.
`
` ‘-
`
`'
`
`'
`
`Mornmg51de H)
`
`450 Seventh Ave.
`
`mm Floor
`
`New York, NY 10123
`(212) 643-8800
`
`Date: January 15, 2018
`
`To whom it may concern:
`
`This is to certify that the attached translation from French and into English is an accurate representation
`of the documents received by this office.
`
`The document is designated as:
`International Application Number: PCT/FR96/00636
`
`Hannah Mendoza, Project Manager in this company, attests to the following:
`
`”To the best of my knowledge, the aforementioned documents are a true, full and accurate translation
`of the specified documents.”
`
`
`
`Signature of Hannah Mendoza
`
`Teoxane S.A.
`
`Exhibit 1071
`
`The Leader in Global lP Solutions
`
`www.morningsidelP.com
`
`l
`
`info@morningsidelP.com
`
`

`

`VVC)96/33751
`
`l
`
`PCT/FR96/00636
`
`Injectable two—phase compositions containing hyaluronic acid,
`
`notably useful
`
`in reconstructive
`
`and
`
`cosmetic surgery
`
`The present
`
`invention relates to:
`
`“
`
`—
`
`—
`
`two—phase compositions containing'
`
`a polymer
`
`selected from
`
`hyaluronic acid and its salts,
`
`a method of preparing said compositions,
`
`a,
`
`filling material, useful
`
`in reconstructive surgery and
`
`in
`
`cosmetic
`
`surgery,
`
`based
`
`on
`
`said.
`
`two—phase
`
`compositions.
`
`10
`
`15
`
`The present
`
`invention notably' proposes
`
`an adequate solution to
`
`the
`
`technical problem of permanent
`
`filling of
`
`volume defects
`
`of
`
`the skin,
`
`such as wrinkles or scars, notably' on
`
`the
`
`face.
`
`More generally,
`
`it proposes
`
`an original
`
`formulation, based, on
`
`hyaluronic acid.
`
`Said
`
`hyaluronic
`
`acid
`
`is
`
`a
`
`glycosaminoglycan,
`
`mucopolysaccharide
`
`of
`
`high molecular weight
`
`that
`
`occurs
`
`or
`
`in
`
`cord,
`
`vitreous
`
`humor,
`
`animal
`
`tissues
`
`such
`
`as
`
`the umbilical
`
`synovial
`
`fluid,
`
`rooster's
`
`combs,
`
`the
`
`skin,
`
`the connective
`
`tissues
`
`(joints,
`
`tendons,
`
`etc.).
`
`Said
`
`acid
`
`can
`
`thus
`
`be
`
`obtained naturally by extraction from certain of
`
`said animal
`
`tissues
`
`(notably from rooster's combs and umbilical cords).
`
`it
`
`can also be obtained by
`
`bacterial
`
`fermentation.
`
`The
`
`chemical
`
`structure
`
`of
`
`said acid is
`
`that
`
`of
`
`a polymer
`
`having disaccharide monomers of N—acetyl—D—glucosamine
`
`and
`
`D—
`
`glucuronic acid,
`
`said.
`
`amine
`
`and said acid being joined together
`
`by
`
`a
`
`Bl
`
`4»
`
`3
`
`glycosidic
`
`bond.
`
`The
`
`disaccharide monomers
`
`themselves
`
`are joined together
`
`by’
`
`$1 —+ 4 glycosidic bonds
`
`to
`
`generate the unbranched, non—crosslinked polysaccharide chain.
`
`20
`
`25
`
`30
`
`{00578302;vl}
`
`Pagel
`
`

`

`VVC)96/33751
`
`2
`
`PCTYFR96/00636
`
`However,
`
`said chain includes,
`
`at
`
`the
`
`level of
`
`its monomers,
`
`functions that allow it to be chemically crosslinked to create a
`
`network of varying density.
`
`It
`
`is
`
`generally
`
`known
`
`that
`
`any molecule
`
`is
`
`much more
`
`resistant
`
`to degradation and to heat when
`
`it
`
`is
`
`crosslinked.
`
`Thus,
`
`it
`
`is
`
`known
`
`that
`
`there
`
`is
`
`interest
`
`in
`
`crosslinking
`
`crosslinked hyaluronic
`
`acid.
`
`Said
`
`crosslinked hyaluronic acid
`
`is much more
`
`stable
`
`10
`
`molecule
`
`itself.
`
`It
`
`in
`
`is
`
`the
`
`body
`
`than the hyaluronic
`
`acid
`
`also more
`
`resistant
`
`to
`
`autoclave
`
`sterilization.
`
`Hyaluronic acid is known for its viscoelastic properties as well
`
`as
`
`its very great
`
`tendency to absorb water.
`
`Its properties
`
`Its
`
`15
`
`20
`
`25
`
`30
`
`largely
`
`explain
`
`the
`
`elasticity
`
`of
`
`the
`
`skin.
`
`biocompatibility,
`
`tolerance and toxicity have been extensively
`
`investigated,
`
`and cosmetic
`
`since this molecule has been used.
`
`for more than 10 years. Its use is notably
`
`in the medical
`
`fields
`
`known in ophthalmological
`
`surgery,
`
`for
`
`treating osteoarthritis,
`
`and for treating patients with major burns.
`
`In the state of
`
`the art, many compositions of various
`
`types
`
`containing hyaluronic acid, have therefore already been described.
`
`Notably the following have been described:
`
`a
`
`in the patent US—A—5,137,875:
`
`injectable collagen solutions
`
`or dispersions containing hyaluronic acid in solution as well
`
`as use thereof for filling voids in soft tissues;
`
`—
`
`—
`
`in the patent US—A—4,716,154: a gel of crosslinked. hyaluronic
`
`acid, as a substitute for the vitreous humor;
`
`in
`
`the
`
`application
`
`EPeAeO 466 300:
`
`a viscoelastic
`
`gel
`
`comprising a gelatinous phase
`
`(that has undergone 21
`
`low
`
`degree
`
`of
`
`crosslinking)
`
`dispersed
`
`in
`
`a
`
`liquid
`
`phase
`
`(that
`
`has
`
`not
`
`undergone crosslinking);
`
`said two phases
`
`{00578302;vl}
`
`PageZ
`
`

