USOO8633194B2
`
`(12) United States Patent
`US 8,633,194 B2
`(10) Patent N0.:
`Fanara et al.
`
`(45) Date of Patent: Jan. 21, 2014
`
`(54) PHARMACEUTICAL COMPOSITION OF
`PIPERAZINE DERIVATIVES
`
`(75)
`
`Inventors: Domenico Fanara, Wanze (BE); Jean
`Scouvart, Brussels (BE); Claire
`Poulain, Brussels (BE); Michel Deleers,
`Linkebeek (BE)
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`WO
`W0
`WO
`WO
`
`0 605 203 A
`0605203 A
`0605203 A2
`2002/047689 A2
`WO 2004/004705 A
`2004/050094 A1
`2005/107711 A2
`
`7/1994
`7/1994
`7/1994
`6/2002
`1/2004
`6/2004
`11/2005
`
`(73) Assignee: UCB Pharma, S.A., Brussels (BE)
`
`OTHER PUBLICATIONS
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 832 days.
`
`(21) Appl.No.:
`
`10/599,451
`
`(22) PCT Filed:
`
`Jul. 6, 2005
`
`(86) PCTNo.:
`
`PCT/EP2005/007340
`
`§ 371 (0X1),
`(2), (4) Date:
`
`Jul. 18, 2007
`
`(87) PCT Pub. No.: W02006/005507
`
`PCT Pub. Date: Jan. 19, 2006
`
`(65)
`
`Prior Publication Data
`
`US 2007/0275974 A1
`
`NOV. 29, 2007
`
`(30)
`
`Foreign Application Priority Data
`
`Jul. 14,2004
`
`(EP) ..................................... 04016519
`
`(51)
`
`Int. Cl.
`A61K 31/48
`
`(2006.01)
`
`(52) us. Cl.
`USPC ..................................................... 514/252.12
`
`(58) Field of Classification Search
`None
`
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,525,358 A
`4,705,683 A
`4,728,509 A
`5,368,852 A
`5,419,898 A
`5,504,113 A
`5,891,913 A
`6,004,968 A
`6,258,814 B1
`6,319,927 B1
`6,432,961 B1
`6,436,924 B2
`7,094,429 B2
`B2
`7,157,464
`B1
`7,198,800
`2004/0058896 A1*
`2009/0137645 A1
`
`6/1985 Baltes et al.
`11/1987 Dettmar
`3/1988 Shimizu et al.
`11/1994 Umemoto et al.
`5/1995 Ikejiri
`4/1996 Lucero et al.
`4/1999 Sallmann et al.
`12/1999 Casey et al.
`7/2001 Martin
`11/2001 Martin
`8/2002 De Longueville et al.
`8/2002 Poppe et al.
`8/2006 Kiel et al.
`1/2007 Pennell et al.
`4/2007 K0
`3/2004 Dietrich et al.
`5/2009 Zhang et al.
`
`............... 514/171
`
`Doron et al.; “Antibacterial effect ofparabens against planktonic and
`biofilm Streptococcus sobrinus”; 2001 International Journal ofAnti-
`microbial Agents; 18: 575-578.*
`Gilliland et al.; “The bactericidal activity of a methyl and propyl
`parabens combination:
`isothermal and non-isothermal studies”;
`1992; Journal oprplied Bacteriology; 72: 252-257.*
`Gilliland et al. “Kinetic evaluation of claimed synergistic paraben
`combinations using a factorial design”; 1992; Journal of Applied
`Bacteriology; 72: 258-261.*
`Routledge et al.; “Some Alkyl Hydroxy Benzoate Preservatives
`(Parabens) Are Estrogenic”; 1998; Toxicology and Applied Pharma-
`cology; 153: 12-19.*
`Database WPI, Section Ch, Week 198551, Derwent Publications
`Ltd., London, GB; Class A96, AN 1985-319295, XP002309643, &
`JP 60 204712 A (SS Pharmaceutical KK), (Oct. 16, 1985) *abstract*.
`Handbook of Pharmaceutical Manufacturing Formulations, Par
`Sarfaraz Niazi, CRC Press, 2004 (5 pages).
`Formulation in Pharmacy Practices, 2nd edition, 2001 (2 pages).
`Definition of Levo-Dromoran. Internet document http://wwwrxlist.
`c0m/lev0-dr0m0ran-drug.htm, 1 sheet.
