molecules
`
`
`
`Article
`
`mp:
`
`Interaction between Different Pharmaceutical
`
`Excipients in Liquid Dosage Forms—Assessment of
`Cytotoxicity and Antimicrobial Activity
`
`, Renato Kovacs 2, Fruzsina Nagy 2, Mirtill Mezfi 1, Nikolett Poczok 1,
`Daniel Nemes 1
`Zoltan Ujhelyi 1, Agata Pet6 1, Palma Fehér 1, Ferenc Fenyvesi 1, Judit Varadi 1,
`Miklés Vecsernyés 1 and Ildiké Bacskay 1*
`
`1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Debrecen 4032,
`Hungary; nemes.daniel@pharm.unideb.hu (D.N.); mmirtill95@gmail.com (M.M.);
`poczok.nil<i@freemail.hu (NP); ujhelyi.zoltan@pharm.unideb.hu (Z.U.); agota713@gmail.com (AP);
`feher.palma@pharm.unideb.hu (P.F.); fenyvesi.ferenc@pharm.unideb.hu (FE);
`varadi.judit@pharm.unideb.hu (].V.); vecsernyes.miklos@pharm.unideb.hu (M.V.)
`Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, Debrecen 4032, Hungary;
`l<ovacs.renato@med.unideb.hu (R.K.); nagyfru@freemail.hu (F.N.)
`Correspondence: bacskay.ildil<o@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. albicuns, C. pampsilosis, C. glabrutu, E. 6011, P. aeruginosa and S. aureus.
`Two complex systems (glycerol—Polysorbate 20; ethanol—Capryol PGMCTM) 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. Flavouring is a crucial part 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 [l ]. 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/molecule523071827
`
`www.mdpi.com /journal/molecules
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 1
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 1
`
`

`

`Molecules 2018, 23, 1827
`
`2 of 19
`
`amount of preservatives must be used, which can kill or inhibit the growth of bacteria, fungi and
`other unicellular. The exact mechanism of action of preservatives is unclear in some cases, but as the
`cell membrane is the only common subcellular component in these microbes, they mostly distort the
`structure of the membrane resulting 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 and their respective sodium salts. The longer the alkyl
`chain, the lower the solubility in water is. Hence, some co-solvent such as ethanol is usually required
`to increase their solubility and it must also be noted, that the sodium salts are less frequent in 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 antimicrobial activity [7].
`
`CM
`
`OH
`
`if}
`[kg/‘0 VA {:83
`”9%
`‘xfi’r"
`0H
`
`0
`%‘O\vx\v’CH3
`/11$
`1
`Lg?
`53:»;
`
`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 [8,9] however, the latest results
`are controversial, describing a low occurrence of allergic reactions caused by parabens [10,11] or a
`severe influence on sensitization [12]. Recent studies have indicated the carcinogenic effect 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
`through the skin from cosmetic products [16,17]. Their direct cytotoxic behaviour has been reported
`on corneal epithelial cells [18], on dermal fibroblasts [19] and on liver cells [20]. Paraben exposure is
`not only restricted to the users of cosmetics [21], as they can pass through the 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 additives i.e., the quantity of preservatives may also
`be reduced. However, serious cytotoxicity values may be measured, if the excipients can potentiate
`their harmful effects [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
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 2
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 2
`
`