`

`WO 96/33751
`
`3
`
`PCT/FR96/00636
`
`advantageously
`
`having' been prepared.
`
`
`from fibers of Hylan
`
`(chemically
`
`in situ modified natural hyaluronic
`
`acid in
`
`order to facilitate its extraction from tissues).
`
`It
`
`advisable
`
`to use
`
`said compositions
`
`in many
`
`contexts
`
`is
`
`in
`
`the medical field.
`
`According to the first
`
`ainl of
`
`the invention,
`
`as
`
`stated.
`
`above,
`
`two—phase compositions are proposed that contain hyaluronic acid
`
`or
`
`a
`
`salt
`
`thereof
`
`{called
`
`the
`
`polymer)
`
`and
`
`that
`
`have
`
`an
`
`original structure. Said compositions consist of
`
`10
`
`suspension
`
`whose
`
`dispersed
`
`phase
`
`consists
`
`an
`
`of
`
`injectable
`
`insoluble
`
`fragments of a hydrogel of said highly crosslinked polymer
`
`and
`
`whose
`
`continuous phase consists of an aqueous solution of said
`
`polymer and/or of another biocompatible polymer,
`
`selected from
`
`slightly crosslinked or not crosslinked proteins, polysaccharides
`
`15
`
`and, derivatives
`
`thereof.
`
`The
`
`term “hyaluronic
`
`acid”
`
`is
`
`used hereinafter
`
`as
`
`a generic
`
`name
`
`to denote both hyaluronic acid per se and its salts,
`
`and
`
`notably hyaluronate
`
`salts.
`
`The
`
`two—phase
`
`compositions
`
`of
`
`the
`
`invention advantageously contain
`
`sodium hyaluronate as
`
`polymer
`
`selected
`
`from hyaluronic
`
`acid
`
`and
`
`its
`
`salts,
`
`at
`
`least
`
`in
`
`their dispersed phase.
`
`It
`
`should be noticed here
`
`that
`
`said
`
`sodium hyaluronate is advantageously of bacterial origin.
`
`The
`
`two—phase
`
`compositions
`
`of
`
`the
`
`invention
`
`are
`
`injectable
`
`compositions. They have been formulated for
`
`this purpose. This
`
`is notably why they contain a continuous phase; said phase serves
`
`as an injection vehicle for the fragments of the dispersed phase.
`
`20
`
`25
`
`30
`
`{00578302;v1]
`
`Page3
`
`

`

`VVC)96/337Sl
`
`4
`
`PCT/FR96/00636
`
`The
`
`term “injectable”,
`
`as
`
`used in the present
`
`text, means
`
`manually
`
`injectable
`
`by means
`
`of
`
`syringes“
`
`equipped with
`
`conventional needles. The
`
`two—phase compositions of the invention
`
`are particularly interesting in that they can be formulated to be
`
`injectable by means of very fine needles {with a diameter between
`
`0.3 and. 0.5mm). Those skilled in the art will
`
`‘understandt
`
`that
`
`the
`
`determining
`
`parameter
`
`is
`
`the
`
`largest
`
`dimension
`
`of
`
`the
`
`fragments in suspension.
`
`In the context of the present
`
`invention,
`
`it
`
`is notably possible to formulate compositions
`
`that
`
`contain
`
`hyaluronic acid and are
`
`injectable through 30 G,
`
`26 Gl/Z,
`
`25 G
`
`hypodermic
`
`needles.
`
`Said
`
`compositions
`
`constitute
`
`the most
`
`advantageous
`
`embodiment of the compositions of the invention.
`
`The main characteristics
`
`of
`
`each, of
`
`the phases of
`
`the two—
`
`phase
`
`compositions
`
`of
`
`the
`
`invention
`
`are
`
`discussed more
`
`in
`
`details below.
`
`10
`
`15
`
`As
`
`stated above,
`
`the
`
`continuous
`
`phase, which
`
`serves
`
`as
`
`an
`
`injection vehicle,
`
`consists
`
`of
`
`an
`
`aqueous
`
`solution,
`
`typically
`
`containing hyaluronic acid {or
`
`a salt
`
`thereof)
`
`and/or another
`
`biocompatible polymer selected from proteins, polysaccharides and
`
`derivatives
`
`thereof.
`
`Said
`
`hyaluronic
`
`acid
`
`and/or
`
`the other
`
`polymer used are not crosslinked or
`
`are
`
`slightly crosslinked
`
`(by means
`
`of
`
`a
`
`crosslinking
`
`agent}.
`
`The viscosity of said
`
`continuous phase should remain
`
`compatible with its function as
`
`an
`
`injection vehicle.
`
`It
`
`should. be noted that said continuous
`
`20
`
`25
`
`phase
`
`also meets
`
`another
`
`function.
`
`After
`
`injection
`
`and
`
`implantation
`
`of
`
`the
`
`two—phase
`
`composition,
`
`it
`
`protects
`
`the
`
`dispersed phase and slows down its degradation.
`
`Said continuous phase can notably be in the form of a solution
`
`30
`
`or a gel.
`
`{00578302:vl}
`
`Page4
`
`

