°
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`rt
`Patentamt
`European
`Patent Office
`desbrewets
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`Mt||||||||| |||||||||||||||ll
`(11)
`EP 1 768 649 B1
`
`(12)
`
`EUROPEANPATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`23.09.2009 Bulletin 2009/39
`
`(21) Application number: 05758582.0
`
`(22) Dateoffiling: 07.07.2005
`
`(51) Int Cl.:
`AG1K 9/08 (7006.01)
`
`AG1K 31/495 (2006.01)
`
`(86) International application number:
`PCT/EP2005/007340
`
`(87) International publication number:
`WO 2006/005507 (19.01.2006 Gazette 2006/03)
`
`(64) PHARMACEUTICAL COMPOSITION OF PIPERAZINE DERIVATIVES
`PHARMAZEUTISCHE ZUSAMMENSETZUNG VON PIPERAZINDERIVATEN
`
`COMPOSITION PHARMACEUTIQUE DE DERIVES DE PIPERAZINE
`
`(84) Designated Contracting States:
`AT BE BG CH CY CZ DE DK EE ES FIFR GB GR
`HU IE IS IT LILT LU LV MC NL PL PT RO SE SI
`SK TR
`
`Designated Extension States:
`AL BA HR MK YU
`
`(30) Priority: 14.07.2004 EP 04016519
`
`(43) Date of publication of application:
`04.04.2007 Bulletin 2007/14
`
`(73) Proprietor: UCB FARCHIM S.A.
`CH-1630 Bulle (CH)
`
`Inventors:
`(72)
`¢ FANARA, Domenico
`B-4520 Wanze(BE)
`
`¢ SCOUVART,Jean
`B-1150 Brussels (BE)
`¢ POULAIN, Claire
`B-1060 Brussels (BE)
`¢ DEELERS, Michel
`B-1630 Linkebeek (BE)
`
`(74) Representative: Lechien, Monique
`UCB, S.A.,
`Intellectual Property Department
`Allée de la Recherche 60
`
`1070 Brussel (BE)
`
`(56) Referencescited:
`EP-A- 0 605 203
`US-A- 5 504 113
`US-B1- 6 432 961
`
`WO-A-20/04004705
`US-B1- 6 319 927
`
`
`
`EP1768649B1
`
`Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
`paid. (Art. 99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
`
`UCB Biopharma SPRL(IPR2019-00400)
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 1
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`

`

`Description
`
`EP 1 768 649 B1
`
`[0001] The present invention relates to a liquid pharmaceutical composition containing an active substance such as
`cetirizine, levocetirizine and efletirizine.
`[0002] A number of substances belonging to the family of substituted benzhydryl piperazines are known to be sub-
`stances with useful pharmacological properties.
`[0003] European Patent EP 58146, filed in the name of UCB, S.A., describes substituted benzhydryl piperazines
`having the general formula
`
`x
`
`——
`
`N—}
`N
`\_/
`
`:
`
`O
`
`(CH,aed
`“
`\L
`
`in which L stands for an -OH or -NH, group, X and X’, taken separately, stand for a hydrogen atom, a halogen atom, a
`linear or branched alkoxy radical at C, or Cy, or a trifluoromethyl radical, m equals 1 or 2, n equals 1 or 2, as well as
`their pharmaceutically acceptable salts.
`[0004] Ofthese compounds, 2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperaziny|]ethoxy] acetic acid, also known un-
`der the nameofcetirizine, and its dichlorohydrate are well knownfor their antihistaminic properties.
`[0005] The active substances belonging to the family of substituted benzhydryl piperazines specifically include
`2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyllethoxy]-acetic acid (cetirizine), 2-[2-[4-[bis(4-fluorophenyl)methyl]-
`1-piperazinyl]ethoxy]acetic acid (efletirizine), their optically active isomers when applicable, as well as their pharmaceu-
`tically acceptable salts.
`[0006]
`Inthe pharmaceuticalfiled, solutions and drops are generally produced as germ-free compositions during their
`production processes. However, once the seal of the containers is broken, and the pharmaceutical compositions are
`completely used over a period oftime, these pharmaceutical compositions are continuously exposed to the risk of being
`contaminated by the microorganismsexisting in the environment or the human body, eachtime the containers are used
`and their covers are openedor closed.
`[0007]
`It has now surprisingly been found that the active substances belonging to the family of substituted benzhydryl
`piperazines possess a preservative effect in aqueous solutions.
`[0008] The purpose of the invention concerns a liquid pharmaceutical composition containing an active substance
`belonging to the family of substituted benzhydryl piperazines chosen among cetirizine, levocetirizine and efletirizine,
`and a reduced amountof preservatives.
