxybenzoic acid derivatives
`alone and in two complex co-solvent systems to explore interferences between the preservatives and
`the componentof the co-solvent systems. Caco-2 cells are widely applied as an in vitro model of
`humangastrointestinal transport and mainly used as a monolayerrather than individual cells, however
`several assays are performed prior to reach complete integrity, such as end point or non-invasive
`cell viability assays (MTT assay, LDH test, RT-CES,etc.) [26]. In our antimicrobial experiments, our
`test solutions were tested on clinically relevant pathogens: S. aureus as a Gram-positive facultative
`anaerobe, E. coli and P. aeruginosa as a Gram-negative aerobes and C.albicans as the most common
`fungal pathogen and C.parapsilosis and C. glabrata as the top Candida species opportunistic pathogens
`different from C.albicans [27].
`The formulations of the investigated systems contain a co-solvent and a surface-active agent.
`The first formulation (S1) consisted of 30% (v/v) glycerol and 0.002% (v/v) Polysorbate 20.
`The surfactant of the second formulation (52) was 0.5% (v/v) Capryol PGMC™and the parabens in
`the form of their 10 (w/w)% solutions, dissolved in 70% (v/v) ethanol. Tables 1 and 2 summarize
`the composition of every solution used in our experiments. The experimental design is presented
`in Figure 2.
`
`MTT-assay Antimicrobial tests 4
`
`@
`y
`Tested solutions:
`Tested solutions:
`‘parabens alone
`sglyceral, ethanal
`‘parabens in complex systems
`SOa“
`0. minute:
`0. minute:
`
`Preparation of solutions Preparation of solutions
`&
`10. minute:
`10. rninute:
`Start of 24 hours long incubation on
`Start of 30 minutes fong incubation on
`Caco-2 cells
`
`fungal and bacterial cells
`se
`os
`"Removal of test solutions from cells
`Measurement of absorbance at 492 and
`Oo nm
`-Addig MTT-solution to cells for 3 hours
`incubation
`
`
`r
`
`‘
`
`*parabens alone
`sparabens in complex systems
`
` @
`
`|
`
` ss
`
`
`
`
`
`
`
`
`
`of MTT-solution
`
`‘Removal
`incubation
`*Dissolving formazan crystals
`
`
`after
`
`Measurement of absorbance at 570 and
`orm
`
`Figure 2. Experimental design.
`
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`Molecules 2018, 23, 1827
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`Table 1. Composition of test solutions for cytotoxicity tests.
`
`
`
`
`Component
`S1
`$2
`Paraben
`0.2 w/w%, 0.02 w/w%, 0.002 w/w%, 0.0002 w/w%
`
`Glycerol - 30 0/0%, 3 v/v%, 0.3 7/0%, 0.03 v/v%
`
`0.002 v/%, 0.0002 0/ 0%, 0.00002 7/0%,
`Polysorbate 20 - 0.000002 0/o%
`
`
`
`
`.Capryol PGMC™ 0.5 v/v%, 0.05 v/v%, 0.005 v/v%, 0.0005
`v/o%
`14 0/0%, 0.14 o/ 0%, 0.014 o/ 0%, 0.0014
`Ethanol
`-
`°
`v/v0%
`
`
`PBS
`
`solvent, used for tenfold, hundredfold, thousand-fold dilution
`
`Table 2. Composition of test solutions for antimicrobialtests.
`
`
`Component
`S1
`$2
`Control
`Paraben
`0.1 w/w%, 0.15 w/w%, 0.25 w/w%
`-
`Glycerol
`-
`-
`Polysorbate 20
`-
`-
`Capryol PEMC™
`0.5 v/v%
`0.7 v/0%, 1.05 v/v%, 1.75 v/o0%
`Ethanol
`0.7 v/v%, 1.05 0/0%, 1.75 v/u%
`RPMI-1640
`solvent for antifungaltests
`Mueller-Hinton broth
`solvent for antibacterialtests
`
`30 v/0%
`0.002 v/o%
`-
`-
`
`Test solutions were preparedin situ, 10 min before the inoculation for antimicrobial investigations.
