(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`7 November2013 (07.11.2013)
`
`WIPO!) PCT
`
`\=
`
`(10) International Publication Number
`WO 2013/166385 Al
`
`Ming; 2900 Glen Avenue, Apt. F, Baltimore, MD 21215
`(US).
`
`Agent: LEE, Jessamine, N.; Wolf, Greenfield & Sacks,
`P.C., 600 Atlantic Avenue, Boston, MA 02210-2206 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, Mx, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`GD)
`
`International Patent Classification:
`A61K 47/34 (2006.01)
`AGIK 9/51 (2006.01)
`A61K 9/10 (2006.01)
`
`QD
`
`International Application Number:
`
`PCT/US2013/039467
`
`(74)
`
`(81)
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`3 May 2013 (03.05.2013)
`
`English
`
`English
`
`Priority Data:
`61/642,227
`
`3 May 2012 (03.05.2012)
`
`US
`
`INC.
`Applicants; KALA PHARMACEUTICALS,
`[US/US]; 135 Beaver Street, Suite 309, Waltham, MA
`02452 (US). JOHNS HOPKINS UNIVERSITY, THE
`[US/US]; 100 N. Charles Street, Sth Floor, Licensing &
`Technology Developement, Baltimore, Maryland 21201
`(US).
`
`Inventors: POPOV, Alexey; 60 Hope Avenue, Waltham,
`MA 02453 (US). ENLOW,Elizabeth, M.; 85 Black Bear
`Drive, Unit 1624, Waltham, MA 02451 (US). BOURAS-
`SA, James; 425 Broadway, Apt 15, Somerville, MA 02145
`(US). GARDNER, Colin, R.; 140 Catering Heights, Con-
`cord, MA 01742 (US). CHEN, Hongming; 16 Birch Hill
`Road, Belmont, MA 02478 (US). ENSIGN, Laura, M.;
`134 Regester Avenue, Baltimore, MD 21212 (US). LAI,
`Samuel, K.; 106 Oak Spring Court, Carrboro, NC 27510
`(US). YU, Tao; Smith Building 6001 Bay H1, 400 N
`Broadway, Baltimore, MD 21231 (US). HANES, Justin;
`6306 Pinehurst Road, Baltimore, MD 21212 (US). YANG,
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW,SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE,ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO,PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR,NE,SN,TD, TG).
`Published:
`
`with international search report (Art. 21(3))
`
`before the expiration ofthe time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`(34) Title: PHARMACEUTICAL NANOPARTICLES SHOWING IMPROVED MUCOSAL TRANSPORT
`
`(57) Abstract: Compositions with improvedparticle transport in mucus are provided. In some embodiments, the compositions and
`methods involve making mucus-penetrating particles (MPP) without any polymeric carriers, or with minimal use of polymeric carri-
`ers. The compositions may include modifying the surface coatings of particles formed of pharmaceutical agents that have a low water
`solubility. Such compositions can be used to achieve efficient transport of particles of pharmaceutical agents though mucusbarriers
`in the body for a wide spectrum of applications, including drug delivery, imaging, and diagnostic applications.
`
`
`
`Wo2013/166385A1|IMTTINMINNIIMTANIMUITTAAAATA
`
`

`

`WO 2013/166385
`
`PCT/U82013/039467
`
`PHARMACEUTICAL NANOPARTICLES SHOWING IMPROVED MUCOSAL TRANSPORT
`
`Related Applications
`
`[0001]
`
`The present application claimspriority under 35 U.S.C. §119(e) to U.S.
`
`Provisional Patent Application No. 61/642,227, filed May 3, 2012 andentitled ““Nanocrystals,
`
`Compositions, and Methods That Aid Particle Transport in Mucus’, which is incorporated
`
`herein by reference.
`
`Field of the Invention
`
`[0002]
`
`The present invention generally relates to nanocrystals, compositions, and
`
`methodsthat aid particle transport in mucus.