`[0009] The present invention is based on the unexpected recognition that a pharmaceutical composition comprising
`an active substance belonging to the family of substituted benzhydryl piperazines and a reduced amount of preservatives
`is stable during a long period of time. Stability means the capacity to resists to microbial contamination.
`[0010] The present invention encompasses a pharmaceutical composition comprising an active substance belonging
`to the family of substituted benzhydryl piperazines and an amountof parahydroxybenzoate esters used as preservatives
`less than 3 mg/ml of the composition, a normal concentration to preserve aqueous solutions.
`[0011] The present invention encompasses a pharmaceutical composition comprising an active substance chosen
`among cetirizine, levocetirizine and efletirizine and at least one preservative, wherein the amountof preservativeis in
`the case of parahydroxybenzoate esters more than 0 and less than 1.5 mg/ml of the composition, and in the case of
`other preservatives correspondsto the bactericidal effect of a parahydroxybenzoate esters concentration of more than
`0 and less than 1.5 mg/ml.
`[0012] Generally, the pharmaceutical composition of the invention is liquid and preferably aqueous.
`[0013]
`In the pharmaceutical composition of the invention, the active substanceis generally selected from the group
`
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`EP 1 768 649 B1
`
`of cetirizine, levocetirizine, efletirizine, and their pharmaceutically acceptable salts. Preferably, the active substanceis
`selected from the group of cetirizine, levocetirizine, and their pharmaceutically acceptable salts.
`[0014] Theterm "cetirizine" refers to the racemate of [2-[4-[(4 chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]-acetic
`acid and its dihydrochloride salt which is well known ascetirizine dihydrochloride; its levorotatory and dextrorotatory
`enantiomers are knownaslevocetirizine and dextrocetirizine. Processes for preparing cetirizine, an individual optical
`isomer thereof or a pharmaceutically acceptable salt thereof have been described in European Patent 0 058 146, Great
`Britain Patent 2.225.320, Great Britain Patent 2.225.321, United States Patent 5,478,941, European Patent application
`0 601 028, European Patent Application 0 801 064 and International Patent Application WO 97/37982. Ophthalmic
`compositions with cetirizine as an active principle and parabens as preservatives are disclosed in EP 605 203 A.
`[0015] The term "levocetirizine" as used herein means the levorotatory enantiomer of cetirizine. More precisely, it
`means that the active substance comprises at least 90%by weight, preferably at least 95% by weight, of one individual
`optical isomer of cetirizine and at most 10%by weight, preferably at most 5% by weight, of the other individual optical
`isomer of cetirizine. Each individual optical isomer may be obtained by conventional means, i.e., resolution from the
`corresponding racemic mixture or by asymmetric synthesis. Each individual optical isomer may be obtained from its
`racemic mixture by using conventional means such asdisclosed in British patent application No. 2,225,321. Additionally,
`eachindividual optical isomer can be prepared from the racemic mixture by enzymatic biocatalytic resolution, such as
`disclosed in U.S. Patents No. 4,800,162 and 5,057,427.
`[0016] The term "efletirizine” as used herein refers to 2-[2-[4-[bis(4-fluorophenyl)methyl]- 1 -piperazinyl]ethoxy]acetic
`acid. Efletirizine is encompassed within general formula | of European patent No. 58146, which relates to substituted
`benzhydrylpiperazine derivatives. Efletirizine has been found to possess excellent antihistaminic properties. It belongs
`to the pharmacological class of histamine H,-receptor antagonists and showsin vitro high affinity and selectivity for
`H,-receptors. It is useful as an antiallergic, and antihistaminic agent. Two pseudopolymorphic crystalline forms of efle-
`tirizine dihydrochloride, namely anhydrousefletirizine dinydrochloride and efletirizine dihydrochloride monohydrate, are
`described in the European patent No. 1 034 171, and another pseudopolymorphic form of efletirizine dihydrochloride is
`describedin the international patent application WO 03/009849. Processesfor preparing efletirizine or a pharmaceutically
`acceptable salt thereof have been described in European Patent 1 034 171, and in the international patent applications
`WO 97/37982 and WO 03/009849.
`
`[0017] The term "pharmaceutically acceptable salts" as used herein refers not only to addition salts with pharmaceu-
`tically acceptable non-toxic organic and inorganic acids, such as acetic, citric, maleic, succinic, ascorbic, hydrochloric,
`hydrobromic, sulfuric, and phosphoric acids and the like, but also its metal salts (for example sodium or potassium salts)
`or ammonium salts, the amine salts and the aminoacid salts. The best results have been obtained with dihydrochloride
`salts.
`
`By preservatives we understand a chemically substance that inhibits the development of microorganismsor,
`[0018]
`in an ideal instance, kills them; so antimicrobial agentable to limit or avoid the growth of microorganisms suchasbacteria,
`yeast and moulds in a solution. Preservatives will comply with Eur P. and USP requirements: for a product incubated
`with a large number of bacteria and fungi, the preservative mustkill and reduce a required amountof bacteria and fungi
`within a prescribed time period.