`Remington the Science and Practice of Pharmacy, 21sted., 2005, pp.
`748-749.
`Communication of a notice of opposition against European Applica-
`tion No. 057585820 dated Jun. 29, 2010 listing cited documents.
`Kibbe A. H., “Handbook of Pharmaceutical Excipients”, 3. edition
`2000, American Pharmaceutical Association, pp. 340,450; ISBN:
`0-917330-96-X.
`Wang, D.Y., “Effect of cetirizine, levocetirizine, and dextrocetirizine
`0n histamine-induced nasal response in healthy adult volunteers”,
`Allergy 56 (2001), pp. 339-343; ISSN: 0105-4538.
`Marketing authorization for Zodac R GTT in Slovakia.
`Marketing authorization for Zodac R SIR in Slovakia.
`Marketing authorization for Zodac R GTT in Czech Republic.
`Marketing authorization for Zodac R SIR in Czech Republic.
`Summary of product characteristics for Zodac R GTT.
`Summary of product characteristics for Zodac R SIR.
`Thomson Reuters Newport Premium: Launched Drug Forms Details.
`Kibbe, Arthur H., “Handbook of Pharmaceutical Excipients”, Third
`Edition 2000, American Pharmaceutical Association, pp. 340-343,
`450-453.
`
`* cited by examiner
`
`Primary Examiner 7 Timothy Thomas
`Assistant Examiner 7 Rayna B Rodriguez
`(74) Attorney, Agent, or Firm 7 McDonnell Boehnen
`Hulbert & Berghoff LLP
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a liquid composition contain-
`ing an active substance belonging to the family of substituted
`benzhydryl piperazines with reduced amounts of preserva-
`tives.
`
`12 Claims, No Drawings
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 001
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`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 001
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`

`

`US 8,633,194 B2
`
`1
`PHARMACEUTICAL COMPOSITION OF
`PIPERAZINE DERIVATIVES
`
`The present invention relates to a liquid pharmaceutical
`composition containing an active substance such as cetiriz-
`ine, levocetirizine and efletirizine.
`A number of substances belonging to the family of substi-
`tuted benzhydryl piperazines are known to be sub stances with
`useful pharmacological properties.
`European Patent EP 58146, filed in the name ofUCB, S.A.,
`describes substituted benzhydryl piperazines having the gen-
`eral formula
`
`/—\
`
`//O
`N—[-(CH2)n—O-]m—H2C—C
`N
`\_/
`\L
`
`in which L stands for an 70H or iNHZ group, X and X',
`taken separately, stand for a hydrogen atom, a halogen atom,
`a linear or branched alkoxy radical at C1 or C4, or a trifluo-
`romethyl radical, m equals 1 or 2, n equals 1 or 2, as well as
`their pharmaceutically acceptable salts.
`Of these compounds, 2-[2-[4-[(4-chlorophenyl)phenylm-
`ethyl]-1-piperazinyl]ethoxy]-acetic acid, also known under
`the name ofcetirizine, and its dichlorohydrate are well known
`for their antihistaminic properties.
`The active substances belonging to the family of substi-
`tuted benzhydryl piperazines specifically include 2-[2-[4-[(4-
`chlorophenyl)phenylmethyl] -1 -piperazinyl] ethoxy] -acetic
`acid (cetirizine), 2-[2-[4-[bis(4-fluorophenyl)methyl]-1-pip-
`erazinyl] ethoxy] acetic acid (efletirizine),
`their optically
`active isomers when applicable, as well as their pharmaceu-
`tically acceptable salts.
`In the pharmaceutical filed, solutions and drops are gener-
`ally produced as germ-free compositions during their produc-
`tion processes. However, once the seal of the containers is
`broken, and the pharmaceutical compositions are completely
`used over a period of time, these pharmaceutical composi-
`tions are continuously exposed to the risk of being contami-
`nated by the microorganisms existing in the environment or
`the human body, each time the containers are used and their
`covers are opened or closed.
`It has now surprisingly been found that the active sub-
`stances belonging to the family of substituted benzhydryl
`piperazines possess a preservative effect in aqueous solu-
`tions.
`
`The purpose of the invention concerns a liquid pharmaceu-
`tical composition containing an active sub stance belonging to
`the family of substituted benzhydryl piperazines chosen
`among cetirizine,
`levocetirizine and efletirizine, and a
`reduced amount of preservatives.
