PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`(51) International Patent Classification 6 :
`
`
`(11) International Publication Number:
`
`WO 95/31211
`
`(43) International Publication Date:
`
`23 November 1995 (23.11.95)
`
`A61K 38/13, 9/00
`
`A1
`
`(81) Designated States: AM, AT, AU, BB, BG, BR, BY, CA, CH,
`CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG,
`KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW,
`MX, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, Tl, 'IT,
`UA, US, UZ, VN, European patent (AT, BE, CH, DE, DK,
`ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
`patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE,
`SN, TD, TG), ARIPO patent (KE, MW, SD, SZ, UG).
`
`Published
`
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`
`
`
`(21) International Application Number:
`PCT/US95/O6302
`
`
`(22) International Filing Date:
`17 May 1995 (17.05.95)
`
`
`
`(30) Priority Data:
`
`08/243,279
`17 May 1994 (17.05.94)
`US
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`08/243,279 (CIP)
`US
`
`
`Filed on
`17 May 1994 (17.05.94)
`
`
`
`‘
`(71) Applicant (for all designated States except US): ALLERGAN,
`
`
`INC. [US/US]; 2525 Dupont Drive, P.O. Box 19534, Irvine,
`CA 92713-9534 (US).
`
`
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): DING, Shulin [US/US];
`
`
`'
`14641 Fir Avenue, Irvine, CA 92714 (US). TIEN, Walter,
`L. [US/US]; 17551 Friends Ct., Irvine, CA 92714 (US).
`OLEJNIK, Orest [US/US]; 21291 Birdhollow Drive, Tra-
`buco Canyon, CA 92679 (US).
`
`(74) Agents: BARAN, Robert, J. et al.; ALLERGAN, INC., 2525
`Dupont Drive, Irvine, CA 92713-9534 (US).
`
`
`
`
`
`
`
`(54) Title: LACRIMAL GLAND SPECIFIC EMULSIONS FOR TOPICAL APPLICATION TO OCULAR TISSUE
`
`0.33 #001?
`
`6 4401/7?
`
`
`
`
`
`
`
`
`24 mm
`
`
`
`
`
`0.2% 4057,4400
`0.27.
`0.27,
`41%;);
`cameo/z
`6345,0514 [rename/rt manor
`snare/v
`
`
`
`
`
`
`A pharmaceutical composition is disclosed in the form Of a nonirrtating emulsion which includes at least one cyclosporin in admixture
`with a higher fatty acid glyceride and polysorbate 80. More particularly, the cyclosporin may be cyclosporing A and the higher fatty acid
`glyceride may be castor oil. Composition has been found to be of a high comfort level and low irritation potential suitable for delivery
`Of medications to sensitive areas such as ocular tissues with enhanced absorption in the lacrimal gland. In addition, the composition has
`stability for up to nine months without crystallization of cyclosporin.
`
`(57) Abstract
`
`F0kMfllfl7/0/V5
`
`
`
`
`
`AKN 1012
`
`
`
`3‘, 3m
`g soon
`£5 2500
`
`2000
`
`
`
` V//////////////fl
`
`1
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Viet Nam
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`
`CS
`CZ
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'lvoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Spain
`Finland
`France
`Gabon
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`United States of America
`Uzbekistan
`
`2
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`LACRIMAL GLAND SPECIFIC EMULSIONS FOR TOPICAL
`
`APPLICATION TO OCULAR TISSUE
`
`This application is a. continuation-in-part of
`
`pending US patent application SN 08/243,279 filed May
`
`17, 1994.
`
`The present invention generally relates to novel
`
`pharmaceutical compositions
`
`incorporating chemicals
`
`which are poorly soluble in water and is more particu-
`
`larly related to a novel ophthalmic emulsion including
`
`cyclosporin in admixture with castor oil and polysor-
`
`bate 80 with high comfort
`
`level and low irritation
`
`potential.
`
`Cyclosporins are a group of nonpolar cyclic
`
`oligopeptides with known immunosuppressant activity.
`
`In
`
`addition,
`
`as
`
`set
`
`forth in U.S. Patent No.
`
`4,839,342, cyclosporin (sometimes referred to in the
`
`literature as
`
`"cyclosporine")
`
`has been
`
`found
`
`as
`
`effective in treating immune medicated keratoconjunc-
`
`tivitis sicca (KCS or dry eye disease)
`
`in a patient
`
`suffering therefrom.
`
`As hereinabove noted, cyclosporin comprises
`
`a
`
`group of cyclic oligopeptides and the major component
`
`thereof is cyclosporin A (C62H111N11012)which has been
`identified along with several other minor metabolites,
`
`cyclosporin B through I.
`
`In addition,
`
`a number of
`
`synthetic analogs have been prepared.
`
`In general, commercially available cyclosporins
`
`may contain a mixture of several
`
`individual cyclo-
`
`sporins which all share a cyclic peptide structure
`
`consisting of eleven amino acid residues with a total
`
`molecular weight of about 1,200, but with different
`
`10
`
`15
`
`20
`
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`
`30
`
`35
`
`3
`
`