`ethyl
`parahydroxybenzoate,
`(methyl
`esters
`p-hydroxybenzoate
`are
`[0019] Examples
`of
`preservatives
`parahydroxybenzoate , propyl parahydroxybenzoate , butyl parahydroxybenzoate , Ci-C20 alkyl parahydroxybenzoate
`and their sodium salts), acrinol, methyl rosaniline chloride, benzalkoniumchloride, benzethonium chloride, cetylpyridinium
`chloride, cetylpyrodium bromide, chlorohexidine, chlorohexidine acetate, benzylalcohol, alcohol, chlorobutanol, isopro-
`panol, ethanol, thimerosal, phenol, sorbic acid, potassium and calcium sorbate, benzoic acid, potassium and calcium
`benzoate, sodium benzoate, calcium acetate, calcium disodium ethylenediaminetetraacetate, calcium propionate, cal-
`cium sorbate, diethyl pyrocarbonate, sulphur dioxide, sodium sulphite, sodium bisulfite, boric acid, sodium tetraborate,
`propionic acid, sodium and calcium propionate, sodium thiosulfate, or a mixture therefore. Preferably the preservative
`is selected from the group of methyl parahydroxybenzoate, ethyl parahydroxybenzoate , propyl parahydroxybenzoate,
`amixture of methyl parahydroxybenzoate and ethyl parahydroxybenzoate or propyl parahydroxybenzoate , andamixture
`of methyl parahydroxybenzoate and propyl parahydroxybenzoate. Best results have been obtained with a mixture of
`methyl parahydroxybenzoate and propyl parahydroxybenzoatein a ratio of 9/1 expressed in weight.
`[0020]
`In aparticular embodimentof the invention, the pharmaceutical composition contains an amountof p-hydroxy-
`benzoate esters (methyl p-hydroxybenzoate/propyl p-hydroxybenzoate in a ratio of 9/1 expressed in weight) selected
`in the range of 0.0001 and 1.5 mg/ml of the composition. Preferably, it contains an amountselected in the range of 0.01
`and 1.125 mg/ml. More preferably it contains an amount of preservatives selected in the range of 0.1 and 1 mg/ml.
`[0021]
`By patient, we understand children, adolescents and adults, preferably of 2 years old. The targeted patients
`are usually old from 2 years and more.
`[0022] Apreferred daily dosage provides from about 0,0005 mg to about 2 mgof levocetirizine or a pharmaceutically
`acceptable salt thereof, per kg of body weight per patient. A particularly preferred daily dosage is from about 0,001 to
`about 2 mg per kg of body weight per patient. The best results have been obtained with a daily dosage from about 0,005
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`UCB Biopharma SPRL (IPR2019-00400)
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`EP 1 768 649 B1
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`to 1 mg per kg of body weight per patient. The dosage may be administered once per day of treatment, or divided into
`smaller dosages, for examples 1 to 4 times a day, and preferably 1 to 3 times a day, and administrated over about a 24
`hours time period to reacha total given dosage. Best results have been obtained with an administration of a composition
`of the invention twice a day for infants; and 5 mg once a dayfor children and adults. The exact dosages in which the
`compositions are administrated can vary according to the type of use, the modeof use, the requirements of the patient,
`as determined by a skilled practitioner. The exact dosage for a patient may be specifically adapted by a skilled person
`in view of the severity of the condition, the specific formulation used, and other drugs which may be involved.
`[0023] The pharmaceutical forms according to the present invention may be prepared according to conventional
`methods used by pharmacists. The forms can be administered together with other components or biologicaly active
`agents, pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.
`[0024] The pharmaceutical compositions of the invention include any conventional therapeutical inert carrier. The
`pharmaceutical compositions can contain inert as well as pharmacodynamically active additives. Liquid compositions
`can for example take the form of a sterile solution which is miscible with water. Furthermore, substances conventionally
`used as preserving, stabilizing, moisture-retaining, and emulsifying agents as well as substances such assalts for varying
`the osmotic pressure, substancesfor varying pH such as buffers, and other additives can also be present. If desired an
`antioxidant can be included in the pharmaceutical compositions. Pharmaceutical acceptable excipients or carriers for
`compositions include saline, buffered saline, dextrose or water. Compositions may also comprise specific stabilizing
`agents such as sugars, including mannose and mannitol. Carrier substances and diluents can be organic or inorganic
`substances,for example water, gelatine, lactose, starch, gum arabic, polyalkylene glycol, cellulose compoundsand the
`like. A prerequisite is that all adjuvants and substances used in the manufacture of the pharmaceutical compositions
`are nontoxic.