`Caco-2 cells were incubated for 30 min with the test solutions, then these solutions were removed and
`the MTT-solution was added for a 3 h long reaction. The converted formazan crystals were dissolved
`in appropriate solvents after the unreacted MTT was removed. Absorbance was measured at two
`different wavelengths andthecell viability was calculated. After seeding the bacterial and fungalcells
`in appropriate concentrations into 96-well microplates, a 24 h long incubation wasstarted. Optical
`density was measured at two wavelengthsat the end of the incubation period.
`
`2. Results
`
`2.1. Cytotoxicity Tests
`
`2.1.1. Cytotoxicity of Parabens
`
`In order to mimic the dilution of samples in the gastrointestinal tract, the cytotoxicity of parabens
`was measured in tenfold, hundredfold and thousand-fold dilutions (Figure 3). The samples were
`diluted by PBS. At 0.2 (w/w)% butyl and ethyl paraben hadsignificantly higher cytotoxicity than
`methyl and ethyl paraben, which had similar toxicity patterns. There wasa linear relationship between
`the cytotoxicity and the dilution ratio of different paraben derivatives. The more concentrated samples
`decreased the cell viability and resulted in significant cytotoxicity. The higherthe ratio of dilution of
`parabens,the better thecell viability of the Caco-2 cell line was.
`
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`Molecules 2018, 23, 1827
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`Cytotoxicity of parabens
`
`Buthyl-paraben
`
`Ethyl-paraben
`Methyl-paraben
`
`100
`
`a
`S 50
`3Oo
`
`25
`
`Propyl-paraben > 75
`
`0
`
`S
`s
`Ss
`o
`
`©
`s
`v
`”
`
`v
`$
`os
`
`Vv
`S
`S
`s
`
`Concentration of parabens “/,%
`
`Figure 3. Cytotoxicity of parabens on Caco-2 cells measured by MTT-assay. Cell viability was expressed
`as the percentage of the absorbance of the untreated control cells. Data expressed as mean + SEM,
`1 = 12. Cell viability of the samples at 0.2000; 0.0200; 0.0020; 0.0002 (w/w)% concentrations: MP:
`
`
`
`
`
`
`81% + 2.4%; 89% + 1.6%; 99% + 3.1%; 100% + 2%; EP: 83% + 3.3%; 88% + 2.6%; 100% + 3.1%;
`
`
`
`
`
`
`100% + 2.5%; PP: 53% + 4.7%; 78% + 4%; 97% + 2.7%; 100% + 2.7%; BP: 41% + 4.6%; 81% + 2.4%;
`
`
`94% + 2.9%; 99% + 2.5%.
`
`
`
`2.1.2. Cytotoxicity of Solvents
`
`Ethanol and glycerol were tested in different concentrations diluted with phosphate buffered
`saline (PBS) for cytotoxicity experiments. As it can be seen on Figures 4 and 5, the cell viability
`decreased in a concentration dependent mannerin the case of these solvents. The ICsg (the inhibitory
`concentration value, where the 50% cell viability was measured by an MTTtest) of glycerol was
`45 (v/v)%. In our complex systems, the concentrations of glycerol were 30 (v/7)%, 3 (v/¥)%, 0.3 (v/v)%,
`0.03 (v/v)% which were lower than this inhibitory concentration.
`The concentrations of ethanol (1.75 (v/v)%, 14 (v/v)%, 0.14 (v/v)%, 0.014 (v/v)%) in complex
`systems were applied for cytotoxicity and antimicrobial tests. Based on this cytotoxicity test, the
`ICs9 value cannot be determined in these concentration ranges. Thecell viability slightly decreased
`according to the concentration, but the highest concentration (1.75 (v/v)%) decreased thecell viability
`
`significantly (80 + 1.7%).