`The present invention is based on the unexpected recogni-
`tion 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.
`
`The present invention encompasses a pharmaceutical com-
`position comprising an active substance belonging to the
`
`2
`
`family of substituted benzhydryl piperazines and an amount
`ofparahydroxybenzoate esters used as preservatives less than
`3 mg/ml of the composition, a normal concentration to pre-
`serve aqueous solutions.
`The present invention encompasses a pharmaceutical com-
`position comprising an active substance chosen among ceti-
`rizine, levocetirizine and efletirizine and at least one preser-
`vative, wherein the amount of preservative is 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 preserva-
`tives corresponds to the bactericidal effect of a parahydroxy-
`benzoate esters concentration ofmore than 0 and less than 1 .5
`
`mg/ml.
`Generally, the pharmaceutical composition of the inven-
`tion is liquid and preferably aqueous.
`In the pharmaceutical composition of the invention, the
`active substance is generally selected from the group of ceti-
`rizine, levocetirizine, efletirizine, and their pharmaceutically
`acceptable salts. Preferably, the active substance is selected
`from the group of cetirizine, levocetirizine, and their pharma-
`ceutically acceptable salts.
`The term “cetirizine” refers to the racemate of [2-[4-[(4
`chlorophenyl)phenylmethyl] -1 -piperazinyl] ethoxy] -acetic
`acid and its dihydrochloride salt which is well known as
`cetirizine dihydrochloride; its levorotatory and dextrorota-
`tory enantiomers are known as levocetirizine and dextroceti-
`rizine. Processes for preparing cetirizine, an individual opti-
`cal
`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, US. Pat. No. 5,478,941, European Patent appli-
`cation 0 601 028, European Patent Application 0 801 064 and
`International Patent Application W0 97/37982.
`The term “levocetirizine” as used herein means the levoro-
`
`tatory enantiomer of cetirizine. More precisely, it means that
`the active substance comprises at least 90% by weight, pref-
`erably at least 95% by weight, of one individual optical iso-
`mer 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 as disclosed in British patent
`application No. 2,225,321 .Additionally, each individual opti-
`cal isomer can be prepared from the racemic mixture by
`enzymatic biocatalytic resolution, such as disclosed in US.
`Pat. Nos. 4,800,162 and 5,057,427.
`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 I of Euro-
`pean patent No. 58146, which relates to substituted benzhy-
`drylpiperazine derivatives. Efletirizine has been found to pos-
`sess excellent antihistaminic properties. It belongs to the
`pharmacological class of histamine Hl-receptor antagonists
`and shows in vitro high affinity and selectivity for Hl-recep-
`tors. It is useful as an antiallergic, and antihistaminic agent.
`Two pseudopolymorphic crystalline forms of efletirizine
`dihydrochloride, namely anhydrous efletirizine dihydrochlo-
`ride and efletirizine dihydrochloride monohydrate, are
`described in the European patent No. 1 034 171, and another
`pseudopolymorphic form of efletirizine dihydrochloride is
`described in the international patent application WO
`03/009849. Processes for preparing efletirizine or a pharma-
`ceutically 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.
`
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`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 002
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 002
`
`

`

`US 8,633,194 B2
`
`3
`The term “pharmaceutically acceptable salts” as used
`herein refers not only to addition salts with pharmaceutically
`acceptable non-toxic organic and inorganic acids, such as
`acetic, citric, maleic, succinic, ascorbic, hydrochloric, hydro-
`bromic, 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 microorganisms or, in an
`ideal instance, kills them; so antimicrobial agent able to limit
`or avoid the growth ofmicroorganisms such as bacteria, 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 must kill and
`reduce a required amount of bacteria and fungi within a
`prescribed time period.