`
`Pharmaceutical compositions can be administered by spray inhalation. Any conventional pharmaceutical com-
`[0025]
`position for spray inhalation administration may be used. Another preferred mode of administration is by aerosol.
`[0026] The pharmaceutical compositions according to the present invention may also be administered orally. They
`may also be administered by nasal instillation, aerosols. The pharmaceutical compositions which can be used for oral
`administration is liquid, for example, in the form of solutions, syrups, drops and the like.
`[0027] The pharmaceutical forms, such as drops, nasal drops, eye drops and ear drops are prepared by conventional
`pharmaceutical methods. The compoundsof the present invention are mixed with a solid or liquid, non-toxic and phar-
`maceutically acceptable carrier and possibly also mixed with a dispersing agent, a stabilizing agent and the like. If
`appropriate,it is also possible to add sweeteners, coloring agents andthe like.
`[0028]
`Preferably, the pharmaceutical composition of the invention is administered in traditional form for oral admin-
`istration, as oralliquid preparation such as syrup.
`[0029] Best results have been obtained with an oral dosage form, in particular liquid formulations such as syrup for
`children.
`
`[0030] An advantage of the invention is that reducing the concentration of the preservative leads to a reduction of the
`risk of an allergic reaction in sensitive patients.
`[0031] Another advantage of the invention is the ability to make easier the manufacturing process avoiding the solu-
`bilization of important amounts of preservatives not freely soluble in water.
`[0032] The invention is further defined by reference to the following examples.
`
`Example 1. Preservative effect of cetirizine.
`
`[0033] An oral solution and drops containing cetirizine are prepared. The compositions are given in table 1.
`
`
`Table 1. - Cetirizine compositions
`
`Oral solution
`Drops
`1
`10
`450
`-
`200
`250
`50
`350
`1
`10
`0.1754
`-
`4.2
`10
`ad pH 5
`ad pH 5
`ad 1
`ad 1
`
`Cetirizine hydrochloride (mg)
`Sorbitol sol. At 70% (mg)
`Glycerine (mg)
`Propyleneglycol (mq)
`Sodium saccharinate (mg)
`Banana flavour (mg)
`Sodium acetate (mg)
`Acetic acid
`Purified water (ml)
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 4
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`EP 1 768 649 B1
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`[0034] The antimicrobial preservative effectiveness tests are realized according to the European Pharmacopoeia
`(Chap. 5.1.3.). Samples of the oral solution and the drops are inoculated with bacterial and yeast suspensions of Pseu-
`domonas aeruginosa ATCC 9027, Escherichia Coli ATCC 8739, Staphylococcus aureus ATC C6538, Candida albicans
`ATCC10231 and Aspergillus niger ATCC 16404. The number of viable microorganisms per ml of preparations under test
`are determined. The results are given in tables 2 and 3.
`
`Time (days)
`
`Inoculum
`0
`7
`14
`21
`28
`
`Time (days)
`
`Inoculum
`0
`7
`14
`21
`28
`
`Table 2. - Microbial content in inoculated sample of the oral solution
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`
`5.5 x 105
`4.9 x 105
`< 100
`<1
`<1
`<1
`
`4.6 x 105
`4.7 x 105
`< 100
`<1
`<1
`<1
`
`4.0 x 105
`3.1 x 106
`< 100
`<1
`<1
`<1
`
`3.7 x 105
`2.6 x 105
`< 100
`2
`<1
`<1
`
`2.3 x 106
`1.7.x 106
`4.8 x 105
`8.2 x 108
`5.5 x 108
`5.0 x 108
`
`Table 3. - Microbial content in inoculated sample of the drops
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`
`4.0x 105
`3.5 x 10°
`< 100
`<1
`<1
`<1
`
`3.4 x 105
`3.8 x 105
`< 100
`<1
`<1
`<1
`
`3.6 x 105
`2.2x 105
`< 100
`<1
`<1
`<1
`
`3.5 x 105
`2.6x 105
`< 100
`<1
`<1
`<1
`
`1.8 x 108
`1.6 x 108
`< 104
`<100
`<1
`<1
`
`In both cases, a rapid disappearance of Pseudomonasaeruginosa, Escherichia Coli, Staphylococcus aureus
`[0035]
`and Candida albicans is observed in the inoculated samples.
`[0036]
`For Aspergillus niger, the number of viable spores is significantly reduced in the oral solution while a rapid
`disappearanceis observedin the drops.
`
`Example 2. Preservative effect of levocetirizine.
`
`[0037] An oral solution and drops containing levocetirizine are prepared. The compositions are given in table 4.