`

`Molecules 2018, 23, 1827
`
`3 of 19
`
`have different biocompatibility profiles and might modify the toxicity of preservatives, increasing their
`effect on the cell membranes by creating a better chemical environment at the site of action [24].
`In this study, our objective was to investigate the cytotoxicity and antimicrobial properties on
`Caco-2 cells and on various pathogenic microorganisms of different 4-hydroxybenzoic acid derivatives
`alone and in two complex co-solvent systems to explore interferences between the preservatives and
`the component of the co-solvent systems. Caco-2 cells are widely applied as an in vitro model of
`human gastrointestinal transport and mainly used as a monolayer rather 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. acruginosa as a Gram-negative aerobes and C. albicans as the most common
`fungal pathogen and C. pm’apsilosis and C. glabmta as the top Candida species opportunistic pathogens
`different from C. albicans [2?].
`
`The formulations of the investigated systems contain a co-solvent and a surface-active agent.
`The first formulation (51) consisted of 30% (v/v) glycerol and 0.002% (0/2)) Polysorbate 20.
`The surfactant of the second formulation (52) was 0.5% (0/2)) Capryol PGMCTM and the parabens in
`the form of their 10 (717/70)% 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.
`
`
` I NWT-assay Antimicrobial tests
`
`.
`'
`.
`'
`Testedsolutions:
`Tested solutions:
` x
`
`,
`
`,
`K
`
`parabens alone
`vglycerol, ethanol
`-parabens in compiex systems
`/7777777777777777777777777777777777777.
`It
`"
`0. minute;
`Preparation of solutions
`
`I
`
`parabens alone
`A-parabens in complex systems
`,7777777777777777777777777777777777777\
`G
`0. minute:
`Preparation of solutions
`
`
`
`!
`f:
`V
`t0. minute:
`19. minute:
`
`
`Start at 30 minutes long incubation on
`Start of 24 hours long incubation an
`Caca»2 cells
`fungaland bacteria! celis
`
`Measurement of absorbance 21492 and
`600 nm
`
`
`
`Removal 01’ test SGMUGI’IS from cells
`
`-Addig MTT—soluttorl to cells for 3 hours
`incubation
`
`V.
`V
`I:
`«Removal
`of MTr-solutian
`after
`
`incubation
`oDissolving formazan crystals
`
`
`Measurement of absorbence at 5m and
`890 nm
`
`
`
`
`
`Figure 2. Experimental design.
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 3
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 3
`
`

`

`Molecules 2018, 23, 1827
`
`40f 19
`
`Table 1. Composition of test solutions for cytotoxicity tests.
`
`
`Component
`81
`S2
`
`Paraben
`0.2 w/w%, 0.02 w/w‘Vn, 0.002 w/w%, 0.0002 w/w“n
`
`Glycerol
`30 v/v%, 3 v/v%, 0.3 z~/v%, 0.03 v/v%
`—
`
`_
`0.002 17/17/0, 0.0002 11/11/0, 0.00002 v/v A),
`Polysorbate 20
`0.000002 v/v"..
`
`0.5 v/v%, 0.05 v/v%, 0.005 v/v%, 0.0005
`Capryol PGMcTM
`
`21/ 21%
`
`1.4 v/v%, 0.14 v/v%, 0.014 v/v%, 0.0014
`Ethanol
`-
`0v/ v /0
`
`solvent, used for tenfold, hundredfold, thousand-fold dilution
`
`PBS
`
`Table 2. Composition of test solutions for antimicrobial tests.
`
`
`Component
`51
`52
`Control
`Paraben
`0.1 w/ 10%, 0.15 w / 20%, 0.25 w /w%
`-
`Glycerol
`-
`-
`Polysorbate 20
`-
`—
`Capryol PGMCTM
`0.5 U/v‘Vo
`0.7 v/v%, 1.05 v/v%, 1.75 U/Un/o
`Ethanol
`0.7 v/v%, 1.05 U/Un/o, 1.75 v/v%
`RPMI-1640
`solvent for antifungal tests
`Mueller-Hinton broth
`solvent for antibacterial tests
`
`30 v / 0%
`0.002 v/v‘fi.
`—
`—
`
`Test solutions were prepared in 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 and the cell viability was calculated. After seeding the bacterial and fungal cells
`in appropriate concentrations into 96-well microplates, a 24 h long incubation was started. Optical
`density was measured at two wavelengths at 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)°/n butyl and ethyl paraben had significantly higher cytotoxicity than
`methyl and ethyl paraben, which had similar toxicity patterns. There was a 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 higher the ratio of dilution of
`parabens, the better the cell viability of the Caco-2 cell line was.
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 4
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 4
`
`