`
`Background of the Invention
`
`[0003]
`
`A mucuslayer present at various points of entry into the body, including the eyes,
`
`nose, lungs, gastrointestinal tract, and female reproductivetract, is naturally adhesive and
`
`serves to protect the body against pathogens, allergens, and debris by effectively trapping and
`
`quickly removing them via mucusturnover. For effective delivery of therapeutic, diagnostic,
`
`or imaging particles via mucus membranes, the particles must be able to readily penetrate the
`
`mucuslayer to avoid mucus adhesion and rapid mucus clearance. Several lines of evidence
`
`suggest that conventional nanoparticles are not capable of crossing mucosalbarriers.
`
`However, it has been recently demonstrated that polymeric nanoparticles (degradable or not)
`
`modified with a special surface coating (covalently or non-covalently) can diffuse in
`
`physiologically think mucus samples nearly as rapidly as they would in water. Such
`
`polymer-based mucus-penetrating particles (MPP) can encapsulate various therapeutic,
`
`imaging, or diagnostic agents to enable drug delivery, diagnostic, or imaging applications.
`
`[0004]
`
`Nevertheless, polymer-based MPP mayhaveseveral inherent limitations
`
`compared to unencapsulated particles of pharmaceutical agents. In particular, in light of drug
`
`delivery applications these limitations may include: 1) Inherently lower drug loading; 2) Less
`
`convenient dosage form, as reconstitution from a dry powderstorage form maybe required
`
`for polymeric nanoparticles; 3) Potentially increased toxicity; 4) Chemical and physical
`
`stability concerns; and 5) Increased manufacturing complexity. Accordingly, improvements
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`in compositions and methods involving mucus-penetrating particles for delivery of
`
`pharmaceutical agents would be beneficial.
`
`Summary of the Invention
`
`[0005]
`
`The present description generally relates to nanocrystals, compositions, and
`
`methodsthat aid particle transport in mucus. In some embodiments, the compositions and
`
`methods involve mucus-penetrating particles without any polymeric carriers, or with minimal
`
`use of polymeric carriers. The subject matter of this application involves, in some cases,
`
`interrelated products, alternative solutions to a particular problem, and/or a plurality of
`
`different uses of structures and compositions.
`
`[0006]
`
`In one set of embodiments, a method of forming coated particles is provided. The
`
`method involves combining core particles with a solution comprising a surface-altering agent,
`
`wherein the core particles comprise a solid pharmaceutical agent or a salt thereof, wherein the
`
`agent or salt has a solubility of less than or equal to about 1 mg/mL in the solution at 25 °C,
`
`and wherein the pharmaceutical agent or salt thereof constitutes at least about 80 wt% of each
`
`of the core particles. The method also involves coating the core particles with the surface-
`
`altering agent to form coated particles, wherein the surface-altering agent comprises a
`
`triblock copolymer comprising a hydrophilic block — hydrophobic block — hydrophilic block
`
`configuration, wherein the hydrophobic block has a molecular weight of at least about 2 kDa,
`
`and the hydrophilic blocks constitute at least about 15 wt% of the triblock copolymer,
`
`wherein the hydrophobic block associates with the surface of the core particles, wherein the
`
`hydrophilic block is present at the surface of the coated particles and renders the coated
`
`particles hydrophilic, and wherein the coated particles have a relative velocity of greater than
`
`0.5 in mucus.
`
`[0007]
`
`In another set of embodiments, a composition comprising a plurality of coated
`
`particles is provided. The coated particle comprises a core particle comprising a solid
`
`pharmaceutical agentor a salt thereof, wherein the agent or salt has an aqueoussolubility of
`
`less than or equal to about 1 mg/mLat 25 °C at any point throughout the pH range, wherein
`
`the pharmaceutical agent or salt thereof constitutes at least about 80 wt% of the core particle.