`

`VVC396/33751
`
`5
`
`PCT/FR96/00536
`
`It
`
`should
`
`not be
`
`excluded
`
`from the
`
`scope
`
`of
`
`the
`
`present
`
`invention that
`
`said continuous
`
`phase
`
`contains
`
`a mixture
`
`of
`
`polymers.
`
`According to an
`
`advantageous
`
`embodiment of
`
`the invention,
`
`it
`
`contains
`
`the
`
`same
`
`polymer
`
`as
`
`the dispersed phase,
`
`namely
`
`hyaluronic acid; however, said hyaluronic acid in said continuous
`
`phase
`
`has
`
`a
`
`far
`
`lower
`
`crosslinking
`
`degree,
`
`or
`
`even
`
`no
`
`crosslinking at all.
`
`The use of hyaluronic acid in the
`
`two
`
`phases
`
`is widely
`
`preferred,
`
`considering
`
`the
`
`advantageous
`
`properties of
`
`this product, which can notably be obtained by a
`
`bacterial
`
`route,
`
`by a cellular
`
`route
`
`(therefore free from any
`
`contaminant of
`
`'the virus
`
`or prion type),
`
`and which has both,
`
`a
`
`highly
`
`gelatinous
`
`character,
`
`an
`
`appreciable
`
`lubricity,
`
`good
`
`biocompatibility and a good behavior in the body.
`
`The other polymers
`
`that may be
`
`used.
`
`in the continuous
`
`phase
`
`the
`
`compositions
`
`of
`
`the
`
`invention are,
`
`as
`
`stated. above,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`of
`
`proteins or polysaccharides
`
`and derivatives
`
`thereof. Advisable
`
`proteins include, non—restrictively:
`
`collagen,
`
`albumin,
`
`elastin
`
`etc.;
`
`advisable polysaccharides
`
`and derivatives
`
`thereof
`
`(other
`
`than
`
`hyaluronic
`
`acid)
`
`include:
`
`chondroitin
`
`sulfates,
`
`keratan
`
`sulfates, heparin,
`etc.
`
`alginic acid,
`
`starch,
`
`carboxymethylcellulose,
`
`Said continuous phase typically contains insoluble
`
`fragments of
`
`a
`
`hydrogel
`
`of
`
`highly
`
`crosslinked
`
`hyaluronic
`
`acid.
`
`These
`
`insoluble
`
`fragments
`
`(of
`
`variable
`
`geometry)
`
`constitute
`
`real
`
`entities
`
`that
`
`can
`
`be
`
`separated from the diluted continuous
`
`phase
`
`by
`
`decanting
`
`or
`
`centrifugation. Hyaluronic
`
`acid
`
`is
`
`highly crosslinked among
`
`said
`
`fragments:
`
`a
`
`relatively dense
`
`network was made
`
`from,
`
`chains
`
`of
`
`hyaluronic
`
`acid
`
`and
`
`a
`
`crosslinking
`
`agent.
`
`This will
`
`be
`
`further
`
`explained
`
`and
`
`quantified in the present text.
`
`The use of
`
`fragments
`
`(or particles)
`
`of
`
`this type,
`
`containing
`
`{00578302;vl}
`
`PageS
`
`

`

`VVC)96/33751
`
`6
`
`PCT/FR96/00636
`
`hyaluronic acid as
`
`a
`
`dispersed. phase
`
`in a continuous phase,
`
`as
`
`described above,
`
`gives
`
`the
`
`two—phase
`
`compositions of
`
`the
`
`invention
`
`their
`
`original
`
`structure.
`
`It
`
`notably makes
`
`said
`
`compositions
`
`original
`
`and
`
`highly
`
`performant
`
`precursors
`
`permanent
`
`filling materials
`
`or
`
`implants
`
`that are
`
`useful
`
`of
`
`in
`
`reconstructive and cosmetic surgery.
`
`The
`
`injectable two—phase compositions of
`
`the invention are most
`
`particularly
`
`designed
`
`for
`
`dermal
`
`injection
`
`(superficial,
`
`medium or
`
`deep)
`
`for
`
`implantation into the dermis.
`
`For
`
`this
`
`10
`
`purpose,
`
`in order to remove
`
`any unpleasant sensation or even any
`
`pain
`
`during
`
`their
`
`injection
`
`and
`
`implantation,
`
`they
`
`are
`
`advantageously
`
`buffered
`
`at
`
`a
`
`pH
`
`between
`
`6.5
`
`and
`
`7.5,
`
`preferably betwean 7
`
`and 7.4,
`
`even more preferably between 7.2
`
`and 7.3.
`
`15
`
`20
`
`Thus,
`
`the
`
`aqueous
`
`solution
`
`{continuous
`
`phase)
`
`on
`
`the
`
`one
`
`hand,
`
`and
`
`the hydrogel
`
`(dispersed phase)
`
`on
`
`the other
`
`hand,
`
`are
`
`advantageously buffered at
`
`these pH values.
`
`A phosphate buffer is generally used.
`
`The advantageous characteristics of the fragments constitutive of
`
`the dispersed, phase within the two—phase
`
`compositions of
`
`the
`
`invention are explained below.
`
`Said fragments
`
`can be
`
`included in said compositions at
`
`steady
`
`state as
`
`under— or over~hydrated fragments.
`
`No matter under— or
`
`over—hydrated,
`
`they
`
`return
`
`to
`
`balance
`
`after
`
`injection,
`
`and
`
`25
`
`implantation.
`
`In any case,
`
`they are used. with dimensions
`
`(and.
`
`in an amount)
`
`compatible with
`
`the means
`
`envisaged.
`
`for
`
`their
`
`injection.
`
`Advantageously, more
`
`than half of
`
`said fragments
`
`have
`
`their
`
`largest
`
`dimension between
`
`40
`
`and
`
`280um, preferably between 75
`
`30
`
`and 250nm. More
`
`advantageously,
`
`almost all of
`
`said fragments
`
`share that characteristic:
`
`then,
`
`they form compositions
`
`that are
`
`{00578302;v1}
`
`PageS
`
`

`

`WO 96/33751
`
`7
`
`PCT/FR96/00636
`
`injectable by means of
`
`fine 0.3 and 0.5mm diameter needles.
`
`Rather than “largest dimension” of
`
`said fragments, we could say
`
`“equivalent diameter”. As
`
`it will be specified later
`
`in the
`
`present
`
`text,
`
`the insoluble fragments of hydrogel of
`
`the two—
`
`phase
`
`compositions
`
`from the invention are obtained by shearing
`
`of
`
`a mass,- hence,
`
`they do not generally consist
`
`in spheres.
`
`The hydrogel of
`
`
`said fragments was obtained from hyaluronic
`
`acid
`
`that
`
`has
`
`been
`
`highly
`
`crosslinked
`
`by means
`
`of
`
`a
`
`crosslinking agent.
`
`To get
`
`fragments
`
`containing a
`
`reasonable
`
`10
`
`amount of
`
`said crosslinking agent,
`
`it is recommended to start
`
`from a
`
`hyaluronic
`
`acid
`
`composition with
`
`a molecular weight
`
`not
`
`less than 1 million Dalton. According
`
`to an
`
`advantageous
`
`a
`
`hyaluronic
`
`acid
`
`embodiment,
`
`it
`
`is
`
`recommended
`
`to
`
`use
`
`composition with a molecular weight
`
`between
`
`1
`
`and 3 million
`
`Dalton.
`
`In
`
`addition,
`
`it
`
`is
`
`advisable
`
`to
`
`implement
`
`said
`
`crosslinking via the hydroxyl
`
`functions of
`
`the hyaluronic acid
`
`by means of
`
`a crosslinking agent,
`
`in conditions
`
`leading to a
`
`degree
`
`of
`
`crosslinking
`
`of
`
`said
`
`hyaluronic
`
`acid
`
`{starting
`
`material)
`
`characterized by the ratio:
`
`total
`
`number of reactive
`
`functions
`
`of
`
`said
`
`crosslinking
`
`agent/total
`
`number
`
`of
`
`disaccharide units of
`0.8 and l.
`
`the molecules of hyaluronic acid between
`
`i5
`
`20
`
`25
`
`30
`
`{00578302;V1]
`
`Page 7
`
`