`
`
`Table 4. - Levocetirizine compositions
`Oral solution
`
`Levocetirizine hydrochloride (mg)
`Maltitol-Lycasin 80-55 (mg)
`Glycerine 85 %(mg)
`Propyleneglycol (mg)
`Sodium saccharinate (mg)
`Tutti frutti flavour (mg)
`Sodium acetate (mg)
`Acetic acid (mg)
`Purified water (ml)
`
`0.5
`400
`235.2
`-
`0.5
`0.15
`3.4
`0.5
`ad 1
`
`Drops
`
`5
`-
`294.1
`350
`10
`-
`5.7
`0.63
`ad 1
`
`[0038] The antimicrobial preservative effectiveness tests are realized according to the European Pharmacopoeia
`(Chap. 5.1.3.). Samples of the oral solution and the drops are inoculated with bacterial and yeast suspensions of Pseu-
`domonas aeruginosa ATCC 9027, Escherichia Coli ATCC 8739, Staphylococcus aureus ATC C6538, Candida albicans
`ATCC10231 and Aspergillus niger ATCC 16404. The number of viable microorganisms per ml of preparations under
`test is determined. The results are given in tables 5 and 6.
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`EP 1 768 649 B1
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`Table 5. - Microbial content in inoculated sample ofthe oral solution
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`
`aeruginosa
`aureus
`3.6 x 10°
`2.7 x 10°
`3.2 x 108
`3.5 x 108
`150
`< 100
`<1
`<1
`<1
`<1
`<1
`<1
`
`3.4 x 10°
`3.9 x 108
`2.8 x 104
`1.4.x 104
`2.6 x 102
`6.2 x 103
`
`1.7 x 108
`1.6 x 10
`1.0x 106
`4.8 x 105
`2.2 x 105
`5.3 x 10°
`
`Inoculum
`0
`7
`14
`21
`28
`
`1.7.x 105
`1.8x 105
`< 100
`<1
`<1
`<1
`
`Time (days)
`
`Inoculum
`0
`7
`14
`21
`28
`
`Table 6. - Microbial content in inoculated sample of the drops
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`
`3.6 x 105
`3.2 x 105
`< 100
`<1
`<1
`<1
`
`1.7.x 105
`1.5x 105
`< 100
`<1
`<1
`<1
`
`2.7 x 105
`3.1 x 105
`< 100
`<1
`<1
`<1
`
`3.4 x 105
`1.8x 105
`< 100
`<1
`<1
`<1
`
`1.7 x 106
`1.7.x 106
`9.0 x 104
`<1000
`<1
`<1
`
`|n both cases, a rapid disappearance of Pseudomonasaeruginosa, Escherichia Coli, Staphylococcus aureus
`[0039]
`is observed in the inoculated samples. A disappearance of Candida albicans and Aspergillus niger is also observed in
`the drops.
`
`Example 3. Efficacy of antimicrobial preservation of cetirizine aqueous solutions by p-hydroxybenzoate esters.
`
`[0040] Oral solutions and drops containing cetirizine according to example 1 but also containing mixtures of p-hy-
`droxybenzoate esters (methyl p-hydroxybenzoate/propyl p-hydroxybenzoate in a ratio of 9/1 expressed in weight) are
`prepared. The total amounts of p-hydroxybenzoate esters are 0.15 mg/ml, 0.45 mg/ml, 0.75 mg/ml and 1.05 mg/ml. The
`efficacy of antimicrobial preservation of these solutions and drops is determined according to the European Pharmaco-
`poeia (Chap. 5.1.3.). The results of the tests are given in tables 7 to 14.
`
`Table 7. - Microbial content in inoculated sample of the oral solution containing 0.15 mg/ml of p-hydroxybenzoate
`esters
`Staphylococcus
`aureus
`
`Time (days)
`
`Pseudomonas
`aeruginosa
`
`Escherichia coli
`
`Candida
`albicans
`
`Aspergillus niger
`
`Inoculum
`
`2.3 x 10
`3.7 x 105
`4.0 x 108
`4.6 x 108
`5.5 x 108
`4.1 x 108
`4.0 x 108
`3.0 x 105
`4.5 x 108
`5.1 x 105
`<1
`<1
`<1
`<1
`4
`9.1 x 108
`
`
`
`
`
`<1 <1 <1 <128 750
`
`0 1
`
`Table 8. - Microbial content in inoculated sample of the oral solution containing 0.45 mg/ml of p-hydroxybenzoate
`esters
`
`Aspergillus niger
`
`2.3 x 106
`1.2 x 106
`<100
`2
`
`Candida
`albicans
`3.7 x 108
`2.9 x 10°
`<1
`<1
`
`Escherichia coli
`
`4.6 x 108
`4.9 x 108
`<1
`<1
`
`Staphylococcus
`aureus
`4.0 x 108
`3.3 x 105
`<1
`<1
`
`Pseudomonas
`aeruginosa
`5.5 x 108
`5.2 x 105
`<1
`<1
`
`Time (days)
`
`Inoculum
`
`0 1
`
`4
`28
`
`6
`