`

`WO 95/31211
`
`PCTfUS95I06302
`
`_ 2 _
`
`substituents or configurations of
`
`some of the amino
`
`acids.
`
`It should be appreciated that reference to the
`
`term "cyclosporin" or "cyclosporins" is used through-
`
`out
`
`the present specification in order to designate
`
`the cyclosporin component
`
`in the composition of the
`
`present invention.
`
`However,
`
`this specific reference is intended to
`
`include any individual member of the cyclosporin group
`
`as well as admixtures of two or more individual cyclo-
`
`sporins, whether natural or synthetic.
`
`The activity of cyclosporins,
`
`as hereinabove
`
`noted,
`
`is as an immunosuppressant and in the enhance-
`
`ment or restoring of lacrimal gland tearing.
`
`This activity can be enhanced if it is possible
`
`to enhance the absorption of the cyclosporin in the
`
`lacrimal gland. The present invention provides for a
`
`formulation and method that produces optimal cyclo—
`
`sporin A concentrations in the lacrimal gland and
`
`other ocular surface tissues.
`
`Unfortunately,
`
`the solubility of cyclosporin in
`
`water
`
`is extremely low and as elaborated in U.S.
`
`Patent No. 5,051,402,
`
`it has been considered not
`
`merely difficult but practically impossible to prepare
`
`a pharmaceutical composition containing cyclosporin
`
`dissolved in an aqueous medium.
`
`As
`
`reported,
`
`the solubility of cyclosporin in
`
`water
`
`is between about
`
`20 ug/ml
`
`to 30 ug/ml
`
`for
`
`cyclosporin A.
`
`Hence, heretofore prepared formula-
`
`tions incorporating cyclosporin have been prepared as
`
`oily solutions containing ethanol.
`
`However,
`
`these
`
`10
`
`15
`
`20
`
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`
`30
`
`35
`
`4
`
`

`

`W0 95/3121 1
`
`PCT/US95/06302
`
`-3—
`
`preparations limit the bioavailability to oral prep-
`arations and this is believed to be due to the separ-
`ation of cyclosporin as a solid immediately after it
`comes into contact with water, such as in the mouth or
`
`eye of a patient.
`
`In the case of injectable preparations of cyclo-
`sporin,
`they first must be diluted with physiological
`saline before intravenous administration but this is
`likely to result in the precipitation of cyclosporin
`and
`therefore may
`be
`considered undesirable
`for
`intravenous administration.
`
`Surface active agents such as polyoxyethylated
`castor oil have been utilized as solubilizers to in—
`ject preparations in order to prevent cyclosporin from
`separating.
`However,
`this also may give rise to
`safety problems (see U.S. Patent No. 5,051,402).
`
`The practical usefulness of cyclosporin would be
`greatly enhanced if administration thereof could be
`effective; for example, cyclosporin's effectiveness in
`the treatment of ocular symptoms of Behcet's Syndrome.
`However, if it is administered orally for the treat—
`ment of these symptoms,
`the accompanying side effects
`due to systemic circulation may cause adverse reac-
`tions such as hypertrichosis or renal dysfunction.
`
`On the other hand, if oily preparations contain-
`ing cyclosporin are applied directly to the eyes,
`ir—
`ritation or a clouding of visual field may result.
`This plus the difficulty in formulating cyclosporin
`limits its use in formulations that would be useful
`during keratoplasty as well
`in the treatment of
`herpetic keratitis and spring catarrh.
`
`10
`
`15
`
`20
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`
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`
`

`

`WO 95/3121]
`
`PCT/US95I06302
`
`_4_
`
`Heretofore, as for example in U.S. Patent No.
`
`5,051,402, attempts have been made to dissolve suffi-
`
`cient cyclosporin in an aqueous solvent system so as
`
`to reach an effective concentration for treatment.
`
`Importantly, this solvent system does not contain any
`
`surface active agent such as polyoxyethylated castor
`
`oil.
`
`Conceptually,
`
`the purpose of dissolving the
`
`10
`
`cyclosporin in an aqueous solvent system is to enable
`
`contact with body fluids which would merely constitute
`
`dilution of the aqueous solvent system which hopefully
`
`would eliminate the immediate precipitation of cyclo—
`
`sporin when contacted with the water content of the
`
`15
`
`body fluids.
`
`20
`
`25
`
`30
`
`35
`
`For direct use in the eye, cyclosporin has been
`
`formulated with a number of pharmaceutically accept-
`
`able excipients,
`
`for example, animal oil, vegetable
`
`oil,
`
`an appropriate organic or aqueous solvent,
`
`an
`
`artificial tear solution, a natural or synthetic poly-
`
`mer or an appropriate membrane.
`
`Specific
`
`examples
`
`of
`
`these pharmaceutically
`
`acceptable excipients, which may be used solely or in
`
`combination, are olive oil, arachis oil, castor oil,
`
`mineral oil, petroleum jelly, dimethyl
`
`sulfoxide,
`
`chremophor,
`
`liposomes, or liposome-like products or a
`
`silicone fluid, among others.
`
`In summary, a great deal of effort has been ex-
`
`pended in order to prepare a pharmaceutical composi—
`
`tion containing cyclosporin dissolved in an aqueous
`
`medium or cyclosporin prepared as an oily solution.
`
`However, successful formulations have yet to be accom-
`
`plished as evidenced by the lack of commercial prod-
`ucts.
`
`6
`
`