`Examples of preservatives are p-hydroxybenzoate esters
`(methyl parahydroxybenzoate, ethyl parahydroxybenzoate,
`propyl parahydroxybenzoate, butyl parahydroxybenzoate,
`C1-C20 alkyl parahydroxybenzoate and their sodium salts),
`acrinol, methyl rosaniline chloride, benzalkonium chloride,
`benzethonium chloride, cetylpyridinium chloride, cetylpy-
`rodium bromide, chlorohexidine, chlorohexidine acetate,
`benzylalcohol, alcohol, chlorobutanol, isopropanol, ethanol,
`thimerosal, phenol, sorbic acid, potassium and calcium sor-
`bate, benzoic acid, potassium and calcium benzoate, sodium
`benzoate, calcium acetate, calcium disodium ethylenedi-
`aminetetraacetate, calcium propionate, calcium 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. Generally,
`the preservative is selected
`from the group of thimero sal, chlorohexidine acetate, benzy-
`lalcohol, benzalkonium chloride, p-hydroxybenzoate esters
`(methyl parahydroxybenzoate, ethyl parahydroxybenzoate,
`propyl parahydroxybenzoate, butyl parahydroxybenzoate,
`C1-C20 alkyl parahydroxybenzoate or a mixture thereof.
`Preferably the preservative is selected from the group of
`methyl parahydroxybenzoate, ethyl parahydroxybenzoate,
`propyl parahydroxybenzoate, a mixture of methyl parahy-
`droxybenzoate and ethyl parahydroxybenzoate or propyl
`parahydroxybenzoate, and a mixture of methyl parahydroxy-
`benzoate and propyl parahydroxybenzoate. Best results have
`been obtained with a mixture ofmethyl parahydroxybenzoate
`and propyl parahydroxybenzoate in a ratio of 9/1 expressed in
`weight.
`In a particular embodiment of the invention, the pharma-
`ceutical composition contains an amount of p-hydroxyben-
`zoate esters (methyl p-hydroxybenzoate/propyl p-hydroxy-
`benzoate in a ratio of 9/1 expressed in weight) selected in the
`range of 0.0001 and 1.5 mg/ml of the composition. Prefer-
`ably, it contains an amount selected in the range of 0.01 and
`1.125 mg/ml. More preferably it contains an amount of pre-
`servatives selected in the range of 0.1 and 1 mg/ml.
`In a particular embodiment of the invention, the pharma-
`ceutical composition contains an amount of thimerosal
`selected in the range of 0.0001 and 0.05 mg/ml of the com-
`position. Preferably, it contains an amount selected in the
`range of 0.005 and 0.035 mg/ml. More preferably it contains
`an amount of preservatives selected in the range of 0.007 and
`0.025 mg/ml.
`In a particular embodiment of the invention, the pharma-
`ceutical composition contains an amount of chlorhexidine
`acetate selected in the range of 0.0001 and 0.05 mg/ml of the
`composition. Preferably, it contains an amount selected in the
`
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`4
`
`range of 0.005 and 0.035 mg/ml. More preferably it contains
`an amount of preservatives selected in the range of 0.007 and
`0.025 mg/ml.
`In a particular embodiment of the invention, the pharma-
`ceutical composition contains an amount of benzylalcohol
`selected in the range of 0.0001 and 10 mg/mi of the compo-
`sition. Preferably, it contains an amount selected in the range
`of 0.05 and 7.5 mg/ml. More preferably it contains an amount
`of preservatives selected in the range of 1 and 5 mg/ml.
`In a particular embodiment of the invention, the pharma-
`ceutical composition contains an amount of benzalkonium
`chloride selected in the range of 0.0001 and 0.05 mg/ml ofthe
`composition. Preferably, it contains an amount selected in the
`range of 0.005 and 0.035 mg/ml. More preferably it contains
`an amount of preservatives selected in the range of 0.01 and
`0.025 mg/ml.
`The amount ofthe selected preservative is defined by com-
`parison with the amount of parahydroxybenzoate ester lead-
`ing to the same preservative effect. The optimum amount of
`preservative used in the invention depends on its nature. The
`preferred amount ofpreservative is such that it gives the same
`preservative effect as an amount of parahydroxybenzoate
`ester in the range of 0.2 and 1.125 mg/ml of the pharmaceu-
`tical composition.
`By patient, we understand children, adolescents and adults,
`preferably of2 years old. The targeted patients are usually old
`from 2 years and more.
`A preferred daily do sage provides from about 0.0005 mg to
`about 2 mg of 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 to 1 mg per kg
`of body weight per patient. The dosage may be administered
`once per day oftreatment, 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 reach
`a 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 day for children and adults. The
`exact dosages in which the compositions are administrated
`can vary according to the type of use, the mode of use, the
`requirements of the patient, as determined by a skilled prac-
`titioner. 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.