`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2035 Page 6
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 6
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`EP 1 768 649 B1
`
`Table 9. - Microbial content in inoculated sample of the oral solution containing 0.75 mg/ml of p-hydroxybenzoate
`esters
`
`Time (days)
`
`Inoculum
`0
`14
`28
`
`Pseudomonas
`aeruginosa
`5.5 x 105
`3.9 x 105
`<1
`<1
`
`Escherichia coli
`
`4.6 x 105
`4.4x 108
`<1
`<1
`
`Staphylococcus
`aureus
`4.0 x 105
`4.0 x 105
`<1
`<1
`
`Candida
`albicans
`3.7 x 10°
`1.9x 105
`<1
`<1
`
`Aspergillus niger
`
`2.3 x 106
`1.9x 106
`<100
`<1
`
`
`Table 10. - Microbial content in inoculated sample of the oral solution containing 1.05 mg/ml of p-hydroxybenzoate
`esters
`
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candidaalbicans
`Aspergillus niger
`aeruginosa
`aureus
`
`
`Inoculum
`0
`14
`28
`
`5.5 x 10°
`3.3 x 108
`<i
`<1
`
`4.6 x 10°
`4.1 x 108
`<1
`<1
`
`4.0 x 10°
`3.1 x 108
`<1
`<1
`
`3.7 x 105
`1.4x 108
`<1
`<1
`
`2.3 x 106
`1.2 x 106
`<100
`<1
`
`Table 11. - Microbial content in inoculated sample of the drops containing 0.15 mg/ml of p-hydroxybenZoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`4.0 x 10
`3.6 x 105
`4.3 x 10°
`2.0x 105
`<1
`<1
`<1
`<1
`
`Inoculum
`0
`14
`28
`
`3.4x 105
`4.0 x 10°
`<1
`<1
`
`3.5 x 105
`2.5x 10°
`<1
`<1
`
`1.8 x 108
`1.5 x 108
`<100
`<1
`
`Table 12. - Microbial content in inoculated sample of the drops containing 0.45 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`4.0 x 105
`3.6 x 108
`3.6 x 105
`1.7 x 105
`<1
`<1
`<1
`<1
`
`3.5 x 105
`2.1 x 105
`<1
`<1
`
`1.8 x 106
`1.4x 106
`<100
`<1
`
`Inoculum
`0
`14
`28
`
`3.4.x 105
`3.6 x 105
`<1
`<1
`
`Table 13. - Microbial content in inoculated sample of the drops containing 0.75 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`4.0 x 10°
`3.6 x 10°
`4.1.x 105
`2.6 x 105
`<1
`<1
`<1
`<1
`
`Inoculum
`0
`14
`28
`
`3.4 x 10°
`3.6 x 106
`<1
`<1
`
`3.5 x 10°
`2.5 x 10°
`<1
`<1
`
`1.8 x 10
`1.6 x 106
`<100
`<1
`
`?
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`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2035 Page 7
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`55
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 7
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`

`

`EP 1 768 649 B1
`
`Table 14. - Microbial content in inoculated sample of the drops containing 1.05 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`
`Inoculum
`0
`14
`28
`
`4.0 x 105
`3.9 x 105
`<1
`<1
`
`3.4.x 105
`3.7.x 105
`<1
`<1
`
`3.6 x 105
`2.8 x 10°
`<1
`<1
`
`3.5 x 105
`2.2 x 106
`<1
`<1
`
`1.8 x 106
`1.3.x 106
`<100
`<1
`
`In all cases, the disappearance of Pseudomonasaeruginosa, Escherichia Coli, Staphylococcus aureus and
`[0041]
`Candida albicans is observedin the inoculated samples. For Aspergillus niger, the number of viable sporesis significantly
`reduced in the oral solution while a rapid disappearance is observedin the drops.
`[0042]
`In all cases the recommended efficacycriteria are achieved.
`
`
`Example 4. Efficacy of antimicrobial preservation of levocetirizine aqueous solutions by p-hydroxybenzoate esters.
`
`[0043] Oral solutions and drops containing levocetirizine according to example 2 but also containing mixtures of p-
`hydroxybenzoate esters (methyl p-hydroxybenzoate/propyl p-hydroxybenzoatein a ratio of 9/1 expressed in weight) are
`prepared. The total amounts of p-hydroxybenzoate esters are 0.375 mg/ml, 0.75 mg/ml and 1.125 mg/ml. The efficacy
`of antimicrobial preservation of these solutions and drops is determined according to the European Pharmacopoeia
`(Chap. 5.1.3.). The results of the tests are given in tables 15 to 20.