`

`WO 96/33751
`
`8
`
`PCT/FR96/00636
`
`In fact,
`
`the network of
`
`insoluble fragments of
`
`the two—phase
`
`compositions
`
`of
`
`the
`
`invention
`
`is
`
`based
`
`on molecules
`
`of
`
`hyaluronic acid joined together by bridges of nmlecules of
`
`the
`
`crosslinking agent;
`
`each
`
`of
`
`the disaccharide units
`
`of
`
`said
`
`molecules of hyaluronic acid shall. have advantageously between
`
`0.8 and l of its hydroxyi
`
`functions used in such bridges.
`
`
`
`The
`
`specified range
`
`for
`
`said degree
`
`of
`
`crosslinking is
`
`an
`
`optimal
`
`range.
`
`The invention does not rule out using
`
`fragments
`
`having
`
`said
`
`degree
`
`of crosslinking under 0.8
`
`(while making
`
`10
`
`sure that said fragments still have their intrinsic properties of
`
`insolubility)
`
`or
`
`above
`
`excessively the nature of
`
`L
`
`
`
`(then
`
`taking
`
`care
`
`not
`
`to
`
`alter
`
`the
`
`fragments based on hyaluronic
`
`acid, which will
`
`include increasing contents of crosslinking
`
`agent)
`
`in compositions
`
`of
`
`the invention.
`
`is
`
`possible
`
`to
`
`use any agent
`
`15'
`
`As
`
`a
`
`crosslinking
`
`agent,
`
`it
`
`known.
`
`to crosslink hyaluronic acid 'via
`
`its
`
`hydroxyl
`
`functions
`
`—
`
`at
`
`least
`
`a
`
`bifunctional crosslinking agent — and notably a
`
`polyepoxide or any derivatives thereof,
`
`to generate the hydrogel
`
`constituting
`
`the
`
`insoluble
`
`fragments
`
`of
`
`the
`
`two—phase
`
`compositions
`
`of
`
`the invention. As
`
`said. crosslinking' agent,
`
`it
`
`is notably possible to use epichlorohydrin, divinylsulfone,
`
`1,4—
`
`bis(2,3—epoxypropoxy)butane
`
`(or
`
`1,4—bis(glycidyloxy)butane
`
`or
`
`l,4—butanediol
`
`diglycidyl
`
`ether
`
`2
`
`BDDE),
`
`l,2—bis(2,3—
`
`epoxypropoxy)ethylene,
`
`l—(2,3eepoxypropyl)—2,3—epoxy‘
`
`cyclohexane
`
`etc. The use of
`
`several
`
`crosslinking agents
`
`is
`
`not
`
`excluded
`
`from the scope of the invention.
`
`Moreover,
`
`the
`
`insoluble hydrogel fragments
`
`in the compositions
`
`of
`
`the
`
`invention can
`
`be
`
`characterized by other parameters,
`
`such as
`
`their dry matter content or
`
`their optical properties.
`
`20
`
`25
`
`30
`
`{00575302;v1}
`
`Page 8
`
`

`

`VVC)96/33751
`
`9
`
`PCT/FR96/00636
`
`The dry' matter content of
`
`said.
`
`fragments was measured in the
`
`context of
`
`the
`
`invention, with said fragments buffered.
`
`(at a
`
`pH vetlue
`
`between
`
`6.5
`
`and 7‘5),
`
`at
`
`steady state.
`
`In
`
`such
`
`conditions, said fragments from the invention have an advantageous
`
`dry matter
`between 5
`
`content between 1,5 and
`and 15%.
`
`20%, more
`
`advantageously
`
`Said fragments,
`
`in the same conditions,
`
`transmit
`
`less than 5%
`
`of light at 400nm.
`
`The
`
`two—phase
`
`compositions of
`
`the
`
`invention generally contain
`
`from 10 to 200mg/ml, advantageously from 20 to l50mg/ml, of said
`
`insoluble
`
`fragments
`
`in suspension in their
`
`continuous
`
`phase.
`
`Introducing an excessive amount of dispersed phase
`
`into said
`
`continuous phase
`
`can compromise
`
`the
`
`injectable
`
`character
`
`of
`
`said compositions.
`
`Introducing‘
`
`an insufficient
`
`amount of
`
`said
`
`reduce
`
`10
`
`15
`
`dispersedv phase into said. continuous phase may greatly
`
`the
`
`advantages
`
`of
`
`said
`
`compositions: They have been designed
`
`to inject
`
`an, effective amount of highly crosslinked hyaluronic
`
`acid (therefore resistant
`
`to degradation).
`
`It
`
`is necessary to
`
`optimize
`
`the
`
`ratio of
`
`transported active mass
`
`/ mass
`
`of
`
`20
`
`injection vehicle.
`
`Advantageous
`
`characteristics
`
`of
`
`the
`
`aqueous
`
`solution of
`
`the
`
`continuous phase in the two—phase compositions of
`
`the
`
`invention
`
`are
`
`noted
`
`below.
`
`Said continuous
`
`phase
`
`contains
`
`hyaluronic
`
`acid
`
`and/or
`
`another
`
`slightly crosslinked or
`
`not crosslinked
`
`25
`
`biocompatible polymer.
`
`Said continuous phase
`
`can
`
`be
`
`characterized by
`
`its
`
`intrinsic
`
`viscosity [n]. This parameter is given by the formula:
`
`30
`
`{00578302;v1}
`
`Page9
`
`