`The pharmaceutical forms according to the present inven-
`tion may be prepared according to conventional methods used
`by pharmacists. The forms can be administered together with
`other components or biologicaly active agents, pharmaceuti-
`cally acceptable surfactants, excipients, carriers, diluents and
`vehicles.
`
`The pharmaceutical compositions of the invention include
`any conventional therapeutical inert carrier. The pharmaceu-
`tical compositions can contain inert as well as pharmacody-
`namically 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 as salts for varying the
`osmotic pressure, substances for varying pH such as buffers,
`and other additives can also be present. If desired an antioxi-
`dant can be included in the pharmaceutical compositions.
`Pharmaceutical acceptable excipients or carriers for compo-
`sitions include saline, buffered saline, dextrose or water.
`Compositions may also comprise specific stabilizing agents
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 003
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`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 003
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`

`

`US 8,633,194 B2
`
`5
`such as sugars, including mannose and mannitol. Carrier
`substances and diluents can be organic or inorganic sub-
`stances, for example water, gelatine, lactose, starch, gum
`arabic, polyalkylene glycol, cellulose compounds and the
`like. A prerequisite is that all adjuvants and substances used in
`the manufacture of the pharmaceutical compositions are non-
`toxic.
`
`Pharmaceutical compositions can be administered by
`spray inhalation. Any conventional pharmaceutical composi-
`tion for spray inhalation administration may be used. Another
`preferred mode of administration is by aerosol.
`The pharmaceutical compositions according to the present
`invention may also be administered orally. They may also be
`administered by nasal instillation, aerosols. The pharmaceu-
`tical compositions which can be used for oral administration
`is liquid, for example, in the form of solutions, syrups, drops
`and the like.
`
`The pharmaceutical forms, such as drops, nasal drops, eye
`drops and ear drops are prepared by conventional pharmaceu-
`tical methods. The compounds of the present invention are
`mixed with a solid or liquid, non-toxic and pharmaceutically
`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 and the like.
`Preferably, the pharmaceutical composition of the inven-
`tion is administered in traditional form for oral administra-
`
`tion, as oral liquid preparation such as syrup.
`Best results have been obtained with an oral dosage form,
`in particular liquid formulations such as syrup for children.
`An advantage of the invention is that reducing the concen-
`tration ofthe preservative leads to a reduction ofthe risk of an
`allergic reaction in sensitive patients.
`Another advantage of the invention is the ability to make
`easier the manufacturing process avoiding the solubilization
`of important amounts of preservatives not freely soluble in
`water.
`
`The invention is further defined by reference to the follow-
`ing examples.
`
`EXAMPLE 1
`
`Preservative Effect of Cetirizine
`
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`An oral solution and drops containing cetirizine are pre-
`pared. The compositions are given in table 1.
`
`45
`
`TABLE 1
`
`Cetirizine compositions
`
`Cetirizine hydrochloride (mg)
`Sorbitol sol. At 70% (mg)
`Glycerine (mg)
`Propyleneglycol (mg)
`Sodium saccharinate (mg)
`Banana flavour (mg)
`Sodium acetate (mg)
`Acetic acid
`Purified water (ml)
`
`Oral solution
`
`1
`450
`200
`50
`1
`0.1754
`4.2
`ad pH 5
`ad 1
`
`Drops
`
`10
`7
`250
`350
`10
`7
`10
`ad pH 5
`ad 1
`
`The antimicrobial preservative effectiveness tests are real-
`ized according to the European Pharmacopoeia (Chap.
`5.1.3.). Samples of the oral solution and the drops are inocu-
`lated with bacterial and yeast suspensions of Pseudomonas
`aeruginosa ATCC 9027, Escherichia Coli ATCC 8739, Sla—
`phylococcus
`aureus ATC C6538, Candida
`albicans
`ATCC10231 andAspergillus niger ATCC16404. The number
`
`50
`
`55
`
`60
`
`65
`
`6
`of viable microorganisms per ml of preparations under test
`are determined. The results are given in tables 2 and 3.