`

`Molecules 2018, 23, 1827
`
`5 of 19
`
`100
`
`5 75
`g
`E‘
`E
`
`50
`
`25
`
`0
`
`-0- Buthyl—paraben
`*- PropyI-paraben
`
`-*' Ethyl-paraben
`~l~ Methyl-paraben
`
`Cytotoxicity of parabens
`
`
`
`639$
`
`0&6"
`e~§$
`o-QQQ
`Concentration of parabens Wlw%
`
`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,
`n = 12. Cell Viabili y 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°41 :: 25°41; PP: 53°41 :: 47°41; 78°41 :: 4°41; 97°41 :: 27°41; 100°41 :: 27°41; BP: 41°41 :: 46°41; 81°41 :: 24°41;
`
`
`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 manner in the case of these solvents. The ICSO (the inhibitory
`concentration Value, where the 50% cell Viability was measured by an MTT test) of glycerol was
`45 (v/ v)%. In our complex systems, the concentrations of glycerol were 30 (v / v)%, 3 (v/ U)O/n, 0.3 (v /v)%,
`0.03 (v / 20% which were lower than this inhibitory concentration.
`The concentrations of ethanol (1.75 (v/v)%, 1.4 (v/v)%, 0.14 (Zr/10%, 0.014 (v/v)%) in complex
`systems were applied for cytotoxicity and antimicrobial tests. Based on this cytotoxicity test, the
`1C50 Value cannot be determined in these concentration ranges. The cell Viability slightly decreased
`according to the concentration, but the highest concentration (1.75 (v/v)%) decreased the cell Viability
`
`significantly (80 :: 1.7%).
`
`Cytotoxicity of ethanol
`
`1 013
`
`g 75
`g63
`"g
`aQ
`
`50
`
`25
`
`O
`
`QQQQQQ
`§§¢§3
`’5
`3‘
`”V
`‘1
`«9
`<5»
`85
`4°
`8 ”$8“$
`g;
`99
`92>
`Concentration of ethanol “LAC;
`
`Figure 4. Cytotoxicity of ethanol 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 the different concentrations: 100% :: 0.2%; 100% :: 1.8%; 100% :: 3.1%; 100% :: 2%;
`
`
`
`
`
`95°41 :: 17°41; 87°41 :: 05°41; 81°41 :: 1.1°41; 72°41 :: 09°41; 66°41 :: 1°41.
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 5
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 5
`
`

`

`Molecules 2018, 23, 1827
`
`60f 19
`
`Cytotoxicity of glycerol
`
`100
`
`q01
`
`
`
`Cellviability 8
`
`25
`
`
`
`Concentration of glycerol vIV%
`
`Figure 5. Cytotoxicity of glycerol 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 the different concentrations: 100% :: 1.1%; 95% :: 2.5%; 90% :: 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 51 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.
`MP had the highest survivability from all esters, the second was EP. The results of the two longer
`parabens were not significantly different from each other at the tested concentrations. Moreover,
`the methyl paraben was not significantly 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 the test solutions diminished at hundredfold and
`ns.
`thousand-fold dilutio
`
`Cytotoxicity of 31
`
`we
`
`..
`
`75
`
`so
`
`
`
`Cellviability
`
`aw Buthyl»paraben
`4‘- Prowtparaben
`a:- Ethyl-paraben
`-I- Methyl-paraben
`Formulated control
`
`Nm
`
`Q
`yN
`
`o“?
`
`c:
`a.
`8
`
`w
`
`’l:
`o
`Q53
`Q‘
`
`Concentration of parabens “Mn
`
`Figure 6. Cytotoxicity of the first formulated system (51) consisting of 30% (v/v) glycerol and 0.002%
`(Zr/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)% concentrations of different formulations containing
`
`
`parabens: formulated control: 48% :: 1.1%; -00% :: 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%;
`
`
`
`
`1000/0 :: 4.6; formulated PP: 60/0 :: 1.30/0; 350/0 :: 4.50/0; 1000/0 :: 4.90/0; 990/0 :
`: 4.6%; formulated BP:
`8% i 1.1%; 30% :t 2.9%; 97% i 2.6%; 100% i 2.3%.
`
`
`
`
`
`In the case of S2 (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 matched the results of the formulated control, while BP had slightly worse cell
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 6
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 6
`
`