`
`Cytotoxicity of ethanol
`
`400
`
`TS
`
`80
`
`25
`
`a
`
`J
`re
`3
`Si
`Me
`FS PO SP SF SP
`ek FPS OP Oe
`Concentration of ethanol */,%
`
`oe
`
`=3o
`
`S>
`
`3oO
`
`Figure 4. Cytotoxicity of ethanol measured by MTT-assay. Cell viability expressed as the percentage of
`
`the absorbanceof the untreated control cells. Data expressed as mean + SEM, n = 12. Cell viability
`
`
`
`
`of the samplesat the different concentrations: 100% + 0.2%; 100% + 1.8%; 100% + 3.1%; 100% + 2%;
`
`
`
`
`
`95% + 1.7%; 87% + 0.5%; 81% + 1.1%; 72% + 0.9%; 66% + 1%.
`
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`Molecules 2018, 23, 1827
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`Cytotoxicity of glycerol
`
`160
`
`75
`
`50
`
`25
`
`
`
`Cellviability
`
`
`Ss
`Sf
`£€
`©
`©
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`EF
`EF
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`LF
`eo Sf
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`ap PP
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`
`Concentration of glycerol “/,%
`
`Figure 5. Cytotoxicity of glycerol measured by MTT-assay. Cell viability expressed as the percentage of
`
`the absorbanceof the untreated control cells. Data expressed as mean + SEM, n = 12. Cell viability
`
`
`
`
`of the samplesat the different concentrations: 100% + 1.1%; 95% + 2.5%; 90% =E 2.5%; 83% + 3.4%;
`
`
`
`
`
`74% + 3.6%; 60% + 2.5%; 50% + 1.7%; 42% + 2%; 35% + 0.4%.
`
`2.1.3. Cytotoxicity of Formulated Systems
`
`The cytotoxicity of S1 can be seen in Figure 6. The formulated control was highly toxic, and
`the cell viability was less than 50% compared to the untreated control at the original concentration.
`MPhad the highest survivability from all esters, the second was EP. The results of the two longer
`parabens werenotsignificantly different from each other at the tested concentrations. Moreover,
`the methyl paraben wasnotsignificantly different from the formulated control, but at the original
`concentration and at tenfold dilution, along with ethyl paraben,these derivatives were different from
`the longer ones. All statistical differences between thetest solutions diminished at hundredfold and
`thousand-fold dilutions.
`
`Cytotoxicity of $1
`
`Buthy!-paraben
`Propy-paraben
`Ethyl-paraben
`Methyl-parahen
`Formulated control
`
`viability $
`
`Cell
`
`ss
`
`oa
`
`e
`
`$
`se
`
`e
`
`+
`&
`s
`
`Concentration of parabens "4%
`
`Figure 6. Cytotoxicity of the first formulated system (S1) consisting of 30% (v/v) glycerol and 0.002%
`(v/v) Polysorbate 20 measured by MTT-assay. Cell viability expressed as the percentage of the
`
`absorbance of the untreated control cells. Data expressed as mean + SEM, n= 12. Cell viability of the
`samples at 0.2000; 0.0200; 0.0020; 0.0002 (w/w)% concentrationsof different formulations containing
`
`
`parabens: formulated control: 48% + 1.1%; 100% + 2.7%; 100% + 4%; 98% + 1.8%; formulated MP:
`
`
`
`
`
`
`36% ~ 1.9%; 88% + 4.1%; 96% + 2.9%; 100% + 2.1%; formulated EP: 5% + 0.3%; 56% = 2%; 92% + 2%;
`
`
`
`
`100% + 4.6; formulated PP: 6% + 1.3%; 35% + 4.5%; 100% + 4.9%; 99% + 4.6%; formulated BP:
`8% + 1.1%; 30% + 2.9%; 97% + 2.6%; 100% + 2.3%.