`

`WO 95/3121]
`
`PCT/US95/06302
`
`_5_
`
`As hereinabove noted, it has been reported that
`
`cyclosporin has demonstrated some solubility in oily
`
`preparations containing higher fatty acid glycerides
`
`such as olive oil, peanut oil, and/or castor oil.
`
`These formulations frequently produce an unpleasant
`
`sensation when applied to the eye because of stimula-
`
`tion or the viscousness which is characteristic of
`
`these oils.
`
`Another drawback of these formulations is that
`
`they contain a high concentration of oils, and oils
`
`exacerbate the symptoms of certain ocular surface
`
`diseases such as dry eyes,
`
`indicated by cyclosporin.
`
`Therefore,
`
`these oily formulations may not be clini—
`
`cally acceptable. Additionally,
`
`these formulations
`
`often suffer from physical instability due to cyclo-
`
`sporin's propensity to undergo conformational change
`
`and crystallize out.
`
`The crystallization problem has
`
`been noticed in formulations containing corn oil or
`
`medium chain triglycerides.
`
`Lastly,
`
`these formula-
`
`tions often have a low thermodynamic activity (degree
`
`of saturation) of cyclosporin which leads to a poorer
`
`drug bioavailability.
`
`It may be possible to minimize the problems
`
`related to unpleasant sensation and syndrome exacerba-
`
`tion by reducing the oil content and dispersing the
`
`oil phase in water into an emulsion. However, it is
`
`not an easy task to formulate an ophthalmic emulsion
`
`because one indispensable class of ingredients in an
`
`emulsion system is emulsifiers, and the majority of
`
`emulsifiers is highly irritating to the eyes.
`
`The present invention is directed to an emulsion
`
`system which utilizes higher fatty acid glycerides but
`
`in combination with polysorbate 80 which results in an
`
`emulsion with a high comfort level and low irritation
`
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`
`7
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`_ 6 -
`
`potential suitable for delivery of medications to sen—
`
`sitive areas such as ocular tissues.
`
`Further,
`
`the
`
`present
`
`invention provides a pharmaceutical composi-
`
`tion and method for causing preferential absorption of
`
`cyclosporin in the lacrimal gland.
`That
`is,
`for a
`given instillation of the composition into an eye, a
`
`greater amount of absorption occurs in the lacrimal
`
`gland for formulations made in accordance with the
`
`present
`
`invention than heretofore utilized formula-
`
`10
`
`tions.
`
`SUMMARY OF THE INVENTION
`
`15
`
`20
`
`25
`
`30
`
`35
`
`In accordance with the present invention, a non—
`
`irritating pharmaceutical composition with high com—
`
`fort level and low irritation potential suitable for
`
`delivery to sensitive areas such as ocular tissues
`
`comprises cyclosporin in admixture with an emulsifying
`
`amount of a higher fatty acid glycerol and polysorbate
`
`80. More particularly,
`
`the composition may comprise
`
`cyclosporin A and the higher fatty acid glyceride may
`
`comprise castor oil.
`
`Preferably, the weight ratio of the castor oil to
`
`the polysorbate 80 is between about 0.3 to about 30
`
`and a weight ratio of the cyclosporin to castor oil is
`
`below 0.16.
`
`More preferably,
`
`the weight ratio of
`
`castor oil to polysorbate 80 is between 0.5 and 12.5,
`
`and the weight ratio of cyclosporin to castor oil is
`between 0.12 and 0.02.
`
`When cyclosporin is dissolved in the oil phase in
`
`accordance with the present invention, the emulsion is
`
`found to be physically stable upon long term storage.
`
`No crystallization of cyclosporin was noticed after
`
`nine months at
`
`room temperature.
`
`Moreover,
`
`the
`
`cyclosporin emulsion is formulated in such a way that
`
`8
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`_7_
`
`the drug has reasonably high thermodynamic activity,
`
`yet without the crystallization problem.
`
`Importantly,
`
`the composition of the present
`
`in-
`
`vention provides for enhanced absorption of the cyclo-
`
`sporin in the lacrimal gland of
`
`the eye.
`
`In this
`
`manner,
`
`the activity of the cyclosporin in restoring
`
`lacrimal gland tearing is increased. That is, since
`
`a greater amount of cyclosporin is absorbed into the
`
`lacrimal gland, more of the cyclosporin is effective
`
`in producing lacrimal gland tearing than heretofore
`
`possible.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The advantages and features of the present inven—
`
`tion will be better understood by the following
`description when considered in conjunction with the
`
`accompanying drawings in which:
`
`Figure l is a bar chart of conjunctival concen-
`
`tration of cyclosporin A after
`
`a
`
`single topical
`
`instillation of various formulations in a rabbit eye;
`Figure 2 is a bar chart of cornea concentration
`
`of cyclosporin A after a single topical instillation
`
`of various formulations in a rabbit eye;
`
`Figure 3 is a bar chart of ciliary body concen-
`
`tration of cyclosporin A after
`
`a
`
`single topical
`
`instillation of various formulations in a rabbit eye;
`and
`
`Figure 4 is a bar chart of lacrimal gland concen-
`
`tration of cyclosporin A after
`
`a
`
`single topical
`
`instillation of various formulations in a rabbit eye.
`
`DETAILED DESCRI PTION
`
`As hereinabove noted, cyclosporin is available as
`
`a mixture in which the principal ingredient is cyclo-
`
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`
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`