`
`The coated particle also includes a coating comprising a surface-altering agent surrounding
`
`the core particle, wherein the surface-altering agent comprises a triblock copolymer
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`comprising a hydrophilic block — hydrophobic block — hydrophilic block configuration,
`
`wherein the hydrophobic block has a molecular weight of at least about 2 kDa, and the
`
`hydrophilic blocks constitute at least about 15 wt% of the triblock copolymer, wherein the
`
`hydrophobic block associates with the surface of the core particle, wherein the hydrophilic
`
`block is present at the surface of the coated particle and renders the coated particle
`
`hydrophilic, and wherein the surface-altering agent is present on the surface of the core
`
`particle at a density of at least about 0.001 molecules per nanometer squared. The coated
`
`particles have a relative velocity of greater than 0.5 in mucus.
`
`[0008]
`
`In another set of embodiments, a method of forming coated comprises providing a
`
`pharmaceutical agent and precipitating the pharmaceutical agent by forming a salt in an
`
`aqueoussolution in the presence of a surface-altering agent to form core particles of the
`
`pharmaceutical agent, wherein the salt has a lower aqueoussolubility than the pharmaceutical
`
`agent in the non-salt form, the aqueous solubility of the salt being less than or equal to about
`
`1 mg/mLat 25 °C at any point throughout the pH range, and wherein the surface-altering
`
`agent is present at a concentration of at least about 0.01% (w/v) in the aqueoussolution. The
`
`method involves coating the core particles with the surface-altering agent to form coated
`
`particles, wherein the surface-altering agent comprises a triblock copolymer comprising a
`
`hydrophilic block— hydrophobic block— hydrophilic block configuration, wherein the
`
`hydrophobic block has a molecular weight of at least about 2 kDa, and the hydrophilic blocks
`
`constitute at least about 15 wt% of the triblock copolymer, wherein the hydrophobic block
`
`associates with the surfaces of the core particles, and wherein the hydrophilic block is present
`
`at the surfaces of the coated particles and renders the coated particles hydrophilic. The
`
`coated particles have a relative velocity of greater than 0.5 in mucus.
`
`[0009]
`
`In another set of embodiments, a method of treatment is provided. The method
`
`comprises administering to a patient or a subject in need thereof, a composition comprising a
`
`plurality of coated particles. The coated particle comprises a core particle comprising a solid
`
`pharmaceutical agentor a salt thereof, wherein the agent or salt has an aqueoussolubility of
`
`less than or equal to about 1 mg/mLat 25 °C at any point throughout the pH range, wherein
`
`the pharmaceutical agent or salt thereof constitutes at least about 80 wt% of the core particle.
`
`The coated particle also includes a coating comprising a surface-altering agent surrounding
`
`the core particle, wherein the surface-altering agent comprises a triblock copolymer
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`comprising a hydrophilic block — hydrophobic block — hydrophilic block configuration,
`
`wherein the hydrophobic block has a molecular weight of at least about 2 kDa, and the
`
`hydrophilic blocks constitute at least about 15 wt% of the triblock copolymer, wherein the
`
`hydrophobic block associates with the surface of the core particle, wherein the hydrophilic
`
`block is present at the surface of the coated particle and renders the coated particle
`
`hydrophilic, and wherein the surface-altering agent is present on the surface of the core
`
`particle at a density of at least about 0.001 molecules per nanometer squared. The coated
`
`particles have a relative velocity of greater than 0.5 in mucus.
`
`[00010]
`
`Other advantages and novel features of the present invention will become
`
`apparent from the following detailed description of various non-limiting embodiments of the
`
`invention when considered in conjunction with the accompanying figures.
`
`In cases where the
`
`present specification and a documentincorporated by reference include conflicting and/or
`
`inconsistent disclosure, the present specification shall control. If two or more documents
`
`incorporated by reference include conflicting and/or inconsistent disclosure with respect to
`
`each other, then the document having the later effective date shall control.