`
`
`
`
`
`
`
`In the case of 52 (Figure 7), BP had the highest cell viability at the original concentration, while
`the other parabens caused nearly total cell death. The tenfold dilution showed another ranking: propyl
`and ethyl paraben matchedthe results of the formulated control, while BP had slightly worsecell
`
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`Molecules 2018, 23, 1827
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`7 of 19
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`viability and MP wasalso significantly more toxic than the formulated control. After further dilution,
`all esters, except BP caused 100%cell viability.
`
`Cytotoxicity of 52
`
`Formulated buthyi-paraben
`Formulated propyl-peraberi
`Formulated ethyl-parabern
`Formulated methyl-paraben
`Formulated contro!
`
`viability Ss
`
`Cell
`
`as
`
`¥
`2
`S
`=
`=
`-
`Concentration of parabens *1,,%
`
`Figure 7. Cytotoxicity of the second formulated system (S2) consisting of 0.5% (v/v) Capryol PEMC™
`and ethanol measured by MTT-assay. Cell viability expressed as the percentage of the absorbanceofthe
`
`untreated control cells. Data expressed as mean + SEM, n = 12. Cell viability of the samples at 0.2000;
`0.0200; 0.0020; 0.0002 (w/w)% concentrationsof different formulations containing parabens: Formulated
`
`
`
`
`
`
`control: 63% + 2.8%; 100% + 2.5%; 100% + 2.5%; 98% + 3.4%; formulated MP: 2% + 0.2%; 90% + 1.8%;
`
`
`
`
`97% + 3.1%; 99% + 2%; formulated EP: 4% + 0.7%; 100% + 2.7%; 100% + 3.1%; 100% + 2.5%;
`
`
`
`
`formulated PP: 5% + 0.6%; 100% + 3.2%; 100% + 2.7%; 100% + 2.7%; formulated BP: 24% + 1.2%;
`
`
`
`81% + 2.6%; 94% + 2.9%; 91% + 2.5%.
`
`
`
`
`
`2.2. Antimicrobial Tests
`
`2.2.1. Antifungal Tests
`
`In orderto test the antimicrobial properties of parabens, three different concentrations wereused.
`Cell viability was expressed as a percent of the absorbance of the positive control in the case of every
`species, respectively. The critical 50% cell viability threshold was presented withaline in each figure.
`Abovethis value a certain compoundis considered ineffective in the case of antimicrobial activity,
`while belowthisline it has an inhibitory effect. We also formulated a control solution for every paraben
`to control their normal antimicrobial effects, without any additives.
`In the case of C.albicans, (Figure 8) there was no significant difference between formulated and
`non-formulated parabens, both the control solutions and the 51 and 82 solutions resulted in the
`same results. However, S1-PP, 51-BP and 52-EP had highercell viability values than their controls, the
`formulations decreased the effectiveness of the parabens. There was no difference between the longer
`and the shorter esters.
`
`The investigation of C. parapsilosis (Figure 9) showed a highcell viability gap between the control
`parabensand the formulations. The control solutions totally eradicated the fungal cells, however,
`both formulations slightly increased their survivability. The increase of dissolved paraben did not
`reduce this gap, but even further weakened the antimicrobial effect of the parabens. At the highest
`concentration, the S1-PP no longer had inhibitoryeffect atall.
`C. glabrata wasalso sensitive to both the control and the formulated solutions (Figure 10), but
`the results of Sl were worse than S2 or the control. This lack of effectiveness increased with the
`
`growing concentration.