`
`Table 15. - Microbial content in inoculated sample of the oral solution containing 0.375 mg/ml of p-hydroxybenzoate
`esters
`
`Escherichia coli
`
`Candidaalbicans Aspergillus niger
`
`Time (days)
`
`Staphylococcus
`Pseudomonas
`aureus
`aeruginosa
`1.7 x 106
`3.4 x 108
`2.7 x 105
`1.7x 108
`3.6 x 105
`Inoculum
`1.6 x 108
`3.8 x 105
`2.8x 105
`1.3x 108
`3.7 x 105
`0
`1.6 x 10°
`1.7 x 104
`<1
`<1
`<1
`14
`
`
`
`
`
`<1 <1 <1 <1es <100
`
`Table 16. - Microbial content in inoculated sample of the oral solution containing 0.75 mg/ml of p-hydroxybenzoate
`esters
`
`Time (days)
`
`Inoculum
`0
`14
`28
`
`Pseudomonas
`aeruginosa
`3.6 x 105
`3.5 x 105
`<1
`<1
`
`Escherichia coli
`
`1.7.x 108
`1.6 x 10°
`<1
`<1
`
`Staphylococcus
`aureus
`2.7.x 105
`2.4x 10°
`<1
`<1
`
`Candidaalbicans
`
`Aspergillus niger
`
`3.4 x 105
`3.4x 10°
`5.5 x 102
`<1
`
`1.7 x 108
`1.6 x 106
`1.4 x 104
`<1
`
`Table 17. - Microbial contentin inoculated sample of the oral solution containing 1.125 mg/ml of p-hydroxybenzoate
`esters
`
`Time (days)
`
`Inoculum
`0
`14
`28
`
`Pseudomonas
`aeruginosa
`3.6 x 10°
`3.9 x 105
`<1
`<1
`
`Escherichia coli
`
`1.7 x 10°
`1.2x 105
`<1
`<1
`
`Staphylococcus
`aureus
`2.7 x 10°
`3.0 x 105
`<1
`<1
`
`Candidaalbicans Aspergillus niger
`
`3.4 x 105
`3.5 x 105
`<10
`<1
`
`1.7 x 108
`1.4.x 106
`< 1000
`<1
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`8
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`UCB Biopharma SPRL(IPR2019-00400)
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`UCB Biopharma SPRL (IPR2019-00400)
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`EP 1 768 649 B1
`
`Table 18. - Microbial content in inoculated sample of the drops containing 0.375 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candidaalbicans Aspergillus niger
`aeruginosa
`aureus
`
`Inoculum
`0
`14
`28
`
`3.6 x 105
`3.1 x 10°
`<1
`<1
`
`1.7 x 105
`1.2x 106
`<1
`<1
`
`2.7 x 105
`2.6 x 105
`<1
`<1
`
`3.4.x 105
`1.7x 105
`<1
`<1
`
`1.7 x 106
`1.8 x 106
`< 1000
`<1
`
`Table 19. - Microbial content in inoculated sample of the drops containing 0.75 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candida albicans Aspergillus niger
`aeruginosa
`aureus
`3.6 x 105
`2.7 x 108
`3.1 x 108
`3.0 x 105
`<1
`<1
`<1
`<1
`
`3.4x 105
`1.8 x 105
`<1
`<1
`
`1.7x 108
`1.4.x 106
`< 1000
`<1
`
`Inoculum
`0
`14
`28
`
`1.7x 108
`1.0 x 105
`<1
`<1
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`Table 20. - Microbial content in inoculated sample of the drops containing 1.125 mg/ml of p-hydroxybenzoate esters
`Time (days)
`Pseudomonas
`Escherichia coli
`Staphylococcus
`Candidaalbicans Aspergillus niger
`aeruginosa
`aureus
`
`Inoculum
`0
`14
`28
`
`3.6 x 105
`2.9 x 105
`<1
`<1
`
`1.7x 108
`6.9 x 104
`<1
`<1
`
`2.7 x 105
`2.7 x 106
`<1
`<1
`
`3.4 x 105
`5.0 x 104
`<1
`<1
`
`1.7 x 106
`1.5 x 106
`< 1000
`<1
`
`In all cases, the disappearance of Pseudomonas aeruginosa, Escherichia Coli, Staphylococcus aureus and
`[0044]
`Candida albicans is observedin the inoculated samples.
`[0045]
`For Aspergillus niger, the number of viable spores is significantly reduced in the oral solution while a rapid
`disappearanceis observedin the drops. In all cases the recommended efficacy criteria are achieved.
`
`Example 5. Nasal solution containing cetirizine and benzalkonium chloride
`
`[0046] A solution containing cetirizine is prepared. The composition is given in table 21.