`
`Brief Description of the Drawings
`
`[00011]
`
` Non-limiting embodiments ofthe present invention will be described by way of
`
`example with reference to the accompanying figures, which are schematic and are not
`
`intended to be drawn to scale. In the figures, each identical or nearly identical component
`
`illustrated is typically represented by a single numeral. For purposes of clarity, not every
`
`componentis labeled in every figure, nor is every component of each embodimentof the
`
`invention shown whereillustration is not necessary to allow those of ordinary skill in the art
`
`to understand the invention. In the figures:
`
`[00012]
`
`FIG. 1 is a schematic drawing of a mucus-penetrating particle having a coating
`
`and a core of a solid pharmaceutical agent according to one set of embodiments;
`
`[00013]
`
`FIG. 2A is a plot showing the ensemble averaged velocity <Vimean> in human
`
`cervicovaginal mucus (CVM)for 200nm carboxylated polystyrene particles (negative
`
`control), 200nm PEGylated polystyrene particles (positive control), and nanocrystal particles
`
`(sample) made by nanomilling and coated with different stabilizers/surface-altering agents
`
`according to one set of embodiments;
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`
`
`[00014] FIG. 2B isaplot showingthe relative velocity <Vmmean>rei in CVM for nanocrystal
`
`particles made by nanomilling and coated with different stabilizers/surface-altering agents
`
`according to one set of embodiments;
`
`[00015]
`
`FIGs. 3A-3D are histograms showingdistribution of trajectory-mean velocity
`
`Vimean 1 CVM within an ensemble of nanocrystal particles coated with different surface-
`
`altering agents according to one set of embodiments;
`
`FIG. 4isa plot showing <Vmean>rei in CVM for nanocrystal particles coated with
`[00016]
`different PEO-PPO-PEOPluronic® triblock copolymers, mapped with respect to molecular
`
`weight of the PPO block and the PEO weight content (%), according to one set of
`
`embodiments:
`
`[00017]
`FIG. 5 isa plot showing the mass transport through CVM forsolid particles
`having different core materials that are coated with either Pluronic® F127 (MPP)or sodium
`
`dodecyl sulfate (CP, a negative control), according to one set of embodiments;
`
`[00018]
`
`FIGs. 6A-6C show drug levels of loteprednol etabonate in the palpebral
`
`conjunctiva (FIG. 6A), bulbar conjunctiva (FIG. 6B), and cornea (FIG. 6C) of New Zealand
`
`white rabbits after administration of prescription loteprednol etabonate, Lotemax®, or
`
`particles of loteprednol etabonate that were coated with Pluronic® F127, according to one set
`
`of embodiments;
`
`[00019]
`
`FIGs. 7A and 7B are physicochemical characterizations of CUR-1% F127
`
`particles according to one set of embodiments. FIG. 7A is a powder X-ray diffraction
`
`(Powder-XRD) diagram of F127, raw curcumin and CUR-1% F127 particles. FIG. 7B is a
`
`representative transmission electron microscope (TEM) image of CUR-1% F127 particles.
`
`[00020]
`
`FIGs. 8A and 8B are ensemble averaged geometric mean-squared displacements
`
`(<MSD>) of CUR-1% F127 particles, 200nm carboxylated polystyrene (PSCOOH)and
`
`200nm PEGylated polystyrene (PSPEG)particles in CVM (FIG. 8A) and humancystic
`
`fibrosis sputum (CFS) (FIG. 8B) as a function of time scale according to one set of
`
`embodiments. Data represent the ensemble average of five independent experiments, with n
`
`=> 100 for each experiment. Error bars indicate geometric standard error.
`
`[00021]
`
`FIG. 9 isa plot showing geometric ensemble effective diffusivity (<Deff>) at a
`
`time scale of 1 s for CUR particles formulated in different concentrations of F127 in human
`
`CVMaccording to one set of embodiments. Data represents the ensemble average ofat least
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`3 independent experiments, with n > 100 for each experiment. Error bars indicate geometric
`
`standarderror.