`
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`=>oOaooQoQoQoQo
`
`yoS
`
`2
`
`
`
`Cellviability
`
`C. albicans
`
`
`
`S\a
`Rs
`Concentration of parabens “/,,%
`
`© si-c
`@ Bp
`@@ Pp
`@2 EP
`@ ve
`
`Figure 8. Cell viability of C. albicans against the control paraben solutions (MP; EP; PP; BP); thefirst
`system (S1-MP; S1-EP; $1-PP; 51-BP) and the second system (S2-MP; $2-EP; $2-PP; 52-BP). Cell viability
`
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n=A4. Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)%concentrations of different formulations
`
`
`
`
`+ 3.2%; 10% a
`c 2.5%; EP: 10% a
`t 3.3%; 10% + 3.4%;
`containing parabens: MP: 11% + 2.2%; 10%
`
`
`
`
`
`10% + 4.1%; PP: 11% + 2.2%; 9% 4 2.3%; 9% + 3%; BP: 10%a
`t 1.7%; 10% + 2.2%; 9%a
`t 3%; formulated
`
` t 1.4%; formula
`
`ed EP
`713% + 2.7%; 12% J
`control of $1: 13% + 1.7%; formulated MP of $1: 12% + 3.1%
`
`
`
`
`
`of S1: 12% + 2.3%; 12% + 1.9%; 16% + 0.9%; formulated PP of S1: 14% + 0.7%; 16% a
`t 0.5%; 27% + 1.3%;
`
`
`
`
`formulated BP of $1: 15% + 1.6%; 16% + 2.5%; 32% + 1%; formulated control of $2: 18% + 3.3%;
`
`
`
`
` t 2.1%; 16% J cr 1.7%; 16% 4
`16% + 2.5%; 16% + 2%; formulated MPof $2: 17%4
`t 1.7%; formulated EP of
`
`r 1.8%; 16% J
`
`
`
`+ 2.2%; 16% + 0.9%;
`$2: 17% + 1.5%; 16% + 0.8%; 32% + 2%; formulated PP of $2: 17%
`
`
`
`formulated BP of $2: 18% + 1.2%; 17% + 1.7%
`722% + 0.8%.
`
`
`
`
`
`
`
`
`
`
`
`
`
`C. parapsilosis
`
` o
`
`s
`os
`ow
`Concentration of parabens “/,,%
`
`=8888
`
`nyoS
`
`°
`
`
`
`Cellviability
`
`Figure 9. Cell viability of C. parapsilosis against the control paraben solutions (MP; EP; PP; BP); the first
`system (S1-MP; S1-EP; S1-PP; S1-BP) and the second system (S2-MP; S2-EP; S2-PP; 52-BP). Cell viability
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n=A4.Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)%concentrations of different formulations
`
`
` cr 0.8%; 1% 4 Cc 0.3%; 0% + 0.1%;
`containing parabens: MP: 5% + 4%; 2% + 1.5%; 0% + 0.5%; EP: 1% a
`PP: 1% + 0.1%; 0% + 0.4%; 0%+ 0.2%; BP: 0% + 0.4%; 0% + 0.3%; 0% + 0.3%; formulated control of
`
`
`
`r 1.4%; 27% a
`S1: 22% + 1.5%; formulated MP of S1: 24% 4
`t 1.2%; 25% + 2%; formulated EP of S1:
`
`
`
`23% + 2.3%; 23% + 1.7%; 29% + 1.9%; formulated PP of $1: 24% 4
`c 1.3%; 33% x 1.2%; 57% a
`Cc 0.7%;
`
`
` formulated BP of S1: 23% + 1.1%; 34% + 2%
`Cc 2.7%;
`7 38% + 0.9%; formulated control of S2: 36%4
`
`
`
`rc 1.6%; 30% + 1.8%; formulated
`t 1.3%; 30% A
`AA% + 1.5%; 44% + 1.8%; formulated MP of S2: 28% 4
`
`
`
`
`EP of $2: 29% + 2.2%; 28% + 1.5%; 31% + 1.8%; formulated PP of S2: 30% + 2.3%; 28% + 2.1%;
`
`
`
`33% xo 1.9%.