`
`
`Table 21. - Cetirizine composition
`Nasal solution
`
`Cetirizine hydrochloride (mg)
`Monobasic sodium phosphate (mg)
`Dibasic sodium phosphate (mg)
`Benzalkonium chloride (mg)
`Purified water (ml)
`
`10
`10.6
`29
`0.025
`ad 1
`
`[0047] The efficacy of antimicrobial preservation of this solution is determined according to the European Pharmaco-
`poeia (Chap. 5.1.3.). The recommendedefficacycriteria are achieved.
`
`Example 6. Nasal solution containing efletirizine and p-hydroxybenzoate esters.
`
`[0048] A solution containing efletirizine is prepared. The composition is given in table 22.
`
`9
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`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2035 Page 9
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 9
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`EP 1 768 649 B1
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`Table 22. - Efletirizine composition
`Nasal solution
`
`Efletirizine hydrochloride (mg)
`Hydroxypropylmethylcellulose (mg)
`Monobasic sodium phosphate (mg)
`Dibasic sodium phosphate (mg)
`Edeteate disodium (mg)
`Sodium chloride (mg)
`Sodium hydroxide
`p-hydroxybenzoate esters (mg)
`Purified water (ml)
`
`6
`5
`8.1
`6.3
`0.5
`1.93
`ad pH 6.5
`0.375
`ad 1
`
`[0049]
`The efficacy of antimicrobial preservation of this solution is determined according to the European Pharmaco-
`poeia (Chap. 5.1.3.). The recommendedefficacycriteria are achieved.
`
`Reference Example 7. Oral solutions and drops containing Levocetirizine and benzylalcohol.
`
`[0050] An oral solution and drops containing levocetirizine are prepared. The compositions are given in table 23.
`
`Table 23. - Levacetirizine compositions
`Oralsolution Drops
`
`Levocetirizine hydrochloride (mg)
`Maltitol-Lycasin 80-55 (mg)
`Glycerine 85 %(mg)
`Propyleneglycol (mg)
`Sodium saccharinate (mg)
`Tutti frutti flavour (mg)
`Sodium acetate (mg)
`Acetic acid (mg)
`Benzylalcohol (mg)
`Purified water (ml)
`
`0.5
`400
`235.2
`-
`0.5
`0.15
`3.4
`0.5
`5.0
`ad 1
`
`5
`-
`294.1
`350
`10
`-
`5.7
`0.53
`5.0
`ad 1
`
`20
`
`25
`
`30
`
`35
`
`[0051] The antimicrobial preservative effectiveness tests are realized according to the European Pharmacopoeia
`(Chap. 5.1.3.). In all cases the recommendedefficacy criteria are achieved.
`
`40
`
`
`Example 8. Oral solutions and drops containing efletirizine
`
`[0052]
`
`An oral solution and drops containing efletirizine are prepared. The compositions are given in table 24.
`
`45
`
`50
`
`55
`
`Table 24. - Efletirizine compositions
`Oral solution
`1
`400
`235.2
`-
`0.5
`0.15
`4.2
`ad pH 5
`0.375
`ad 1
`
`Efletirizine hydrochloride (mg)
`Maltitol-Lycasin 80-55 (mq)
`Glycerine 85 %(mqg)
`Propyleneglycol (mg)
`Sodium saccharinate (mg)
`Tutti frutti flavour (mg)
`Sodium acetate (mg)
`Acetic acid (mg)
`p-hydroxybenzoate esters (mg)
`Purified water (ml)
`
`Drops
`10
`-
`294.1
`350
`10
`-
`10
`ad pH 5
`0.375
`ad 1
`
`10
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`UCB Biopharma SPRL(IPR2019-00400)
`Exhibit 2035 Page 10
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2035 Page 10
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`

`

`EP 1 768 649 B1
`
`[0053] The antimicrobial preservative effectiveness tests are realized according to the European Pharmacopoeia
`(Chap. 5.1.3.). In all cases the recommendedefficacycriteria are achieved.
`
`
`Example 9. Eye drops containing efletirizine and thimerosal (reference), chlorhexidine acetate (reference) and p-
`hydroxvbenzoateesters.
`
`[0054] Three formulations of eye drops containing efletirizine are prepared. The compositions are given in table 25.
`
`Table 25. - Efletirizine compositions
`Eye drops
`
`Efletirizine hydrochloride (mg)
`Boric acid (mg)
`Sodium hydroxide
`Thimerosal (mg)
`Chlorhexidine acetate (mg)
`p-hydroxybenzoate esters (mg)
`Purified water (ml)
`
`10
`20
`adpH7
`0.05
`-
`-
`ad 1
`
`10
`20
`adpH7
`-
`0.05
`-
`ad 1
`
`10
`20
`adpH7
`-
`-
`0.375
`ad 1
`
`5
`
`10
`
`15
`
`20
`
`[0055] The antimicrobial preservative effectiveness tests are realized according to the European Pharmacopoeia
`(Chap. 5.1.3.). In all cases the recomm