`

`VVC)96/33751
`
`
`where:
`
`10
`
`PCT/FR96/00636
`
`no is the viscosity of the solvent,
`
`n is the measured viscosity of the solution,
`
`c is the concentration of said solution.
`
`Said
`
`continuous
`
`phase
`
`of
`
`the
`
`two—phase
`
`compositions
`
`of
`
`the
`
`invention advantageously has an intrinsic viscosity between 1500
`
`and
`
`3200ml/g.
`
`Preferably,
`
`the
`
`intrinsic viscosity is between
`
`2000 and 2700ml/g.
`
`10
`
`15
`
`Said intrinsic viscosity of
`
`said continuous phase
`
`depends of
`
`course
`
`on
`
`the nature
`
`of
`
`the
`
`polymer used,
`
`its concentration
`
`and its degree of crosslinking.
`
`Said. viscosity'
`
`is ix) be optimized for
`
`a
`
`compromise,
`
`since said
`
`continuous phase is to constitute the injection_ vehicle of
`
`the
`
`insoluble fragments while
`
`slowing down
`
`the degradation of said
`
`implanted fragments.
`
`Generally,
`
`this
`
`involves
`
`optimization
`
`at
`
`the
`
`level
`
`of
`
`the
`
`available
`
`20
`
`25
`
`30
`
`composition
`
`of
`
`said
`
`continuous
`
`phase;
`
`the
`
`parameters are the nature of the polymer(s),
`
`their concentration
`
`and optionally their degree
`
`of crosslinking.
`
`No matter the nature of said polymer
`
`in the
`
`continuous phase,
`
`a
`
`person skilled.
`
`in the art
`
`is able 'to control
`
`its crosslinking,
`
`when possible.
`
`It was said that according to an advantageous
`
`invention,
`
`hyaluronic
`
`acid is
`
`also used at
`
`embodiment of
`
`the level of said
`
`the
`
`continuous phase.
`
`In this context, said hyaluronic
`
`acid can
`
`be
`
`non—crosslinked
`
`or
`
`slightly'
`
`crosslinked.
`
`It
`
`is
`
`also
`
`recommended here
`
`to use a hyaluronic
`
`acid composition with a
`
`molecular weight
`
`not
`
`less
`
`than
`
`1 million Dalton, more
`
`advantageously with
`[00578302,‘V1}
`
`a
`
`molecular weight
`
`between
`
`1
`
`and
`
`3
`
`PagelO
`
`

`

`VVC)96/33751
`
`‘
`
`11
`
`PCTYFR96/00635
`
`million
`
`Dalton. When
`
`said
`
`hyaluronic
`
`acid
`
`is
`
`slightly
`
`crosslinked,
`
`it is advisable to implement said. crosslinking' via
`
`the hydroxyl
`
`functions of
`
`the hyaluronic acid. by means
`
`of
`
`a
`
`crossiinking agent,
`
`in
`
`conditions
`
`leading
`
`to
`
`a
`
`degree
`
`of
`
`
`
`crossiinking
`
`of
`
`said
`
`hyaluronic
`
`acid
`
`(starting material}
`
`characterized
`
`by
`
`the
`
`ratio of
`
`total
`
`number
`
`of
`
`reactive
`
`functions
`
`of
`
`said
`
`crosslinking'
`
`agent/total
`
`number
`
`of
`
`disaccharide units
`
`of
`
`the
`
`hyaluronic
`
`acid. molecules between
`
`0.01 and 0.4. It can be seen that
`
`said degree of
`
`crosslinking
`
`10
`
`for
`
`the
`
`hyaluronic
`
`acid used
`
`in
`
`the
`
`continuous
`
`phase
`
`is
`
`generally always
`
`less than half that of
`
`the hyaluronic acid in
`
`the dispersed phase.
`
`The
`
`crosslinking is generally implemented. when preparing the
`
`continuous phase,
`
`the way it
`
`is
`
`implemented when preparing the
`
`15
`
`20
`
`25
`
`30
`
`dispersed phase,
`
`for
`
`'the case where hyaluronic acid :hs used in
`
`both said phases.
`
`Thus,
`
`said
`
`crosslinking
`
`of
`
`the
`
`continuous
`
`phase
`
`also
`
`advantageously
`
`uses
`
`a
`
`polyepoxide
`
`and
`
`notably
`
`l,4—bis{2,3H
`
`epoxypropoxy)butane as a, crosslinking agent.
`
`In this
`
`case,
`
`said polyepoxide
`
`is
`
`attached by ether bridges
`
`to the disaccharide units of
`
`the
`
`chains of hyaluronic
`
`acid,
`
`both in the
`
`continuous
`
`and
`
`the dispersed phases.
`
`Said hyaluronic acid.
`
`in the dispersed phase of
`
`the
`
`tw0wphase
`
`compositions
`
`of
`
`the
`
`invention,
`
`or
`
`advantageously'
`
`in
`
`both
`
`dispersed.
`
`and. continuous phases, of
`
`said compositions
`
`(contrary
`
`to any other
`
`polymer
`
`according
`
`to a particularly preferred
`
`embodiment of the invention)
`
`is preferably a hyaluronic acid that
`
`was obtained by a bacterial route.
`
`According
`
`to the preferred embodiment
`
`of
`
`the
`
`invention,
`
`an
`
`injectable suspension is proposed,
`
`as defined above,
`
`containing
`
`hyaluronic acid from a bacterial
`
`fermentation in both phases.
`
`By means of
`{00578302;v1}
`
`such a highly biocompatible suspension,
`
`fragments
`
`Pagell
`
`