`
`FIGs. 10A-10C are plots showing diffusivity of CUR particles formulated with
`[00022]
`different Pluronics® in human CVM according to one set of embodiments. FIG. 10A shows
`
`distribution of <Deff> at a time scale of 1s with regards to the molecular weight (MW) of
`
`poly(ethylene glycol) (PEG) segment and poly(propylene oxide) (PPO) segmentof
`Pluronics®. Each data point represents a specific type of Pluronics®. PPO and PEG MW were
`
`estimated based on the molecular weight provided by the manufacturer. FIGs. 10 B-C show <
`
`Deff > at a time scale of Is as a function of the MW of (B) PEG or (C) PPO segments. Inset
`
`represents the same plot with linear scale of <Deff>, while R represents the correlation
`
`coefficient. Data represent the ensemble average ofat least three independent experiments,
`
`with n > 100 for each experiment. Error bars indicate geometric standard error.
`
`[00023]
`
`FIG. 11 is a plot showing cumulative release of CUR-1% F127 particles in
`
`phosphate buffered saline (pH=7.4) with octanol extraction according to one set of
`
`embodiments;
`
`[00024]
`
`FIG. 12 isa plot showing cumulative release from a dialysis bag of free TFV in
`
`solution vs. TFV-Zn particles in suspension into normal phosphate buffered saline according
`
`to one set of embodiments;
`
`[00025]
`
`FIGs. 13A-13B are images showing distribution of mucus penetrating / F127-
`
`coated TFV particles (FIG. 13A) and muco-adhesive / PVA-coated TFV particles (FIG. 13B)
`
`on flattened vaginal tissue from human-like estrus phase mice according to oneset of
`
`embodiments. Vaginal tissues were removed within 10 minutes of administration.
`
`[00026]
`
`FIGs. 14A-14D showthe transport rates of CP and MPP in mouseestrus phase
`
`CVM. (FIG. 14A) Representative trajectories for particles exhibiting effective diffusivities
`
`within one SEM ofthe ensemble averageat a time scale of 1 s. (FIG. 14B) Ensemble-
`
`averaged geometric mean square displacements (<MSD>) as a function of time scale. Data
`
`for particles on ex vivo mouse vaginal tissue (mCVM) compared to the sameparticles in ex
`
`vivo human CVM (hCVM) (S. K. Lai, Y. Y. Wang, K. Hida, R. Cone, J. Hanes,
`
`Nanoparticles reveal that human cervicovaginal mucusis riddled with pores larger than
`
`viruses. P Natl Acad Sci USA 107, 598-603 (2010)) and the theoretical diffusion rate of 110
`nm particles in water (~4 um’/s). (FIG. 14C) Distributions of the logarithms of individual
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`particle effective diffusivities (Der) at a time scale of 1 s, Data represent the ensemble
`
`average of three independent experiments, with n > 150 particles for each experiment.
`
`Diffusivity values to the left of the dotted line indicate particles with MSD valuesless than
`
`the particle diameter. (FIG. 14D) Percentage of particles capable of penetrating a 100 um-
`
`thick layer ofmouse CVM overtime, based on Fick’s Second Law of diffusion simulation of
`
`particles undergoing randomdiffusion, with diffusivities equal to the experimentally
`
`measureddiffusivities of the particles. FIG. 14E is a graphical depiction of vaginal drug
`
`delivery from gel, CP, and MPP formulations.
`
`[00027]
`
`FIGs. 15A-15B are plots showing the transport of nanoparticles on IE mouse
`
`vaginal tissue. Ensemble-averaged geometric mean squared displacements (<MDS>) as a
`
`function of time scale. (FIG. 15A) Data are shown for MPP and CPon ex vivo vaginal tissue
`
`of induced estrus (IE) and naturally cycling estrus phase mice. (FIG. 15B) Biodegradable
`
`MPPsonIE tissue were compared to non-degradable MPPs. Data represent the ensemble
`
`average of at least 3 independent experiments, with an average n > 150 particles and at least
`
`130 particles for each experiment. Data are presented as a mean with the standard error of the
`
`mean (SEM).
`
`[00028]
`
`FIG. 16 includes imagesillustrating particle distribution in the mouse vagina.