`
`TABLE 2
`
`Microbial content in inoculated sample of the oral solution
`
`Time
`(days)
`Inoc-
`ulum
`0
`7
`14
`21
`28
`
`Pseudamanas Escherichia
`aeruginasa
`cali
`5.5 x105
`4.6 ><105
`
`4.9><105
`<100
`<1
`<1
`<1
`
`4.7><105
`<100
`<1
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`4.0 ><105
`
`3.1 x 105
`<100
`<1
`<1
`<1
`
`Candida Aspergillus
`albicans
`niger
`3.7 x105
`2.3 ><106
`
`2.6><105
`<100
`2
`<1
`<1
`
`1.7><106
`4.8 x 105
`8.2 x 103
`5.5 x 103
`5.0 x 103
`
`TABLE 3
`
`Microbial content in inoculated sample of the drops
`
`Time
`(days)
`Inoc-
`ulum
`0
`7
`14
`21
`28
`
`Pseudamanas Escherichia
`aeruginasa
`cali
`4.0 x105
`3.4 ><105
`
`3.5 x105
`<100
`<1
`<1
`<1
`
`3.8x105
`<100
`<1
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`3.6 ><105
`
`2.2x105
`<100
`<1
`<1
`<1
`
`Candida Aspergillus
`albicans
`niger
`3.5 x105
`1.8 ><106
`
`2.6><105
`<100
`<1
`<1
`<1
`
`1.6><106
`<104
`<100
`<1
`<1
`
`In both cases, a rapid disappearance of Pseudomonas
`aeruginosa, Escherichia Coli, Staphylococcus aureus and
`Candida albicans is observed in the inoculated samples.
`For Aspergillus niger, the number of viable spores is sig-
`nificantly reduced in the oral solution while a rapid disappear-
`ance is observed in the drops.
`
`EXAMPLE 2
`
`Preservative Effect of Levocetirizine
`
`An oral solution and drops containing levocetirizine are
`prepared. The compositions are given in table 4.
`
`TABLE 4
`
`Levocetirizine compositions
`
`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)
`
`Oral solution
`
`0.5
`400
`235.2
`7
`0.5
`0.15
`3.4
`0.5
`ad 1
`
`Drops
`
`5
`7
`294.1
`350
`10
`7
`5.7
`0.53
`ad 1
`
`The antimicrobial preservative effectiveness tests are real-
`ized according to the European Pharmacopoeia (Chap.
`5.1.3.). Samples of the oral solution and the drops are inocu-
`lated with bacterial and yeast suspensions of Pseudomonas
`aeruginosa ATCC 9027, Escherichia Coli ATCC 8739, Sla—
`phylococcus
`aureus ATC C6538, Candida
`albicans
`ATCC10231 andAspergillus nigerATCC16404. The number
`of viable microorganisms per ml of preparations under test is
`determined. The results are given in tables 5 and 6.
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 004
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 004
`
`

`

`7
`TABLE 5
`
`8
`TABLE 8
`
`US 8,633,194 B2
`
`Microbial content in inoculated sample of the oral solution
`containing 0.45 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`
`Slaphyla-
`caccus
`aureus
`
`Candida Aspergillus
`aZbicans
`niger
`
`Inoc-
`ulum
`0
`14
`28
`
`5.5 ><105
`
`4.6 ><105
`
`4.0 ><105
`
`3.7 ><105
`
`2.3 ><106
`
`5.2 ><105
`<1
`<1
`
`4.9 ><105
`<1
`<1
`
`3.3 ><105
`<1
`<1
`
`2.9 ><105
`<1
`<1
`
`1.2 ><106
`<100
`2
`
`TABLE 9
`
`Microbial content in inoculated sample of the oral solution
`containing 0.75 mg/ml of p-hydroxybenzoate esters
`
`Microbial content in inoculated sample of the oral solution
`
`Slaphyla-
`Time
`Pseudamanas Escherichia
`caccus
`Candida Aspergillus
`(days)
`aeruginasa
`caZi
`aureus
`aZbicans
`niger
`
`
`5
`
`Inoc-
`ulum
`
`0
`7
`14
`21
`28
`
`3.6x 105
`
`1.7><105
`
`2.7><105
`
`3.4x 105
`
`1.7><106
`
`3.2x105
`150
`<1
`<1
`<1
`
`1.8><105
`<100
`<1
`<1
`<1
`
`31.5x105
`<100
`<1
`<1
`<1
`
`3.9x105
`2.8><104
`1.4x104
`2.6x102
`6.2 ><103
`
`1.6x106
`1.0><106
`4.8x105
`2.2><105
`5.3 X105
`
`TABLE 6
`
`Microbial content in inoculated sample of the drops
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`3.6x 105
`1.7><105
`
`3.2x105
`<100
`<1
`<1
`<1
`
`1.5><105
`<100
`<1
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`2.7><105
`
`31.1x105
`<100
`<1
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.4x 105
`1.7><106
`
`1.8x105
`<100
`<1
`<1
`<1
`
`1.7><106
`9.0 x 104
`<1000
`<1
`<1
`
`10
`
`U
`
`20
`
`25
`
`Slaphyla-
`Time
`Pseudamanas Escherichia
`caccus
`Candida Aspergillus
`(days)
`aeruginasa
`caZi
`aureus
`aZbicans
`niger
`
`Inoc-
`5.5 ><105
`4.6 ><105
`4.0 ><105
`3.7 ><105
`2.3 ><106
`ulum
`0
`14
`28
`
`4.0><105
`<1
`<1
`
`1.9x105
`<1
`<1
`
`1.9x106
`<100
`<1
`
`3».9><105
`<1
`<1
`
`4.4)(105
`<1
`<1
`
`Time
`(days)
`Inoc-
`ulum
`0
`7
`14
`21
`28
`
`.