`

`Molecules 2018, 23, 1827
`
`7 of 19
`
`viability and MP was also significantly more toxic than the formulated control. After further dilution,
`all esters, except BP caused 100% cell viability.
`
`tiii
`
`Fmrmuiated Mimi-paraben
`Furmuiated prowl-paraben
`Farmuiaied ethyl-paraben
`Farmuiated methyiuparaben
`Formulated mntroé
`
`Cytotoxicity of $2
`
`
`
`Cellviability
`
`50
`
`N4::
`
`Concentration of parabens “($6
`
`Figure 7. Cytotoxicity of the second formulated system (52) consisting of 0.5% (27/27) Capryol PGMCTM
`and ethanol 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)% concentrations of different formula ions containing parabens: Formulated
`
`
`
`
`
`
`control: 63% :: 2.870; 100% :: 2.5%; 100% :: 2.5%; 98%: :: 3.4%; formulated MP: 2% :: 020/0; 90%: :: 1.8%;
`
`
`
`
`97% :: 3.10/0; 99% :: 2‘70; formulated EP: 4% :: 0.7%; 100°/o :: 2.7%; 100°/o :: 3.1°/o; 100"/o :: 2.5°/o;
`
`
`
`
`
`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 order to test the antimicrobial properties of parabens, three different concentrations were used.
`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 with a line in each figure.
`Above this value a certain compound is considered ineffective in the case of antimicrobial activity,
`while below this line 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. albicrms, (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, Sl-PP, Sl-BP and SZ-EP had higher cell 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. pampsilosis (Figure 9) showed a high cell viability gap between the control
`parabens and 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 inhibitory effect at all.
`C. glabmta was also sensitive to both the control and the formulated solutions (Figure 10), but
`the results of 51 were worse than S2 or the control. This lack of effectiveness increased with the
`
`growing concentration.
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 7
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2031 Page 7
`
`