`
`Distribution of red fluorescent non-biodegradable and biodegradable CPs and MPPsin
`
`transverse cryosections of estrus phase and IE mouse vaginal tissue.
`
`Imagesare
`
`representative of n > 3 mice.
`
`[00029]
`
`FIG. 17 includes images and plots showing the quantification of vaginal
`
`nanoparticle coverage. Distribution of red fluorescent non-biodegradable and biodegradable
`
`CPs and MPPson flattened estrus phase mouse vaginaltissue. Insets are images of dark
`
`areas at higher magnification. Images are representative of the averages calculated for n > 3
`
`mice. Data are means + SEM. *P < 0.05, Student's t test.
`
`[00030]
`
`FIG. 18 includes images showing the cervical nanoparticle coverage. Cervical
`
`distribution of red fluorescent non-biodegradable and biodegradable CPs and MPPs onestrus
`
`phase mouse cervical tissue. Insets are images of dark areas at higher magnification. Images
`
`are representative of n> 3 mice. Data are means + SEM. *P < 0.05, Student’s t test.
`
`[00031]
`
`FIG. 19 includes images showing the effects of mucus removal on mucoadhesive
`
`nanoparticles. Distribution of red fluorescent non-biodegradable and biodegradable CPs in
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`transverse cryosections of mouse vaginal tissue with either an intact mucus layer (No
`
`treatment) or mucus removed by lavage and swabbing (Mucus removed). Images are
`
`representative of n > 3 mice.
`
`[00032]
`
`FIG. 20 includes images showing the particle distribution in the TE mouse vagina.
`
`Distribution of red fluorescent non-biodegradable CP and MPPintransverse cryosections of
`
`IE mousevaginaltissue. Images are representative of n > 3 mice.
`
`[00033]
`
`FIGs. 21A-21B are imagesand a plot showing the retention of non-biodegradable
`
`MPPsand CPsin the IE mouse cervicovaginal tract. (FIG. 21A) Overlay of particle
`
`fluorescence intensity and bright-field images for CPs and MPPs in whole cervicovaginal
`
`tract tissue. (FIG. 21B) Fraction of particles remaining over time based on quantification of
`
`particle fluorescence, Data are means + SEM (n = 7), *P < 0.05, Student’s t test.
`
`[00034]
`
`FIG. 22 includes images showingthe distribution and retention of a model drug,
`
`FITC, in the estrus mouse vagina delivered in gel form or encapsulated in biodegradable
`
`MPPs. Fluorescent images were taken of flattened mouse vaginal tissue after 24 h. Images
`
`are representative of n = 3 mice. Data are means + SEM. *P < 0.05, Student’s t test.
`
`[00035]
`
`FIG. 23 includes images showing the acute toxicity and cytokine concentrations
`
`with daily administration. Hematoxylin and eosin (H&E)-stained cross-sections of mouse DP
`
`vaginal tissue excised 24 h after intravaginal administration of 5% N9, PBS, CPs, MPPs, BD-
`
`CPs, and BD-MPPs. Scale bar applies to all images. Arrowheads point to clusters of
`
`neutrophils. Images are representative of n = 5 mice.
`
`[00036]
`
`FIG. 24 is a plot showing the cytokine concentrations with daily administration.
`
`Cytokine concentrations in DP mouse cervicovaginal lavage (CVL)after daily vaginal
`
`treatments for 7 days. Data are means + SEM. *P < 0.05, Student’s t test.
`
`[00037]
`
`FIG. 25 is a bar graph showing the relationship between the relative velocity of
`
`polystyrene particles coated with Pluronic® F127 in mucus and the density of the Pluronic®
`
`F127 molecules on the particle surface.