`WO 95/31211
`
`PCT/US95/06302
`
`-8—
`
`sporin A with significant, but smaller, quantities of
`
`other cyclosporins such as cyclosporin B through I.
`
`However, as also hereinabove noted, the present inven-
`
`tion may be applied to either a pure cyclosporin or to
`
`a mixture of individual cyclosporins.
`
`The discovery on which the present
`
`invention is
`
`founded relates to a combination of a higher fatty
`
`acid glyceride and an emulsifier and dispersing agent,
`
`polysorbate 80.
`
`The selection of
`
`these components
`
`could not have been anticipated on the basis of con-
`
`ventional thinking.
`
`For example,
`
`although it
`
`is well
`
`known
`
`that
`
`cyclosporin may be used in combination with castor
`
`oil,
`
`this combination is
`
`irritating to sensitive
`
`tissues such as the eye. Thus, conventional teaching
`
`in the art is away from a formulation which utilizes
`
`a higher fatty acid glyceride, such as castor oil, and
`
`cyclosporin.
`
`Stated another way,
`
`there is no way of deducing
`
`that
`
`the use of an emulsifier and dispersing agent
`
`such as polysorbate 80 will reduce the irritation po~
`
`tential of an emulsion utilizing castor oil.
`
`There
`
`are no examples of polysorbate in combination with
`
`castor oil which, when admixed to cyclosporin, pro-
`
`duces an emulsion with a high comfort
`
`level and low
`
`irritation potential suitable for
`
`the delivery of
`
`medication to sensitive areas such as ocular tissues.
`
`The present invention achieves a stable solution
`
`state of cyclosporin. This stable solution state is
`
`another important performance characteristic differ-
`
`entiating the present invention from the conventional
`
`oil systems. Cyclosporin is notorious for its ten-
`
`10
`
`15
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`
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`
`35
`
`10
`
`10
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`_ 9 ..
`
`dency to precipitate out
`
`in conventional oil systems
`
`in which it is fully dissolved initially.
`
`In accordance with the present
`
`invention,
`
`the
`
`emulsions can be further stabilized using a polyelec-
`
`trolyte, or polyelectrolytes if more than one,
`
`from
`
`the family of cross-linked polyacrylates,
`carbomers and Pemulen®.
`
`such as
`
`Pemulen® is a polymeric emulsifier having a CTFA
`
`name of Acrylates/C10-30 Alkyl Acrylate Cross-Polymer
`
`and is discrived in th "Carbomer 1342" monograph in
`
`the USPXXII/NFXVII .
`
`’
`
`In addition, the tonicity of the emulsions can be
`
`further adjusted using glycerine, mannitol, or sorbi-
`
`tol if desired.
`
`The Ph of the emulsions can be ad—
`
`justed in a conventional manner using sodium hydroxide
`
`to a near physiological pH level and while buffering
`
`agents are not required, suitable buffers may include
`
`phosphates, citrates, acetates and borates.
`
`While the preferable medications in accordance
`
`with the present invention include cyclosporin, other
`
`chemicals which are poorly soluble in water such as
`
`indomethacin and steroids such as androgens, predniso-
`
`lone,
`
`prednisolone
`
`acetate,
`
`fluorometholone,
`
`and
`
`dexamethasones, may be emulsified with castor oil and
`
`polysorbate 80 resulting in a composition with similar
`low irritation potential.
`
`The
`
`invention is
`
`further
`
`illustrated by the
`
`following examples with all parts and percentages
`
`expressed by weight.
`
`The cyclosporin used in the
`
`examples was supplied by Sandoz.
`
`10
`
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`
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`
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`
`11
`
`