`.
`In both cases, a rap1d d1sappearance of Pseudomonas
`aeruginosa, Escherichia Cali, Staphylococcus aureus is
`observed in the inoculated samples. A disappearance of Can—
`dida albicans and Aspergillus niger is also observed in the
`drops.
`
`EXAMPLE 3
`
`Efficacy of Antimicrobial Preservation of Cetirizine
`Aqueous Solutions by p-hydroxbenzoate Esters
`
`m
`
`35
`
`40
`
`Oral solutions and drops containing cetirizine according to
`example 1 but also containing mixtures of p-hydroxyben- 45
`zoate esters (methyl p-hydroxybenzoate/propyl p-hydroxy-
`benzoate 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/mland 1.05 mg/ml. The efficacy
`of antimicrobial preservation of these solutions and drops is so
`determined according to the European Pharmacopoeia
`(Chap. 5.1.3.). The results of the tests are given in tables 7 to
`14.
`
`TABLE 10
`
`Microbial content in inoculated sample of the oral solution
`containing 1.05 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`5.5 ><105
`4.6 ><105
`
`3.3x 105
`<1
`<1
`
`4.1 x 105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`4.0 ><105
`
`31.1x105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.7 ><105
`2.3 ><106
`
`1.4x105
`<1
`<1
`
`1.2><106
`<100
`<1
`
`TABLE 11
`
`Microbial content in inoculated sample of the drops
`containing 0.15 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`4.0 ><105
`3.4 ><105
`
`4.3 ><105
`<1
`<1
`
`4.0 ><105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`3.6 ><105
`
`2.0 ><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.5 ><105
`1.8 ><106
`
`2.5 ><105
`<1
`<1
`
`1.5 ><106
`<100
`<1
`
`TABLE 7
`
`Microbial content in inoculated sample of the oral solution
`containing 0.15 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`5.5)(105
`4.6x105
`
`5.1x105
`<1
`<1
`
`4.5)(105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`4.0><105
`
`31.0x105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3».7><105
`2.3x106
`
`4.0><105
`<1
`<1
`
`4.1x106
`9.1 x 103
`750
`
`55
`
`60
`
`65
`
`TABLE 12
`
`Microbial content in inoculated sample of the drops
`containing 0.45 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`4.0 ><105
`3.4 ><105
`
`3.6x 105
`<1
`<1
`
`3.6x 105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`3.6 ><105
`
`1.7><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.5 ><105
`1.8 ><106
`
`2.1 x 105
`<1
`<1
`
`1.4><106
`<100
`<1
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 005
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 005
`
`

`

`US 8,633,194 B2
`
`9
`TABLE 13
`
`10
`TABLE 16
`
`Microbial content in inoculated sample of the drops
`containing 0.75 mg/ml of p-hydroxybenzoate esters
`
`Microbial content in inoculated sample of the oral solution
`containing 0.75 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`4.0><105
`3.4x105
`
`4.1 x 105
`<1
`<1
`
`3.6x105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`3.6x105
`
`2.6><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.5x105
`1.8><106
`
`2.5><105
`<1
`<1
`
`1.6><106
`<100
`<1
`
`TABLE 14
`
`Microbial content in inoculated sample of the drops
`containing 1.05 mg/ml of p-hydroxybenzoate esters
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`4.0><105
`3.4x105
`
`Slaphyla-
`caccus
`aureus
`3.6x105
`
`Candida Aspergillus
`aZbicans
`niger
`3.5x105
`1.8><106
`
`Time
`(days)
`Inoc-
`ulum
`1.3><106
`2.2><105
`2.8><105
`3.7><105
`3.9)(105
`0
`<100
`<1
`<1
`<1
`<1
`14
`
`
`
`
`
`<1 <1 <1 <128 <1
`
`In all cases, the disappearance of Pseudomonas aeruginosa,
`Escherichia Cali, Staphylococcus aureus and Candida albi—
`cans is observed in the inoculated samples. For Aspergillus
`niger, the number of viable spores is significantly reduced in
`the oral solution while a rapid disappearance is observed in
`the drops.