`
`Detailed Description
`
`[00038]
`
`Nanocrystals, compositions, and methodsthat aid particle transport in mucusare
`
`provided. In some embodiments, the compositions and methods involve making mucus-
`
`penetrating particles (MPP) without any polymeric carriers, or with minimal use of polymeric
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`carriers. The compositions and methods mayinclude, in some embodiments, modifying the
`
`surface coatings of particles formed of pharmaceutical agents that have a low water/aqueous
`
`solubility. Such methods and compositions can be used to achieve efficient transport of
`
`particles of pharmaceutical agents though mucusbarriers in the body for a wide spectrum of
`
`applications, including drug delivery, imaging, and diagnostic applications. In certain
`
`embodiments, a pharmaceutical composition including such particles is well-suited for
`
`administration routes involving the particles passing through a mucosalbarrier.
`
`[00039]
`
`In some embodiments, the particles described herein have a core-shell type
`
`arrangement. The core may comprise a solid pharmaceutical agent or a salt thereof having a
`
`relatively low aqueoussolubility. The core may be coated with a coating or shell comprising
`
`a surface-altering agent that facilitates mobility of the particle in mucus. As described in
`
`more detail below, in some embodiments the surface-altering agent may comprise a triblock
`
`copolymer comprising a hydrophilic block — hydrophobic block — hydrophilic block
`
`configuration. The molecular weights of each of the hydrophilic and hydrophobic blocks
`
`may be chosen to impart certain transport characteristics to the particles, such as increased
`
`transport through mucus.
`
`[00040]
`
`Non-limiting examples of particles are now provided. As shownin the illustrative
`
`embodiment of FIG.1, a particle 10 includes a core 16 (which maybe in the form of a
`
`particle, referred to herein as a core particle) and a coating 20 surrounding the core.
`
`In one
`
`set of embodiments, a substantial portion of the core is formed of one or more solid
`
`pharmaceutical agents (e.g., a drug, therapeutic agent, diagnostic agent, imaging agent) that
`
`can lead to certain beneficial and/or therapeutic effects. The core may be, for example, a
`
`nanocrystal (i.e., a nanocrystal particle) of a pharmaceutical agent. The core includes a
`
`surface 24 to which one or more surface-altering agents can be attached. For instance, in
`
`somecases, core 16 is surrounded by coating 20, which includes an inner surface 28 and an
`
`outer surface 32. The coating may be formed,at least in part, of one or more surface-altering
`
`agents 34, such as a polymer(e.g., a block copolymer), which may associate with surface 24
`
`of the core. Surface-altering agent 34 may be associated with the core particle by, for
`
`example, being covalently attached to the core particle, non-covalently attached to the core
`
`particle, adsorbed to the core, or attached to the core through ionic interactions, hydrophobic
`
`and/or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`10
`
`combinations thereof. In one set of embodiments, the surface-altering agents, or portions
`
`thereof, are chosen to facilitate transport of the particle through a mucosal barrier(e.g.,
`
`mucus or a mucosal membrane).
`
`[00041]
`
`In certain embodiments described herein, one or more surface-altering agents 34
`
`are oriented in a particular configuration in the coating of the particle. For example, in some
`
`embodiments in which a surface-altering agentis a triblock copolymer, such as a triblock
`
`copolymer having a hydrophilic block — hydrophobic block — hydrophilic block
`
`configuration, a hydrophobic block 36 may be oriented towards the surface of the core, and
`
`hydrophilic blocks 38 may be oriented away from the core surface (e.g., towards the exterior
`
`of the particle). The hydrophilic blocks may have characteristics that facilitate transport of
`
`the particle through a mucosal barrier, as described in more detail below.
`
`[00042]
`
`Particle 10 may optionally include one or more components40 suchastargeting
`
`moieties, proteins, nucleic acids, and bioactive agents which may optionally impart
`
`specificity to the particle. For example, a targeting agent or molecule (e.g., a protein, nucleic
`
`acid, nucleic acid analog, carbohydrate, or small molecule), if present, may aid in directing
`
`the particle to a specific location in the subject’s body. The location may be, for example, a
`
`tissue, a particular cell type, or a subcellular compartment. One or more components 40, if
`
`present, may be associated with the core, the coating, or both; e.g., they may be associated
`
`with surface 24 of the core, inner surface 28 of the coating, outer surface 32 of the coating,
`
`and/or embeddedin the coating. The one or more components 40 may be associated through
`
`covalent bonds, absorption, or attached through ionic interactions, hydrophobic and/or
`
`hydrophilic interactions, electrostatic interactions, van der Waals interactions, or
`
`combinationsthereof. In some embodiments, a component maybeattached (e.g., covalently)
`
`to one or more of the surface-altering agents of the coated particle using methods knownto
`
`those of ordinary skill in theart.