`

`Molecules 2018, 23, 1827
`
`80f 19
`
`C. albicans
`
`
`
`
`
`
`............................................................
`
`100
`
`J}09noGOO
`
`N1:
`
`
`
`Cellviability
`
`
`
`' SZ-BP
`SZvPP
`SZvEP
`m SZ-MP
`$243
`I S1-BP
`S1-PP
`S1-EP
`S1—MP
`
`g?
`9"
`5.9
`Concentration of parabens WIW%
`
`I s1-c
`I BP
`8 PP
`m EP
`I MP
`
`Figure 8. Cell Viability of C. albicans against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl-MP; S1-EP; S1-PP; Sl-BP) and the second system (S2-MP; SZ-EP; SZ-PP; S2-BP). Cell viability
`
`expressed as the percentage of the absorbance of the untreated control. Data expressed as mean :: SEM,
`n = 4. Cell viability of fungal cells at 0.1 ; 0.15 ; 0.25 (70/7v)% concentrations of different formulations
`
`
`
` : 3.4%;
`containing parabens: MP: 11% :: 2.2%; 10% :: 3.2%; 10% :: 2.5%; EP: 10% :: 3.3%; 10% :
`
`
`
`
`
`
`10% :: 4.10/0; PP: 11% :: 2.2%; 9% :: 2.30/0; 90/0 :: 3%; BP: 10% :: 1.7%; 100/0 :: 2.2%; 9% :: 3%.; formulated
`
`
`
`control of 81: 13% :: 1.7%; formulated MP of $1: 12% :: 3.1%; 13% :: 2.7%; 12% :: 1.4%; formula ed EP
`
`
`
`
`
`
`of 81: 12% :: 2.3%; 12% :: 1.9%;16% :: 0.9%; formulated PP of $1: 14% :: 0.7%; ;6% :
`: 0.5%; 27% :: 1.3%;
`
`
`
`
`formulated BP of S1: 15% :: 1.6%; 16% :: 2.5%; 32% :: 1%; formulatec. control of S2: 18% :
`: 3.30/0;
`
`
`
`
`16% :: 2.5%; 16% :: 2%; formulated MP of 52: 17% :: 2.1%; 16% :: 1.7%; 16% :: 1.7%; formulated EP of
`
`
`
`
`
`:: 2.2%; 16% :: 0.9%;
`S2: 17% 2: 1.5%; 16% 2: 0.8%; 32% Z: 2%; formulated PP of S2: 17% I: 1.8%; 16%
`
`
`
`formulated BP of S2: 18%) :: 1.2%;; 170/0 :: 1.7%;; 22%) :: 0.8%).
`
`
`
`
`
`
`
`100
`
`C. parapsilosis
`
`
`
`Cellviability
`
`6}09oo
`is
`
`No
`
`Concentration of parabens “’Iw%
`
`Figure 9. Cell Viability of C. pampsilosis against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl-MP; Sl-EP; Sl-PP; Sl-BP) and the second system (S2-MP; SZ-EP; S2-PP; S2-BP). Cell Viability
`expressed as the percentage of the absorbance of the untreated control. Data expressed as mean :: SEM,
`n = 4. Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)% concentrations of different formulations
`
`
`
` I 0.30/0, 00/0 " 0.10/0,
`containing parabens: MP: 5% :: 4%; 2% :: 1.5%; 0% + 0.5%; EP: 1% :: 0.8%; 1% :
`PP: 1% :: 0.1%; 0% + 0.4%; 0% + 0.2%; BP: 0% + 0.4%; 0% + 0.3%; 0% + 0.3%; formulated control of
`
`
`
`
`S1: 22% :: 1.5%; formulated MP of 81: 24% :: 1.4%; 27% :: 1.2%; 25% :: 2%; formulated EP of 81:
`
`
` I 1.20/0; 570/0 2: 0.70/0;
`23% ::
`.3%; 23% :: 1.7%; 29% :: 1.9%; formulated PP of S1: 24% :: 1.3%; 33% :
`
`
`
`formula ed BP of S1: 23% :: 1.1%; 34% :: 2%; 38% :: 0.9%; formulated contro
`l of S2: 36% :: 2.7%;
`
`
`
`44% :: 1.5%; 44% :: 1.8%; formulated MP of $2: 28% :: 1.3%; 30% :: 1.6%; 30%
`:: 1.8%; formulated
`
`
`
`EP of S2: 29% :: 2.2%; 28% :: 1.5%; 31% :: 1.8%; formulated PP of S2: 300/0 :
`: 2.3%; 28% :
`
`
`
`27% :: 1.9%; formulated BP of S2: 31% :: 1.2%; 31% :: 1.5%; 33% :: 1.9%.
`
`
`
`
`
`
`
` Z 2.10/0;
`
`
`
`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
`
`100
`
`C. glabrata
`
`
`
`Cellviability
`
`80
`
`60
`
`40
`
`20
`
`I SZ<BP
`SZ-PP
`SZ-EP
`w SZ—MP
`32-0
`I S1-BP
`1—PP
`% S1-EP
`S1—MP
`I S1—C
`I BP
`I PP
`m EP
`I MP
`
`Concentration of parabens Wlw%
`
`Figure 10. Cell Viability of C. glabmm against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl—MP; Sl—EP; S1—PP; Sl—BP) and the second system (S2—MP; SZ—EP; SZ—PP; S2—BP). Cell viability