`

`WO 95/31211
`
`PCT/U895/06302
`
`-10-
`
`Example 1
`
`polysorbate so
`
`5.00%
`
`2.50%
`
`0.625%
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`_nn-I-n
`0.05%
`
`l—m-“m
`“a nun
`
`a “an
`a
`
`
`_ 7 2 7 6 7.2—7.6 7.2-7.6 7.2-7.6 7.2-7.5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`
`
`Example 2
`
`
`
`
`—BB-
`
`
`
`
`
`
`
`
`
`-ulgu
`n
`_7.2-7.6 7.2-7.6 7.2-7.6
`
`7.2-7.6
`
`_--m
`
`
`
`Example 3
`
`Polysorbate 80
`
`
`
`
` Castor 011
`Carbomer 1382
`
` Purlfled water
`
`7.2-7.6
`
`12
`
`12
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`-11-
`
`Example 4
`
`_—
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`_n
`
`——
`
`_-
`
`The formulations set forth in Examples 1-4 were
`
`made for treatment of keratoconjunctivitis sicca (dry
`
`eye)
`
`syndrome with Examples 2,
`
`3 and 4 without
`
`the
`
`active ingredient cyclosporin utilized to determine
`
`the toxicity of the emulsified components.
`
`The formulations in Examples 1-4 were applied to
`
`rabbit eyes eight times a day for seven days and were
`
`found to cause only slight
`
`to mild discomfort and
`
`slight hyperemia in the rabbit eyes. Slit lamp exam-
`
`ination revealed no changes in the surface tissue.
`
`In
`
`addition,
`
`the cyclosporin containing castor oil emul—
`
`sion, as hereinabove set forth in Examples lA-lD, was
`
`also tested for ocular bioavailability in rabbits; and
`
`the therapeutic level of cyclosporin was found in the
`
`tissues of interest after dosage. This substantiates
`
`that cyclosporin in an ophthalmic delivery system is
`
`useful
`
`for treating dry eye as set
`
`forth in U.S.
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`Patent No. 4,839,342.
`
`In addition, no difference in toxicity was found
`
`between formulations with cyclosporin (Examples lA—lD)
`
`and formulations without cyclosporin (Examples 2-4).
`
`35
`
`The formulations set forth in Examples 1-4 were
`
`found to be physically stable upon long term storage.
`
`13
`
`13
`
`