`In all cases the recommended eflicacy criteria are achieved.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Time
`(days)
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`
`Slaphyla-
`caccus
`aureus
`
`Candida Aspergillus
`aZbicans
`niger
`
`Inoc-
`ulum
`0
`14
`28
`
`3.6x105
`
`1.7><105
`
`2.7x105
`
`3.4x105
`
`1.7><106
`
`3.5><105
`<1
`<1
`
`1.6><105
`<1
`<1
`
`2.4><105
`<1
`<1
`
`3.4><105
`5.5 x102
`<1
`
`1.6><106
`1.4><104
`<1
`
`TABLE 17
`
`Microbial content in inoculated sample of the oral solution
`containing 1.125 mgfml of p-hydroxybenzoate esters
`
`Slaphyla-
`Time
`Pseudamanas Escherichia
`caccus
`Candida Aspergillus
`(days)
`aeruginasa
`caZi
`aureus
`aZbicans
`niger
`
`Inoc-
`3.6x105
`1.7><105
`2.7x105
`3.4x105
`1.7><106
`ulum
`0
`14
`28
`
`1.2><105
`<1
`<1
`
`3.0x105
`<1
`<1
`
`3.5x105
`<10
`<1
`
`1.4><106
`<1000
`<1
`
`3.9x105
`<1
`<1
`
`TABLE 18
`
`Microbial content in inoculated sample of the drops
`containing 0.375 mgfml of p-hydroxybenzoate esters
`
`EXAMPLE 4
`
`Efficacy of Antimicrobial Preservation of
`Levocetirizine Aqueous Solutions by
`p-hydroxybenzoate Esters
`
`Oral solutions and drops containing levocetirizine accord-
`ing to example 2 but also containing mixtures of p-hydroxy-
`benzoate esters (methyl p-hydroxybenzoate/propyl p-hy-
`droxybenzoate in 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 eflicacy 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 mgfml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`3.6x105
`1.7><105
`
`3.7x105
`<1
`<1
`
`1.3><105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`2.7><105
`
`2.8><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.4><105
`1.7><106
`
`3.8x105
`1.7><104
`<1
`
`1.6><106
`1.6><105
`<100
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`3.6x105
`1.7><105
`
`3.1 x 105
`<1
`<1
`
`1.2><105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`2.7x105
`
`2.6><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.4x105
`1.7><106
`
`1.7><105
`<1
`<1
`
`1.8><106
`<1000
`<1
`
`TABLE 19
`
`Microbial content in inoculated sample of the drops
`containing 0.75 mg/ml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`3.6x105
`1.7><105
`
`3.1 x 105
`<1
`<1
`
`1.0><105
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`2.7x105
`
`3.0><105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.4x105
`1.7><106
`
`1.8><105
`<1
`<1
`
`1.4><106
`<1000
`<1
`
`TABLE 20
`
`Microbial content in inoculated sample of the drops
`containing 1.125 mgfml of p-hydroxybenzoate esters
`
`Time
`(days)
`Inoc-
`ulum
`0
`14
`28
`
`Pseudamanas Escherichia
`aeruginasa
`caZi
`3.6x105
`1.7><105
`
`2.9x105
`<1
`<1
`
`6.9x104
`<1
`<1
`
`Slaphyla-
`caccus
`aureus
`2.7><105
`
`2.7x105
`<1
`<1
`
`Candida Aspergillus
`aZbicans
`niger
`3.4><105
`1.7><106
`
`5.0><104
`<1
`<1
`
`1.5 x106
`<1000
`<1
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p. 006
`
`Apotex, Inc. (IPR2019-00400), Ex. 1001, p