`expressed as the percentage of the absorbance of the untreated control. Data expressed as mean :: SEM,
`n = 4. Cell viability of fungal ce.ls at 0.1; 0.15; 0.25 (70/70)% concentra ions 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
`
`
`
`
`$1: 6% :: 1.6%; formulated MP of 51: 14% :: 3.4%; 16% :: 2.5%; 15% :: 2.5%; formulated EP of 51:
`
`
`
`
`
`14%: :: 1.1%;; 13%: :: 1.4%); 19%: :: 2%); formulated PP of S1: 14%: :: 0.7%;; 15%: :: 1.4%); 21%; :: 1%;;
`
`
`
`formulated BP of 81: 14% :: 3.4%; 19% :: 3.2%; 24% :: 2.9%; formulated control of S2: 6% :: 2.9%;
`
`
`
`
`6% :: 2.5%; 6% :: 2%; formulated MP of S2: 5% :: 3.4%; 5% :: 1.4%; 5% :: 2%; formulated EP of S2:
`
`
`
`
`
`5% :: 1.4%; 5% :: 2.8%; 5% :: 1.7%; formulated PP of 52: 5% :: 4.1%; 5% :: 3.2%; 5% :: 1%; formulated
`
`
`
`BP of 52: 1% :: 0.9%; 1% :: 0.8%; 1% :: 0.9%.
`
`
`
`
`
`
`
`2.2.2. Antibacterial Tests
`
`5. aurcus (Figure 11) was not sensitive to the control solutions, and increasing doses of MP, EP
`and PP did not decrease the cell viability. Meanwhile, the ethanolic solution of BP and 51 proved to
`be highly effective. 52 containing Capryol PGMCTM apart from ethanol was also 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 81, which was very effective
`against it. The addition of a surface-active agent in 52 could increase the antimicrobial properties of BP
`and PP as they showed greater inhibitory effect than the normal ethanolic solutions. 51 showed higher
`efficacy than the other solutions.
`P. aeruginosa (Figure 13) showed the widest spectrum of resistance. The ethanolic EP, PP and BP
`could not inhibit its growth at all, like PP and BP in 52. The presence of Capryol PGMCTM was also
`advantageous for the EP and BP, their effectiveness was highly increased, but they still could not reach
`a 50% inhibitory rate. All derivatives formulated in 51 were totally effective in every concentration,
`and methyl paraben was also effective at the highest dose in 52 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
`
`100
`
`0’no0O
`
`asc
`
`No
`
`
`
`Cellviability
`
`S. aureus
`
`
`
`Concentration of parabens wIW%
`
`Figure 11. Cell viability of S. uureus against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl-MP; Sl-EP; S1-PP; Sl-BP) and the second system (S2-MP; SZ-EP; S2-PP; S2-BP). Cell viability
`expressed as the percentage of the absorbance of the untreated control. Data expressed as mean :: SEM,
`
`rations of different formu.ations
`n = 4. Cell Viability of bacterial ce.ls at 0.1; 0.15; 0.25 (70/70)% concen
`
`
`
`EP: 700/0 :: 2
`.40/0; 700/0
`:: 1.30/0;
`containing parabens: MP: 80% :: 2.2%; 66% :: 1.9%; 59% :: 1.5%;
`
`
`
`
`
`0.90/0; 10/0
`62% :: 1.6%; PP: 83% :: 2.3%;
`: 1.10/0; 10/0 ::
`:: 0.40/0;
`
`
`0.5%; 1% :: 0.8%;
`formulated control of 51: 1% :: 0.3%; formulated MP of 81: 2% :: 1.4%; 1% ::
`
`
`
`PP of S1: 2% :
`Z 1.60/0; 40/0 I
`formulated EP of 51: 1% :: 0.2%; 1% :: 0.4%; 4% :: 2.8%; formulated
`
`
`
`
`ated control
`1.5%; formu
`6% :: 2.4%; formulated BP of
`51: 40/0 :: 2.10/0; 50/0 :: 1.60/0; 30/0 ::
`
`
`
`
`2.60/0; 10/0 I: 0.50/0,
`: 1.20/0; 200/0 ::
`37% :: 1.6%; 4% :: 2.7%; 4% :: 1.1%; formulated MP of S2: 61% :
`
`
`
`formulated EP of S2: 28% :: 2.4%; 2% :: 1.3%; 1% :: 0.9%; formulated PP of 82: 22% :: 1.7%; 4% :: 2.1%;