`27% + 1.9%; formulated BP of S2: 31% + 1.2%; 31% + 1.5%;
`
`
`
`
`
`
`
`
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2031 Page 8
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 8
`
`

`

`Molecules 2018, 23, 1827
`
`9 of 19
`
`Cell
`
`C. glabrata
`
`viability
`
`Concentration of parabens “/,%
`
`Figure 10. Cell viability of C. glabrata against the control paraben solutions (MP; EP; PP; BP); thefirst
`system (S1-MP; S1-EP; S1-PP; 51-BP) and the second system (S2-MP; S2-EP; $2-PP; S2-BP). Cell viability
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n =A. Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)% concentrations of different formulations
`
`
`
`containing parabens: MP: 11% + 1.1%; 5% + 0.7%; 5% + 2%; EP: 5% + 2.3%; 4% £ 1.2%; 4% + 0.9%;
`
`
`
`
`
`
`PP: 5% + 3.2%; 4% + 1%; 5% + 1.2%; BP: 5%+3%; 5%+1.1%; 4% + 1.2%; formulated control of
`
`
`S1: 6% + 1.6%; formulated MP of S51: 14% + 3.4%; 16% + 2.5%; 15% + 2.5%; formulated EP of S1:
`
`
`
`
`
`14% + 1.1%; 13% + 1.4%; 19% + 2%; formulated PP of $1: 14% + 0.7%; 15% + 1.4%; 21% + 1%;
`
`
`
`formulated BP of S1: 14% + 3.4%; 19% + 3.2%; 24% + 2.9%; formulated control of $2: 6% + 2.9%;
`
`
`
`
`6% + 2.5%; 6% + 2%; formulated MP of 82: 5% + 3.4%; 5% + 1.4%; 5% + 2%; formulated EP of $2:
`
`
`
`
`5% + 14%; 5% + 2.8%; 5% + 1.7%; formulated PP of $2: 5% + 4.1%; 5% + 3.2%; 5% + 1%; formulated
`
`
`
`BP of $2: 1% + 0.9%; 1% + 0.8%; 1% + 0.9%.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`2.2.2. Antibacterial Tests
`
`S. aureus (Figure 11) was not sensitive to the control solutions, and increasing doses of MP, EP
`and PP did not decreasethecell viability. Meanwhile, the ethanolic solution of BP and $1 proved to
`be highly effective. 52 containing Capryol PGMC™apart from ethanol wasalso effective, with the
`exception of the formulated MP, which only passed the 50% limit at 1.5 (w/w)%.
`E. coli (Figure 12) had resistance against EP and BP, except for the $1, which wasveryeffective
`againstit. The addition of a surface-active agent in $2 could increase the antimicrobial properties of BP
`and PP as they showedgreater inhibitory effect than the normal ethanolic solutions. 51 showed higher
`efficacy than the othersolutions.
`P. aeruginosa (Figure 13) showedthe widest spectrum of resistance. The ethanolic EP, PP and BP
`could not inhibit its growthatall, like PP and BP in $2. The presence of Capryol PGMC™wasalso
`advantageousfor the EP and BP, their effectiveness was highly increased, but they still could not reach
`a 50%inhibitory rate. All derivatives formulated in S1 weretotally effective in every concentration,
`and methyl] paraben wasalso effective at the highest dose in $2 and the control ethanolic solutions.