`

`W0 95/3121]
`
`PCT/U895/06302
`
`-12-
`
`With regard to formulations 1A-1D, no crystallization
`
`of cyclosporin was noticed after nine months at room
`
`temperature.
`
`Further, other higher fatty acid glycerides such
`
`as olive oil, peanut oil and the like may also be
`
`utilized with the polysorbate 80 with similar results
`
`regarding biotoxicity.
`
`10
`
`The following examples demonstrate the activity
`
`of
`
`the composition in accordance with the present
`
`invention for enhanced absorption of cyclosporin A in
`
`the lacrimal gland.
`
`15
`
`Materials
`
`20
`
`25
`
`The [Mebmt-3H]—cyclosporin-A (lot #TRQ6553) was
`
`prepared by AmerSham International
`
`(Buckinghamshire,
`
`England) with
`
`radiochemical purity of
`
`-98% (by
`
`reversed phase HPLC)
`
`and specific activity of 2.6
`
`Ci/mmol
`
`(2.16 mCi/mg).
`
`The 3H-label is a metabolic-
`
`ally stable position as shown by the asterisk.
`
`The
`
`radiolabeled CsA was supplied as an ethanol solution
`
`(1 mCi/ml).
`
`All organic
`
`solvents used in the
`
`procedures described in this study were "HPLC grade".
`
`all other chemicals and reagents were analytical grade
`unless otherwise noted.
`
`The compositions of the six formulations tested
`
`30
`
`are listed in Table A.
`
`14
`
`14
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`_13_
`
`IABLE_A
`
`
`
`Ingredients
`
`Ciififr
`
`
`
`Castor
`
`
`Miglyol
`
`
`Oil-in-
`Aqueous-
`. _. _
`Polyoxyl
`
`
`a Cyclo- Caitég
`Polygxyl 40 with
`Water
`
`
`
`Emulsion dextrin Emulsion
`Edetate
`_mm
`--—----
`------
`
`
`
`
`
`
`
`
` Miguel on -----
`HPMC Butylated
`
`1.00 ---
`2.20
`
`Pluronic L121+P123 _
`Tween 80
`
`Glycerin
`
`Pemulen® TR—2
`
`Carbopol 981
`
`Polyoxyl 40
`Stearate (mg)
`
`H drox toluene
`
`Ethanol(9200 proof)
`
`Sodium Chloride
`
`Sodium
`Monophosphate
`
`Disodium Edetate
`
`Water
`
`Batch Size
`
`5.:
`
`in
`
`
`
`
`
`The radiolabeled formulations were formulated to
`
`ensure
`
`that
`
`the
`
`radioactivity was
`
`homogeneous
`
`throughout
`
`the vehicle.
`
`The expected radioactivity
`
`concentrations of the radiolabeled drug formulations
`
`5
`
`were 1—2 mCi/ml.
`
`The expected specific activity of
`
`radiolabeled cyclosporin A (CsA) formulations was 0.5-
`
`2 mCi/mg. All test articles were stored at ambient
`
`temperature.
`
`15
`
`15
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`-14—
`
`Analysis of Test Drug Formulations
`
`The test formulations were analyzed in triplicate
`
`by HPLC to determine the concentration of CsA and
`
`radiochemical purity of
`
`the CsA dosing solutions
`
`(>93%) before dosing. The radioactive concentrations
`
`of the test
`
`formulations were quantified by liquid
`
`scintillation counting (LSC).
`
`Chromatographic Conditions
`
`Pump:
`
`(Beckman
`126
`Model
`Beckman
`Instruments, San Ramon, CA)
`
`Mobile phase:
`
`Acetonitrile: 0.03% H3PO4 in water,
`pH 3
`(65:35 v/v)
`
`Flow rate:
`
`1.0 ml/min
`
`Column:
`
`Injector:
`
`l4C detector:
`
`Scintillant:
`
`Supercosil C8, 7.5 cm x 4.6 mm,
`3 um (Supelco, Bellefonte, PA)
`Superguard LC-8 (Supelco)
`Column heater
`(Bio-rad, Richmond,
`CA) at 60-70°C
`
`712B
`WISP
`Milford, MA)
`
`(Waters Associates,
`
`171
`Isotope
`Radio
`(Beckman Instruments)
`
`Detector
`
`Flow
`Ready
`Instruments),
`'4 ml/min
`
`III
`Flow
`
`(Beckman
`Rate
`of
`
`UV detector:
`
`Model
`202 nm
`
`166
`
`(Beckman Instruments),
`
`Data processor:
`
`System Gold
`Beckman
`Instruments)
`
`(Beckman
`
`Run Time:
`
`15 min
`
`Retention Time:
`
`6 min (cyclosporin A)
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`40
`
`45
`
`16
`
`16
`
`

`

`WO 95/3121]
`
`PCT/US95/06302
`
`_ 1 5 _
`
`Mag
`
`Female New Female New Zealand albino rabbits were
`
`obtained and quarantined for at least five days before
`
`procedures. Animals were housed in temperature— and
`
`humidity-controlled rooms.
`
`Food and tap water were
`
`provided ad libitum.
`
`Fifty-eight rabbits (2-3 kg)
`
`were selected from the colony to minimize bias.
`
`They
`
`were individually identified by ear tags and appeared
`
`10
`
`to be healthy.
`
`Dosing
`
`15
`
`20
`
`The animals were divided into six groups of nine
`
`rabbits; each group was treated with one of the six
`
`CsA formulations. During dosing,
`
`the lower eyelid of
`
`each rabbit was gently pulled away from the eye and
`
`35 pl of
`
`the formulation were administered in the
`
`lower conjunctival cul-de-sac of each eye.
`
`After
`
`dosing,
`
`the upper
`
`and lower eyelid were handheld
`
`closed for ~5 seconds and released.
`
`The animals were
`
`observed visually for any signs of tearing or ocular
`discomfort.
`
`25
`
`Sampling
`
`30
`
`35
`
`Tissues were collected at 20-min.,
`
`6—hr.
`
`and
`
`24-hr. post-dose for each group. Three rabbits (six
`
`eyes) were used at each time point. At the specific
`
`sampling times,
`
`the animals were euthanized by an
`
`intravenous injection of 0.5-1 ml Eutha—6
`
`(Western
`
`Supply Co., Arcadia, California) via marginal ear
`
`vein.
`
`Each eye was then rinsed with normal saline.
`
`The aqueous humor
`
`(“200 p1) was removed by means of a
`
`0.5 m1 tuberculin syringe. The orbital lacrimal gland
`
`('400 mg), upper and lower bulbar conjunctivae (”50 mg
`
`each), corneal
`
`(”50 mg) and iris—ciliary body ('50 mg)
`
`17
`
`17
`
`