`
`
`
`
`0% :: 0% +03%; formulated BP
`Of 52. 30/0 I: 2.50/0; 30/0 I: 1.80/0; 30/0 I: 2.9O/u.
`
`66% :: 2.5%; 63% :: 1.4%; BP: 2% :
`
`
`
`
` Z 1.30/0;
`
`
`of S2:
`
`100
`
`E. coli
`
`
`
`Cellviability h0')00oDO
`
`NO
`
`
`
`Concentration of parabens WIW%
`
`Figure 12. Cell Viability of E. coli against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl-MP; Sl-EP; Sl-PP; Sl-BP) and the second system (S2-MP; SZ-EP; SZ-PP; S2-BP). Cell Viability
`expressed as the percentage of the absorbance of 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 formu.ations
`
`
`
`
`: 1.7%; 10% : 2%; 0% + 01%; EP; '
`6% :: 1.20/0; 80/0 :
`I 160/0, 70/0 I
`: 1.2%;
`containing parabens: MP: 20% :
`
`
`
`
`
`
`ulated
`PP: 59% :: 1.6%; 46% :: 2.5%;
`40% :: 0.9%; BP: 59% :: 1.8%; 50%
`:: 0.5%; 54% :: 1%; form
`
`control of S1: 0% + 0.4%; formulated MP of 81: 0% :: 0.3%; 0% :: 0.1%; 0% :
`: 0.2%; formulated EP
`
`of 51: 0% + 0.1%; 00/0 + 0.2%; 1
`% :: 0.4%; formulated PP of 81: 0% + 0.2%; 0% + 0.4%; 1% :
`: 0.4%;
`
`
`: 0.6%;
`formulated BP of S1: 0% + 0.3%; 7% :: 2.4%; 1%
`,: 1%; formulated control of 52: 40% :
`
`
`
`
`40% :: 1.1%; 37% :: 0.9%; formulated MP of S2: 12% :: 1.2%; 7% :: 1.
`6%; 1% ::0.2%; formulated EP of
`
`
`
`
`
`52: 15%: :: 1.7%); 3‘70 :: 2.4%); 10/0 :: 0.7%); formulated PP Of 52: 51%) :: 0.8%); 17%: ::
`1.50/0} 23U/u Z: 1.90/0;
`
`
`
`formulated BP of S2: 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
`
`1 00
`
`P. aureginosa
`
`
`
`Cellviability
`
`#-monaco
`
`No
`
`
`
`Concentration of parabens WIW%
`
`ll SZ-BP
`sz-PP
`82-139
`m SZ-MP
`sz-c
`I 81-813
`m S1-PP
`
` . S1-MP
`
`I S1-C
`I 8P
`a PP
`m EP
`- MP
`
`Figure 13. Cell Viability of P. aeruginosn against the control paraben solutions (MP; EP; PP; BP); the first
`system (Sl—MP; Sl—EP; S1—PP; Sl—BP) and the second system (S2—MP; S2—EP; SZ—PP; S2—BP). Cell viability
`
`expressed as the percentage of the absorbance of the untreated control. Data expressed as mean :: SEM,
`n = 4. Cell viability of fungal cells at 0.1; 0.15; 0.25 (w/w)% concentrations of different formula ions
`
`
`
`
`
`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 81: 0% + 0.4%; formulated MP of 51: 0% + 0.2%; 0% + 0.3%; 0% + 0.1%; formu.ated
`EP of 81: 0% + 0.2%; 0% + 0.2%; 0% + 0.3%; formulated PP of 51: 1% + 0.5%; 1% + 0.3%; 1.8% + 0.4%;
`
`formulated BP of 51: 1% + 0.1%; 8% :: 2.1%; 4% :: 1.7%; formulated control of 52: 100% :: 2.6%;
`
`
`
`
`
`
`99% :: 1.4%; 99% :: 1.7%; formulated VIP of S2: 96% :: 2.5%; 62% :: 1.5%; 4% :: 1.3%; formulated
`
`
`
`EP of S2: 100% :: 2%; 92% :: 1.7%; 72% :: 1.3%; formulated PP of S2: 100% :: 2.1%; 100% :: 1.6%;
`
`
`
`
`100%: :: 1.8%); Formulated BP 01:52: 100%: :: 2‘70; 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:
`S1 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 PGMCTM (HLB value: 5) and parabens in the form of their 70%
`(v/ v) ethanolic solutions [25].
`
`The basis of selection was to use one co-solvent, different surfactants with high and moderate
`HLB values 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/v)%
`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 and it
`showed cytotoxic properties at 10% (71/0) on Caco-2 cells [31,32].
`The effective glycerol concentration for enhancing the solubility of different active pharmaceutical
`ingredients (APIS) was proved from 20% (22/22) [33].