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2031 Page 9
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 9
`
`

`

`Molecules 2018, 23, 1827
`
`10 of 19
`
`S. aureus
`
`Cell
`
`viability
`
`Concentration of parabens “/,,%
`
`Figure 11. Cell viability of 5. aureus against the control paraben solutions (MP; EP; PP; BP); thefirst
`system (S1-MP; S1-EP; S1-PP; 51-BP) and the second system (S2-MP; S2-EP; S2-PP; S2-BP). Cell viability
`
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n= 4. Cell viability of bacterial cells at 0.1; 0.15; 0.25 (w/w)% concentrations of different formulations
`
`
`
`containing parabens: MP: 80% + 2.2%; 66% + 1.9%; 59% + 1.5%; EP: 70% + 2.4%; 70% + 1.3%;
`
`
`
`
`
`62% + 1.6%; PP: 83% + 2.3%; 66% + 2.5%; 63% + 1.4%; BP: 2% + 1.1%; 1% + 0.9%; 1% + 0.4%;
`
`formulated control of $1: 1% + 0.3%; formulated MP of S1: 2% + 1.4%; 1% + 0.5%; 1% + 0.8%;
`
`
`
`formulated EP of $1: 1% + 0.2%; 1% + 0.4%; 4% + 2.8%; formulated PP of S1: 2% + 1.6%}; 4% + 1.3%;
`
`
`
`
`6% + 2.4%; formulated BP of S1: 4% + 2.1%; 5% + 1.6%; 3% + 1.5%; formulated control of $2:
`
`
`
`
`37% + 1.6%; 4% + 2.7%; 4% + 1.1%; formulated MP of 52: 61% + 1.2%; 20% + 2.6%; 1% + 0.5%;
`
`
`
`formulated EP of $2: 28% + 2.4%; 2% + 1.3%; 1% + 0.9%; formulated PP of $2: 22% + 1.7%; 4% + 2.1%;
`
`
`
`
`0% + 0% +0.3%; formulated BP of $2: 3% + 2.5%; 3% + 1.8%; 3% + 2.9%.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`E. coli
`
`Cell
`
`viability e
`
`wv
`o
`Concentration of parabens “/,,%
`
`8 S1-C
`i BP
`am PP
`@@ EP
`= wp
`
`Figure 12. Cell viability of E. coli against the control paraben solutions (MP; EP; PP; BP); the first
`system (S1-MP; S1-EP; S1-PP; S1-BP) and the second system (S2-MP; S2-EP; S2-PP; 52-BP). Cell viability
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n= 4. Cell viability of bacterial cells at 0.1; 0.15; 0.25 (w/w)%concentrationsof different formulations
`
`
`
`
`containing parabens: MP: 20% + 1.7%; 10% += 2%; 0% + 0.1%; EP: 16% + 1.2%; 8% + 1.6%; 7% + 1.2%;
`
`
`
`
`
`
`PP: 59% + 1.6%; 46% + 2.5%; 40% + 0.9%; BP: 59% + 1.8%; 50% + 0.5%; 54% + 1%; formulated
`
`control of S1: 0% + 0.4%; formulated MP of $1: 0% + 0.3%; 0% + 0.1%; 0% + 0.2%; formulated EP
`
`of S1: 0% + 0.1%; 0% + 0.2%; 1% + 0.4%; formulated PP of 51: 0% + 0.2%; 0% + 0.4%; 1% + 0.4%;
`
`formulated BP of 51: 0% + 0.3%; 7% + 2.4%; 1% + 1%; formulated control of S2: 40% + 0.6%;
`
`
`
`
`40% + 1.1%; 37% + 0.9%; formulated MP of $2: 12% + 1.2%; 7% + 1.6%; 1% +0.2%; formulated EP of
`
`
`
`
`
`$2: 15% + 1.7%; 3% + 2.4%; 1% + 0.7%; formulated PP of $2: 51% + 0.8%; 17% + 1.5%; 23% + 1.9%;
`
`
`
`formulated BP of $2: 58% + 2.9%; 51% + 0.4%; 29% + 1.8%.