`

`W0 95/31211
`
`PCT/US95/06302
`
`_ 1 6 _
`
`were dissected.
`
`The tissues dissected were blotted
`
`dry and weighed.
`
`Ocular
`
`tissue and aqueous humor
`
`samples
`
`from both eyes were collected from four
`
`untreated animals to be used as blank samples.
`
`Analysis of Radioactivity
`
`An aliquot of aqueous humor
`
`(50-175 pl) was
`
`counted directly in 10 m1 of Ready-Solv HP by LSC.
`
`Tissue and blood samples were weighed into combustion
`
`cones prior to combustion in a Model
`
`307 Packard
`
`Tissue Oxuduzer
`
`(Packard
`
`Co.,
`
`Downers Grove,
`
`Illinois). After combustion of the tissue samples,
`3H20 was trapped in the Monophase-S solution (Packard)
`
`and the radioactivity of the samples was determined by
`
`LSC fix:
`
`a Beckman Model 1801 or 3801 scintillation
`
`counter (Beckman Instruments, San Ramon, California).
`
`Data Analyses
`
`Excel
`
`software
`
`(version 4.0, Microsoft Corp,
`
`Redmond, Washington) was used for data analysis.
`
`concentrations of total radioactivity in the tissue
`
`samples were
`
`expressed as
`
`dpm/g or
`
`dpm/ml
`
`and
`
`converted to ng equivalents (eq) of CsA/g or ml, using
`
`the specific activity of
`
`the dosing formulations.
`
`Mean, standard deviation (SD) or standard error of the
`
`mean
`
`(SEM) was calculated according to standard
`
`methods.
`
`Radioactivity levels were not considered
`
`significant unless the dpm was greater than twice that
`
`of background b=(blanks).
`
`Comparisons of tissue drug concentrations at each
`
`time point for the formulations were determined by
`
`one-factor ANOVA. All statistical comparisons were
`
`made using StatView® (version 1.03, Abacus Concepts,
`
`Inc., Berkeley, California).
`
`the Fisher and Scheffe
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`18
`
`18
`
`

`

`WO 95/31211
`
`PCT/US95I06302
`
`-17-
`
`F tests were used to determine significant differences
`
`between formulations at the 95% level
`
`(a = 0.05). The
`
`rejection criteria for excluding any outlier data was
`
`based on standard outlier tests.
`
`No more than one
`
`outlier was eliminated from any data set.
`
`Results and Discussion
`
`The
`
`radioactivity
`
`concentrations
`
`in
`
`ocular
`
`tissues at 20 minutes,
`
`6 hours, and 24 hours after a
`
`single topical application of various formulations are
`
`depicted in Figures 1—4.
`
`In general,
`
`the concentra-
`
`tions in the ocular tissues were greatest at the earl-
`
`iest time point of 20 minutes as reported in previous
`
`single dose studies (2, 3). The radioactivity concen-
`
`tration was highest in the conjunctiva and cornea for
`
`each formulation.
`
`The relatively low aqueous humor
`
`and iris-ciliary body concentrations suggest
`
`low in-
`
`traocular absorption of CsA, consistent with the low
`
`CsA corneal permeability of -1.0 x 10‘6 cm/sec (6).
`
`The decline of radioactivity concentrations from the
`
`cornea was slower than those from the conjunctiva,
`
`lacrimal gland, and aqueous humor. The observed blood
`
`radioactivity concentrations (<3 ng—eq/ml) were much
`
`lower
`
`than
`
`trough plasma
`
`CsA concentrations
`
`of
`
`80—250 ng/ml observed after oral dosing to humans (1).
`
`The dependence of CsA corneal and conjunctival
`
`penetration on the formulation was
`
`interpreted in
`
`terms of CsA concentration in formulation and the
`
`release rate of CsA from formulation into tear film.
`
`The
`
`aqueous
`
`formulations
`
`demonstrated
`
`a greater
`
`propensity to release CsA for diffusion across the
`
`surface tissue epithelia.
`
`The 0.2% straight castor
`
`oil was
`
`formulated below the CsA solubility and
`
`therefore the release rate could be hampered by the
`
`less than maximal CsA thermodynamic activity (5).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`19
`
`19
`
`