`
`[00043]
`
`It should be understood that components and configurations other than those
`
`shownin FIG. 1 or described herein may be suitable for certain particles and compositions,
`
`andthat not all of the components shown in FIG. 1 are necessarily present in some
`
`embodiments.
`
`[00044]
`
`In one set of embodiments, particle 10, when introduced into a subject, may
`
`interact with one or more componentsin the subject such as mucus,cells, tissues, organs,
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`11
`
`particles, fluids (e.g., blood), portions thereof, and combinations thereof. In some such
`
`embodiments, the coating of particle 10 can be designed to include surface-altering agents or
`
`other components with properties that allow favorable interactions (e.g., transport, binding,
`
`adsorption) with one or more materials from the subject. For example, the coating may
`
`include surface-altering agents or other components having a certain hydrophilicity,
`
`hydrophobicity, surface charge, functional group, specificity for binding, and/or density to
`
`facilitate or reduce particular interactions in the subject. One specific example includes
`
`choosing a certain hydrophilicity, hydrophobicity, surface charge, functional group,
`
`specificity for binding, and/or density of one or more surface-altering agents to reduce the
`
`physical and/or chemical interactions betweenthe particle and mucusof the subject, so as to
`
`enhance the mobility of the particle through mucus. Other examples are described in more
`
`detail below.
`
`[00045]
`
`In some embodiments, once a particle is successfully transported across a mucosal
`
`barrier (e.g., mucus or a mucosal membrane) in a subject, further interactions between the
`
`particle in the subject may take place. Interactions may take place, in some instances,
`
`through the coating and/or the core, and may involve, for example, the exchange of materials
`
`(e.g., pharmaceutical agents, therapeutic agents, proteins, peptides, polypeptides, nucleic
`
`acids, nutrients, e.g.) from the one or more components ofthe subject to particle 10, and/or
`
`from particle 10 to the one or more components of the subject. For example, in some
`
`embodiments in which the core is formed of or comprises a pharmaceutical agent, the
`
`breakdown,release and/ortransport of the pharmaceutical agent from the particle can lead to
`
`certain beneficial and/or therapeutic effects in the subject. As such, the particles described
`
`herein can be used for the diagnosis, prevention, treatment or managementof certain diseases
`
`or bodily conditions.
`
`[00046]
`
`Specific examples for the use of the particles described herein are provided below
`
`in the context of being suitable for administration to a mucosal barrier (e.g., mucusor a
`
`mucosal membrane) in a subject. It should be appreciated that while many of the
`
`embodiments herein are described in this context, and in the context of providing a benefit for
`
`diseases and conditionsthat involve transport of materials across a mucosal barrier, the
`
`invention is not limited as such andthe particles, compositions, kits, and methods described
`
`herein may be usedto prevent, treat, or manage other diseases or bodily conditions.
`
`

`

`WO 2013/166385
`
`PCT/US2013/039467
`
`12
`
`[00047] Mucusisa sticky viscoelastic gel that protects against pathogens, toxins, and
`
`debris at various points of entry into the body, including the eyes, nose, lungs, gastrointestinal
`
`tract, and female reproductive tract. Many synthetic nanoparticles are strongly mucoadhesive
`
`and becomeeffectively trapped in the rapidly-cleared peripheral mucuslayer, vastly limiting
`
`their distribution throughout the mucosal membrane as well as penetration toward the
`
`underlying tissue. The residence time of these trapped particles is limited by the turnoverrate
`
`of the peripheral mucus layer, which, depending on the organ, ranges from secondsto several
`
`hours. To ensure effective delivery of