`
`
`
`
`
`
`
`
`
`
`
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 10
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 10
`
`

`

`Molecules 2018, 23, 1827
`
`11 of 19
`
`
`
`Cellviability
`
`P. aureginosa
`
`y°
`=eadQaQooaoO3
`
`Concentration of parabens “/,,%
`
`Figure 13. Cell viability of P. aeruginosa against the control paraben solutions (MP; EP; PP; BP); the first
`system (S1-MP; S1-EP; S1-PP; 51-BP) and the second system (S2-MP; S2-EP; $2-PP; S2-BP). Cell viability
`
`expressed as the percentage of the absorbanceof the untreated control. Data expressed as mean + SEM,
`n =A. Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)% concentrations of different formulations
`
`
`
`
`
`containing parabens: MP: 100% + 2.2%; 72% + 2.0%; 7% + 1.2%; EP: 100% + 2.1%; 100% + 2.3%;
`
`
`
`
`
`
`100% + 1.7%; PP: 100% + 1.7%; 100% + 1.2%; 100% + 1.8%; BP: 100% + 1.3%; 100% + 1.4%; 98% + 2%;
`formulated control of $1: 0% + 0.4%; formulated MP of $1: 0% + 0.2%; 0% + 0.3%; 0% + 0.1%; formulated
`EP of S1: 0% + 0.2%; 0% + 0.2%; 0% + 0.3%; formulated PP of $1: 1% + 0.5%; 1% + 0.3%; 1.8% + 0.4%;
`
`formulated BP of $1: 1% + 0.1%; 8% + 2.1%; 4% + 1.7%; formulated control of $2: 100% + 2.6%;
`
`
`
`
`
`99% + 1.4%; 99% + 1.7%; formulated MP of $2: 96% + 2.5%; 62% + 1.5%; 4% + 1.3%; formulated
`
`
`
`EP of 52: 100% + 2%; 92% + 1.7%; 72% + 1.3%; formulated PP of $2: 100% + 2.1%; 100% + 1.6%;
`
`
`
`100% + 1.8%; Formulated BP of $2: 100% + 2%; 96% + 1.4%; 72% + 1.9%.
`
`
`
`
`
`
`
`
`
`3. Discussion
`
`The microbial stability of any oral pharmaceutical product until its expiry date is essential
`regardless if the product was contaminated during its application. However, the use of preservatives
`is a cheap way to protect any product, there are authorized drugs on the market with ineffective
`microbial protection [28]. In our study, we formulated two different co-solvent systems:
`51 which contained 30%(v/v) glycerol and 0.002% (v/v) Polysorbate 20 (HLB value: 16.7) and 52
`which contained 0.5%(v/v) Capryol PGMC™(HLBvalue: 5) and parabensin the form of their 70%
`(v/v) ethanolic solutions[25].
`Thebasis of selection was to use one co-solvent, different surfactants with high and moderate
`HLBvalues and preservatives (parabens) in our investigations, because these excipients are officially
`widely applied in liquid, oral pharmaceutical formulations. In order to comply with EMEA guidelines,
`these authorized excipients were used in safe concentrations [29]. The cytotoxicity of ethanol and
`glycerin as co-solvents were also tested on Caco-2 cell line and their cytocompatible concentrations
`were determined. The safe 0.5 (v/v)% ethanol concentration was controlled in OTC products for
`children [30]. We applied ethanol as co-solvent in 1.75 (v/v)%, 1.4 (v/v)%, 0.14 (v/v)%, 0.014 (v/2)%
`concentration range and these concentrations proved to be cytocompatible on Caco-2 cells. Ethanol
`can increase the solubility of several drugs, such as COX-2 inhibitors even at low concentration andit
`showedcytotoxic properties at 10% (v/v) on Caco-2 cells [31,32].
`Theeffective glycerol concentration for enhancing the solubility of different active pharmaceutical
`ingredients (APIs) was proved from 20% (v/v) [33]. The concentration of glycerol of our complex
`systems wasused from 30 (v/v)%, 3 (u/v)%, 0.3 (v/0)%, to 0.03 (v/v)% which were lower than the
`inhibitory concentration.
`The selection of Polysorbate 20 and Capryol PGMCbased on our previous experiments [25].
`It was found that the safe concentration range of Polysorbate 20 was 0.002 (v/v)%, 0.0002 (v/v)%,
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2031 Page 11
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 11
`
`

`

`Molecules 2018, 23, 1827
`
`12 of 19
`
`0.00002 (v/v)% and 0.000002 (v/v)%. The HLB value of Polysorbate 20 is high andeffective solubilizing
`properties can be presented. Surfactant with lower HLB value was Capryol PGMC and cytocompatible
`concentration range (0.5 (v/7)%, 0.05 (9/v)%, 0.005 (v/v)%, 0.0005 (v/v)%) was confirmed in our study.
`Based on our experimental design, we tested two complex formulations containing different
`parabensto evaluate their cytotoxic and antimicrobial interference and to investigate how these
`materials influence the safety and efficacy of para