`

`VVC)96/33751
`
`12
`
`PCTYFR96/00636
`
`are
`
`injected very' easily and
`
`said fragments are particularly
`
`resistant
`
`to
`
`degradation,
`
`once
`
`implanted,
`
`thanks
`
`to
`
`their
`
`intrinsic
`
`structure
`
`and
`
`because
`
`they
`
`are protected by
`
`the
`
`continuous phase with which they were injected.
`
`According to the second aim of
`
`the present
`
`invention,
`
`a nethod
`
`is
`
`proposed.
`
`for
`
`preparing'
`
`the
`
`two—phase
`
`compositions
`
`described above.
`
`Said method
`
`comprises, characteristically:
`
`—
`
`—
`
`—
`
`preparing and purifying an
`
`insoluble hydrogel of highly
`
`crosslinked hyaluronic acid;
`
`fragmenting said hydrogel by shearing;
`
`suspending the fragments of
`
`said shearedt hydrogel
`
`into a
`
`suitable continuous phase.
`
`During the first
`
`step above,
`
`a hydrogel of highly crosslinked
`
`hyaluronic acid should be prepared. For this purpose, generally,
`
`the raw material used,
`
`(often fibers of
`
`sodium hyaluronate)
`
`dissolved and reacted with a
`
`suitable
`
`crosslinking agent
`
`is
`
`in
`
`suitable proportions. A hyaluronic acid with a molecular weight
`
`of
`
`less than
`
`1 million Dalton, preferably between
`
`1
`
`and
`
`3
`
`is advantageously used
`
`as
`
`starting material,
`
`10
`
`15
`
`20
`
`million Dalton,
`
`and
`
`crosslinked so that
`
`the ratio of
`
`total
`
`number‘ of
`
`reactive
`
`functions
`
`of
`
`said,
`
`crosslinking
`
`agent/total
`
`number
`
`disaccharide
`
`units
`
`of
`
`the
`
`hyaluronic
`
`acid molecules
`
`of
`
`is
`
`between 0.8
`
`and
`
`l. At
`
`the end of
`
`the crosslinking reaction,
`
`the
`
`reaction mixture
`
`is purified
`
`to
`
`remove
`
`the
`
`unreacted
`
`reactants. This purification,
`
`can be carried. out by extraction
`
`with
`
`deionized water
`
`in
`
`a
`
`Soxhlet
`
`extractor.
`
`The purified
`
`hydrogel
`
`obtained
`
`is
`
`then
`
`fragmented
`
`by
`
`shearing.
`
`Said
`
`shearing
`
`generates
`
`fragments
`
`of
`
`non~uniform geometry
`
`and
`
`size.
`
`A
`
`person
`
`skilled
`
`in
`
`the
`
`art will
`
`be
`
`able
`
`to
`
`25
`
`30
`
`optimize
`
`this operation to obtain suitable fragments.
`
`{00578302;v1)
`
`Pagelz
`
`

`

`VVC)96/33751
`
`13
`
`PCTYFR96/00636
`
`Said
`
`fragments
`
`are
`
`then
`
`suspended
`
`into the
`
`continuous phase
`
`that was prepared previously or in parallel.
`
`Said continuous phase shall be prepared in a similar manner' if
`
`crosslinking
`
`is
`
`to
`
`be
`
`implemented.
`
`Advantageously,
`
`said
`
`continuous
`
`phase
`
`is
`
`prepared
`
`from a hyaluronic
`
`acid with a
`
`molecular weight
`
`of
`
`less
`
`than
`
`1 million Dalton,
`
`preferably
`
`between
`
`1
`
`and
`
`3 million Dalton,
`
`and said hyaluronic acid.
`
`is
`
`crosslinked so
`
`that
`
`the
`
`ratio of
`
`total
`
`number
`
`of
`
`reactive
`
`functions
`
`of
`
`the
`
`crosslinking
`
`agent/total
`
`number
`
`10
`
`hyaluronic
`
`acid molecules,
`
`of
`
`is
`
`disaccharide
`
`units
`
`of
`
`the
`
`between 0.01 and 0.4.
`
`If
`
`such crosslinking is
`
`implemented to
`
`a
`
`lesser degree,
`
`the purification of
`
`the
`
`crosslinked product
`
`shall
`
`generally
`
`be
`
`implemented
`
`by’
`
`techniques
`
`other
`
`than
`
`extraction.
`
`It
`
`is notably possible
`
`to process by
`
`successive
`
`15
`
`cycles
`
`of
`
`solubilization/precipitation,
`
`or even byr dialysis.
`
`If
`
`no
`
`crosslinking is
`
`carried out,
`
`the
`
`continuous
`
`phase
`
`is
`
`preparedv by simple mixing of
`
`the
`
`raW' material
`
`in an
`
`aqueous
`
`Solution.
`
`The
`
`resulting phases
`
`at
`
`the
`
`end of
`
`their preparation, process
`
`20
`
`are more or less hydrated.
`
`25
`
`{00578302;v1]
`
`Page13
`
`

`

`VVC)96/33751
`
`14
`
`PCTVFR96/00636
`
`For maxing them and suspending the fragments of hydrogel
`
`in the
`
`continuous phase, either one or both the following processes can
`
`be adopted:
`
`—
`
`notably the
`
`suitably hydrated fragments
`
`can be _mixed
`
`in
`
`a
`
`continuous
`
`phase prepared with
`
`the desired. degree of
`
`hydration,
`
`—
`
`—
`
`it is also possible to mix said dry fragments as well as a
`
`dry
`
`precursor
`
`of
`
`the
`
`continuous
`
`phase
`
`and
`
`suitably
`
`hydrate
`
`said dry mixture,
`
`it is also possible to consider mixing of
`
`the two phases,
`
`one
`
`in the
`
`dry state,
`
`and
`
`the other
`
`in the
`
`hydrated
`
`state,
`
`and
`
`adjusting'
`
`the
`
`degree
`
`of hydration of
`
`the
`
`mixture if necessary.
`
`The first of these three processes is preferential.
`
`Compositions of
`
`the invention prepared that way can be packaged,
`
`notably in syringes,
`
`and then sterilized, e.g.
`
`in an autoclave.
`
`Earlier
`
`in
`
`the
`
`text,
`
`we.
`
`saw that
`
`such compositions
`
`are
`
`recommended.
`
`for
`
`injection.
`
`into the skin as
`
`a
`
`filling' material,
`
`more precisely' as
`
`a precursor of
`
`such filling material, which
`
`guarantees its long—term efficiency.
`
`According
`
`to
`
`its
`
`last
`
`aim,
`
`the
`
`invention
`
`therefore relates
`
`to a
`
`filling material useful
`
`in reconstructive
`
`and cosmetic
`
`surgery, based on the two—phase compositions as described above.
`
`characteristically,
`
`said. material has
`
`a
`
`structure
`
`that evolves
`
`after injection.
`
`and implantation into the dermis.
`
`10
`
`15
`
`20
`
`25
`
`{00578302;v1}
`
`Page14
`
`