`

`WO 95/31211
`
`PCT/US95/06302
`
`.— l 8 _
`
`The ocular surface tissues contained a higher
`fraction of the CsA dose than the other tissues and
`
`was used to discriminate among the aqueous, emulsion
`
`The poly-
`and the straight castor oil formulations.
`oxyl 40 formulation produced higher ocular surface
`
`5
`
`tissue concentrations than the emulsions and straight
`castor oil.
`The emulsions were also effective in
`
`delivery of CsA to the tissues of interest,
`
`lacrimal
`
`gland, cornea, and conjunctiva.
`
`The castor oil emul—
`
`10
`
`sion showed higher lacrimal gland concentrations than
`
`the modified Santen and the miglyol emulsion.
`
`The
`
`straight castor oil showed the lowest concentrations
`
`the factors
`in surface ocular tissues. Apparently,
`influencing CsA penetration into the lacrimal gland
`and the surface tissues are different.
`
`Although there has been hereinabove described a
`
`particular pharmaceutical composition in the form of
`
`a nonirritating emulsion for the purpose of illustrat-
`ing the manner in which the invention may be used to
`advantage, it should be appreciated that the invention
`is not limited thereto. Accordingly, any and all mod-
`ifications, variations, or equivalent arrangements,
`which may occur to those skilled in the art, should be
`considered to be within the scope of the present in-
`vention as defined in the appended claims.
`
`15
`
`20
`
`25
`
`20
`
`20
`
`

`

`WO 95/31211
`
`PCT/US9SI06302
`
`-19-
`
`WHAT IS CLAIMED IS:
`
`1.
`
`A composition comprising a nonirritating
`
`emulsion of at least one cyclosporin in admixture with
`
`a higher fatty acid glyceride, polysorbate 80 and an
`
`emulsion-stabilizing amount of
`
`Pemulen® in water
`
`suitable for topical application to ocular tissue.
`
`2.
`
`The pharmaceutical composition according to
`
`claim 1 wherein the cyclosporin comprises cyclosporin
`A.
`
`3.
`
`The pharmaceutical composition according to
`
`claim 2 wherein the weight ratio of the higher fatty
`
`acid glyceride to the polysorbate 80 is between about
`
`0.3 and about 30.
`
`4.
`
`The pharmaceutical composition according to
`
`claim 3 wherein the higher fatty acid glyceride com-
`
`prises castor oil and the weight ratio of cyclosporin
`to castor oil is below about 0.16.
`
`5.
`
`A pharmaceutical composition comprising a
`
`nonirritating emulsion of at least one cyclosporin in
`
`admixture with castor oil and polysorbate 80 in water
`
`suitable for topical application to ocular tissue.
`
`6.
`
`The pharmaceutical composition according to
`
`claim 5 wherein the cyclosporin comprises cyclosporin
`A.
`
`7.
`
`The pharmaceutical composition according to
`
`claim 6 wherein the weight ratio of castor oil to the
`
`polysorbate 80 is between about 0.3 and about 30.
`
`21
`
`21
`
`

`

`W0 95/31211
`
`PCT[U895/06302
`
`-20-
`
`8.
`
`The pharmaceutical composition according to
`
`claim 7 wherein the weight ratio of cyclosporin to
`
`castor oil is below about 0.16.
`
`9.
`
`The composition according to claim 1 wherein
`
`the higher fatty acid glyceride and polysorbate 80 are
`
`present in amounts sufficient to prevent crystalliza—
`
`tion of cyclosporin for a period of up to about nine
`months.
`
`10.
`
`A stable, nonirritating ophthalmic composi-
`
`tion comprising cyclosporin in admixture with an
`
`emulsifying amount of a higher fatty acid glyceride
`
`and polysorbate 80.
`
`i
`
`11.
`
`A
`
`pharmaceutical
`
`emulsion
`
`comprising
`
`cyclosporin A, castor oil, Pemulen®, glyceride and
`
`water in amounts sufficient to prevent crystallization
`
`of cyclosporin A for a period of up to about nine
`
`months, said pharmaceutical emulsion being suitable
`
`for topical application to ocular tissue.
`
`12.
`
`The pharmaceutical
`
`emulsion according to
`
`claim 11 wherein the cyclosporin A is present
`
`in an
`
`amount of between about 0.05 to about 0.40%,
`
`by
`
`weight,
`
`the castor oil
`
`is present
`
`in ana mount of
`
`between about 0.625%, by weight, the polysorbate 80 is
`
`present
`
`in an amount of about 1.0%, by weight,
`
`the
`
`Pemulen® is present
`
`in an amount of about 0.05%, by
`
`weight, and the glyceride is present
`
`in an amount of
`
`about 2.2%, by weight.
`
`13.
`
`A pharmaceutical
`
`emulsion consisting of
`
`between about 0.05% and about 0.40%, by weight, cyclo-
`
`sporin A, between about 0.625% and about 5.0%, by
`
`weight, castor oil, about 1.0%, by weight, polysorbate
`
`80, about 0.05%, by weight, Pemulen®, and about 2.2%,
`
`22
`
`22
`
`

`

`WO 95/31211
`
`PCT[U895/06302
`
`-21-
`
`by weight, glycerine in water with a pH of between
`
`about 7.2 and 7.6 suitable for topical application to
`ocular tissue.
`
`14.
`
`A pharmaceuti