(54)
`
`(75)
`
`Inventors: Raffaele Cammarano, Mount Hawthorn
`(AU); Felix Meiser, Kew (AU); Almar
`Postma, Balwyn (AU); Frank Caruso,
`Preston (AU)
`
`(73)
`
`Assignee: Iceutica Pty Ltd., Balcatta, Western
`Australia (AU)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 278 days.
`
`(21)
`
`(22)
`
`(86)
`
`(87)
`
`(65)
`
`(30)
`
`Appl. No.:
`
`12/306,948
`
`PCT Filed:
`
`Jun. 29, 2007
`
`PCT No.:
`
`PCT/AU2007/000910
`
`§ 371 (00)’
`(2), (4) Date:
`
`Dec. 7, 2009
`
`PCT Pub. No.: WO2008/000042
`
`PCT Pub. Date: Jan. 3, 2008
`
`Prior Publication Data
`
`US 2010/0092563 A1
`
`Apr. 15, 2010
`
`Foreign Application Priority Data
`
`Jun. 30, 2006
`
`(AU) .............................. .. 2006903527
`
`(51)
`
`(52)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`A61K 9/16
`A61K 31/40
`A61K 31/192
`A61K 31/196
`A61K 31/496
`A61K 31/4535
`A61K 31/551
`A61K 9/14
`US. Cl.
`CPC ........... .. A61K 31/192 (2013.01); A61K 31/496
`(2013.01); A61K 31/4535 (2013.01); A61K
`31/551(2013.01);A61K9/1617(2013.01);
`A61K 9/143 (2013.01); A61K31/196 (2013.01)
`USPC ......... .. 424/493; 514/420; 514/567; 514/569;
`514/570
`
`(58)
`
`Field of Classi?cation Search
`None
`See application ?le for complete search history.
`
`(12) United States Patent
`Cammarano et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,808,751 B2
`Aug. 19, 2014
`
`US008808751B2
`
`METHODS FOR THE PREPARATION OF
`BIOLOGICALLY ACTIVE COMPOUNDS IN
`NANOPARTICULATE FORM
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,145,684 A *
`5,202,129 A
`5,298,262 A *
`2002/0047058 Al *
`
`9/1992 Liversidge et al. ......... .. 424/489
`4/1993 Samejima et al.
`3/1994 Na et al. ...................... .. 424/489
`4/2002 Verhoffet al. .
`241/26
`
`. . . . . .. 264/5
`2003/0137067 A1* 7/2003 Cooper et al. . . . . .
`424/465
`2003/0228357 A1* 12/2003 Johnson et al. ...... ..
`241/21
`2004/0173696 A1* 9/2004 Cunningham et al. .
`2007/0059356 A1* 3/2007 Almarsson et al. ......... .. 424/464
`
`FOREIGN PATENT DOCUMENTS
`
`0600528
`EP
`WO WO2007/070851
`
`6/1994
`6/2007
`
`OTHER PUBLICATIONS
`
`Tsuzuki, T.; Pethick, K.; McCormick, P. Synthesis of CaCO3
`nanoparticles by mechanochemical processing. Journal of
`Nanoparticle Research, v01. 2, p. 375-380, 2000.*
`Tsuzuki, T.; Pirault, E.; McCormick, P. Mechanochemical Synthesis
`of Gadolinium Oxide Nanoparticles. Nanostructured Materials, v01.
`11,N0.1,p.125-131,1999.*
`Tsuzuki, T.; McCormick, P. Mechanochemical synthesis of
`nanoparticles. Journal of Materials Science, vol. 39, p. 5143-5146,
`2004*
`Grigorieva, T. F; Barinova, A. P.; Lyakhov, N. Z. Mechanosynthesis
`ofnanocomposites. Journal ofNanopaIticle Research, v01. 5, p. 439
`453, 2003.*
`Tsuzuki, T.; McCormick, P. Mechanochemical Synthesis of Metal
`Sulphide Nanoparticles. Nanostructured Materials, v01. 12, p. 75-78,
`1999.*
`Of?ce Action in corresponding Canadian Application 2,653,384,
`dated Mar. 10, 2014, pp. 1-3.
`Juhnke, M. et al., “Nanoparticles of soft materials by high-energy
`milling at low temperatures,” 7”“ world congress of chemical engi
`neering, Glasgowzpp. 1-10 (2005).