`

`VVC)96/33751
`
`15
`
`PCT/FR96/00636
`
`After
`
`absorption
`
`of
`
`the
`
`continuous
`
`phase
`
`(provided
`
`for
`
`protecting
`
`the
`
`dispersed
`
`phase),
`
`the
`
`fragments
`
`of
`
`the
`
`dispersed. phase gather' and generate a stable film. This film is
`
`a completely original structure.
`
`It
`
`is
`
`recommended
`
`to use
`
`this
`
`filling material notably
`
`for
`
`filling wrinkles
`
`on
`
`the
`
`face,
`
`such as
`
`the glabellar wrinkle,
`
`the perioral wrinkles,
`
`the nasolabial
`
`folds,
`
`for
`
`reducing the
`
`crow's feet, etc.
`
`10
`
`15
`
`The invention is illustrated by the following examples.
`
`Example 1
`
`A two—phase
`
`composition of
`
`the
`
`invention is prepared from, a
`
`single polymer:
`
`fibers of
`
`sodiunl hyaluronate
`
`(molecular weight:
`
`Mw = 2.106), of bacterial origin.
`
`said fibers
`
`in 0.25
`
`M sodium
`
`Two
`
`solutions of
`
`
`ll.8 wt% of
`
`hydroxide are prepared first.
`
`20
`
`25
`
`30
`
`430ul
`
`of
`
`1,4-bis(2,3-epoxypropoxy)butane
`
`(butanediol
`
`diglycidyl ether: BDDE)
`
`is added to one of said solutions.
`
`The homogenized deture is heated in a. water bath an: 50“C
`
`for
`
`2 hours.
`
`The hydrogel obtained is
`
`a
`
`solid.
`
`It
`
`is
`
`purified
`
`(by
`
`extraction with
`
`deionized water
`
`in
`
`a
`
`Soxhlet extractor)
`
`in order to remove both the crosslinking
`
`agent
`
`(BDDE)
`
`and
`
`the
`
`unreacted ' polymer
`
`from its
`
`structure.
`
`In said hydrogel,
`
`the ratio of
`
`total number of
`
`reactive functions of
`
`said. crosslinking agent/total
`
`number
`
`of disaccharide units
`
`of
`
`the molecules
`
`of
`
`the
`
`polymer
`
`amounts to 0.84. At steady state in phosphate buffer,
`
`said
`
`crosslinked
`hydrogel
`contains
`9.7
`wt%
`of
`In this state,
`it transmits less than 5% of
`
`dry
`light
`
`highly
`matter.
`at 400nm.
`
`(00578302;vl}
`
`Page 15
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`

`

`VVC)96/33751
`
`16
`
`'
`
`PCT/FR96/00636
`
`Said purified hydrogel
`
`is
`
`then sheared to obtain solid
`
`fragments with an average size between 75 and 250nm. These
`
`fragments are suspended in a phosphate buffer at pH 7.2, at a
`
`rate of 6g per 100ml of phosphate buffer.
`
`In
`
`parallel,
`
`Blul
`
`of
`
`1,4~bis(2,3—epoxypropoxy)butane
`
`(butanediol
`
`diglycidyl
`
`ether:
`
`BDDE)
`
`is
`
`added.
`
`to
`
`the
`
`other
`
`one of
`
`said two solutions.
`
`The
`
`homogenized mixture
`
`is also heated in a. water bath at
`
`50°C for
`
`2 hours. At
`
`the end of
`
`this heating' phase,
`
`a very viscous
`
`fluid is
`
`10
`
`obtained, which has to be purified.
`
`In this viscous
`
`fluid,
`
`the
`
`ratio of
`
`total
`
`number of
`
`reactive functions of
`
`said
`
`crossiinking agent/total
`
`number
`
`of disaccharide units of
`
`of
`
`the
`
`polymer
`
`is
`
`0.12.
`
`The
`
`continuous
`
`composition is
`
`prepared
`
`the molecules
`
`of
`
`phase
`
`the desired tw0mphase
`
`15
`
`20
`
`from said
`
`fluid.
`
`Said
`
`fluid is
`
`in
`
`fact purified by
`
`solubilization/precipitation.
`
`At
`
`the
`
`end.
`
`of
`
`said
`
`purification,
`
`the precipitate
`
`is
`
`dried
`
`and
`
`then
`
`re—
`
`hydrated with a phosphate buffer at
`
`pH 7.2,
`
`at
`
`a
`
`rate
`
`of
`
`6g of
`
`said. precipitate per
`
`100ml of
`
`said buffer.
`
`The
`
`suspension containing the
`
`fragments
`
`and the viscous
`
`aqueous solution are then ndxed in proportions of 1/1. An
`
`injectable
`
`suspension
`
`is
`
`obtained,
`
`notably
`
`injectable
`
`through needles from 26 G 1/2 to 30 G.
`
`25
`
`Example 2
`
`As a rule,
`
`the process is the same as for example 1, except that:
`
`1)
`
`only 250ul of BDDE are used for preparing the dispersed
`
`phase;
`
`30
`
`{00578302;Vl}
`
`Page16
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`

`WO 96/33751
`
`17
`
`PCT/FR96/00636
`
`2)
`
`for
`
`preparing
`
`the
`
`continuous
`
`phase,
`
`the
`
`fibers
`
`of
`
`hyaluronic acid are dissolved directly into the phosphate
`
`buffer at pH 7.2 (no crosslinking is implemented);
`
`3)
`
`for the final mixing of dispersed phase and continuous phase,
`
`said two phases are used in a ratio of 2/1.
`
`Such suspensions (according to the examples 1 and 2) were injected
`
`In fact,
`
`for filling facial wrinkles in 10 volunteers.
`
`less than
`
`a milliliter of
`
`these suspensions was
`
`injected each time.
`
`The
`
`product, when injected in the medium or deep dermis,
`
`did not
`
`cause
`
`any undesirable
`
`reaction,
`
`in particular no
`
`inflammatory
`
`reaction,
`
`no
`
`redness
`
`and
`
`no pain. After
`
`three months,
`
`the
`
`implant
`
`is still present
`
`and provides
`
`an effective filling of
`
`the
`
`cutaneous
`
`defect
`
`treated.
`
`The
`
`suspensions
`
`—
`
`two—phase
`
`compositions H of
`
`the invention are effective for
`
`longmlasting
`
`treatment of cutaneous depressions.
`
`10
`
`15
`
`[00578302;v1}
`
`Page 17
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`

`

`VVC)96/33751
`
`Claims
`
`18
`
`PCT7FR96/00636
`
`1.
`
`Two—phase
`
`composition containing a polymer
`
`selected from
`
`hyaluronic acid.
`
`and its salts,
`
`characterized in that
`
`it
`
`consists
`
`of
`
`an
`
`injectable
`
`suspension,
`
`whose
`
`dispersed
`
`phase
`
`consists
`
`of
`
`insoluble
`
`fragments
`
`of
`
`a hydrogel of
`
`said, highly crosslinked polymer,
`
`and whose continuous phase
`
`consists
`
`of
`
`an
`
`aqueous
`
`solution of
`
`said polymer and/or
`
`of
`
`another
`
`slightly
`
`crosslinked
`
`or
`
`not
`
`crosslinked
`
`biocompatible
`
`polymer,
`
`selected
`
`from
`
`proteins,
`
`polysaccharides and derivatives thereof.
`
`
`
`Two—phase
`
`composition according to claim 1, characterized
`
`in that
`
`said suspension is buffered, at a Efll between 6.5
`
`and 7.5.
`
`Two—phase
`
`composition
`
`according
`
`to
`
`claim 1
`
`or
`
`2,
`
`characterized in that more than one half of
`
`said fragments
`
`their
`
`largest
`
`dimension
`
`between
`
`40
`
`and
`
`280nm,
`
`10
`
`15
`
`have
`
`advantageously between 75 and 250mm.
`
`Two—phase composition according to any of claims
`
`1
`
`to 3,
`
`characterized,
`
`in
`
`that
`
`the
`
`hydrogel
`
`constituting
`
`said
`
`fragments
`
`is obtained from said polymer with a. molecular
`
`weight of
`
`less than 1 million Dalton, preferably between
`
`1
`
`and
`
`I3 million Dalton,
`
`and
`
`said hyaluronic
`
`acid is
`
`crosslinked via
`
`the
`
`hydroxyl
`
`functions
`
`of
`
`said polymer,
`
`by means of
`
`a crosslinking agent,
`
`in a
`
`ratio of
`
`total
`
`number
`
`of
`
`reactive
`
`functions
`
`of
`
`said
`
`crosslinking
`
`agent/total
`
`number
`
`of
`
`disaccharide
`
`units
`
`of
`
`the
`
`molecules of
`
`the polymer between 0.8 and l.
`
`Two—phase composition according to any' of claims
`
`2
`
`to 4,
`
`characterized in that
`
`said fragments,
`
`contain
`
`between
`
`1.5
`
`and
`
`20
`
`wts
`
`advantageously between 5 and 15 wt%.
`
`at
`
`of
`
`steady state,
`
`dry matter,
`
`20
`
`25
`
`30
`
`(00578302iv11
`
`Page 18
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`

`

`WO 96/33751
`
`-
`
`19
`
`PCT/FR96/00636
`
`6.
`
`Two—phase composition according to any of claims
`
`1
`
`to 5,
`
`characterized in that
`
`it
`
`contains
`
`from 10
`
`to 200mg/ml,
`
`advantageously
`
`from 20
`
`to 150mg/ml of
`
`said fragments
`
`in
`
`suspension in said continuous phase.
`
`5
`
`7
`
`8.
`
`10
`
`Two—phase composition according to any of claims
`
`1
`
`to 5,
`
`characterized
`
`in
`
`that
`
`said continuous
`
`phase
`
`has
`
`an
`
`intrinsic viscosity between 1500 and 3200ml/g.
`
`Two~phase composition according to any of claims
`
`1
`
`to 7,
`
`characterized
`
`in
`
`that
`
`said
`
`continuous
`
`phase
`
`is
`
`an
`
`aqueous
`
`solution of
`
`said polymer with a molecular weight
`
`of
`
`less than 1 million Dalton, preferably between
`
`1
`
`and
`
`3 million
`
`Dalton,
`
`and
`
`which
`
`has
`
`optionally
`
`been
`
`via the hydroxyl
`
`functions of said polymer,
`
`crosslinked,
`
`by means of
`
`a crosslinking agent,
`
`in a
`
`ratio of
`
`total
`
`number
`
`of
`
`reactive
`
`functions
`
`of
`
`said
`
`crosslinking
`
`agent/total
`
`number
`
`of disaccharide units of the molecules
`
`of the polymer between 0.01 and 0.4.
`
`9.
`
`10 .
`
`Two—phase composition according to any of claims
`
`1
`
`to 8,
`
`characterized in that
`
`the dispersed phase,
`
`and optionally
`
`the
`
`continuous
`
`phase,
`
`are
`
`crosslinked
`
`with
`
`a
`
`polyepoxide, notably l, 4—bis (.2 , 3—epoxypropoxy) butane.
`
`Two—phase composition according to any of claims
`
`1
`
`to 9,
`
`characterized
`
`in
`
`that
`
`the hyaluronic
`
`acid or
`
`a
`
`salt
`
`thereof in the suspension,
`
`i.e.
`
`in the dispersed phase and
`
`optionally
`
`the
`
`continuous
`
`phase,
`
`was
`
`obtained
`
`by
`
`a
`
`bacterial route .
`
`15
`
`20
`
`25
`
`30
`
`(00578302;V1}
`
`Page 19
`
`

`

`VVC396/33751
`
`20
`
`‘
`
`PCT/FR96/00636
`
`11. Method of preparing a
`
`two—phase
`
`composition according to
`
`any of
`
`the preceding claims,
`
`characterized in that
`
`it
`
`comprises the st