`
`* cited by examiner
`
`Primary Examiner * Susan Tran
`Assistant Examiner * Jessica Worsham
`(74) Attorney, Agent, or Firm * Fish & Richardson PC.
`
`ABSTRACT
`(57)
`A method for producing a composition comprising nanopar
`ticles of a biologically active compound, comprising the step
`of: dry milling a solid biologically active compound and a
`millable grinding compound in a mill comprising a plurality
`of milling bodies, for a time period suf?cient to produce a
`solid dispersion comprising nanoparticles of the biologically
`active compound dispersed in an at least partially milled
`grinding compound is described as are various compositions
`produced using such methods.
`
`14 Claims, 26 Drawing Sheets
`
`LUPIN EX. 1020
`Lupin v. iCeutica
`US Patent No. 8,999,387
`
`Page 1
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 1 0f 26
`
`US 8,808,751 B2
`
`141
`
`12
`
`101
`
`00 1
`
`surface area mzlg Q
`
`
`
`
`
`N
`
`O
`
`50
`
`1O
`15
`3O
`volume % of DCA in salt milling (washed samples)
`
`5
`
`FIG. 1
`
`Page 2
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 2 0f 26
`
`US 8,808,751 B2
`
`—'number weighted
`—- — Intensity weighted
`
`Page 3
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 3 0f 26
`
`US 8,808,751 B2
`
`Page 4
`
`

`
`U S. Patent
`
`Aug. 19, 2014
`
`Sheet 4 0f 26
`
`US 8,808,751 B2
`
`Vol %
`
`SEM post washing
`
`TEM post washing
`
`15
`
`30
`
`50
`
`Page 5
`
`

`
`U S. Patent
`
`Aug. 19, 2014
`
`Sheet 5 0f 26
`
`US 8,808,751 B2
`
`FIG. 6
`
`Page 6
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 6 0f 26
`
`US 8,808,751 B2
`
`FIG. 7
`
`Page 7
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 7 0f 26
`
`US 8,808,751 B2
`
`20.00
`
`15.00-l
`
`/
`
`
`
`mglL dissolved 10.00 f/
`
`"0— Nana—Drug
`+ APl - commercial
`
`
`
`mgIL dissolved
`
`5.00
`
`0.00 I
`O
`
`100
`
`200
`time (min)
`
`300
`
`460
`
`50
`
`100
`
`200
`>150
`time (min)
`
`250
`
`300
`
`350
`
`FIG. 8A
`
`FIG. SB
`
`Comparison of the dissolution of particulate raloxifene HCI (4-) with commercial
`raloxifene HCI(AF) in simulated gastric fluid (a) and in simulated intestinal fluid (b).
`
`Page 8
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 8 0f 26
`
`US 8,808,751 B2
`
`FIG. 9A
`
`FIG 9B
`
`FIG. 9C
`
`SEM images of the micron and nano structure of (a) commercial raloxifene HCl,
`(b) milled after 15 min, (0) and (d) washed and dried prior to milling.
`
`Page 9
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 9 0f 26
`
`US 8,808,751 B2
`
`Hamster {rim}
`FIG. 10
`
`EB
`
`
`
`Number (*8)
`
`
`
`Heat FIow(W/g)
`
`Temperature (EC)
`
`Un'welsalV 4.06TA1nstrums1ts
`
`FIG. 11
`
`Page 10
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 10 0f 26
`
`US 8,808,751 B2
`
`— ~— ~— Ralax HG!
`
`-— Relax HCI washed
`
`2
`
`Page 11
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 11 0f 26
`
`US 8,808,751 B2
`
`H20
`
`1
`
`1
`
`1
`
`14
`dsnmso
`
`ems
`
`CTAB
`
`47 5
`
`12
`
`ll,
`
`1(1
`
`,
`
`9
`
`‘
`
`7
`
`6
`
`8
`
`__
`
`3
`
`2
`
`1
`
`0
`
`4
`
`5
`ppm
`
`1H NMR (d6-DMSO) of particulate raloxifene HCl after milling, washing and drying
`(residual CTAB surfactant observed in spectrum at a level of 2.1 mol%; water
`present in d6—DMSO).
`
`FIG. 13
`
`Page 12
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 12 0f 26
`
`US 8,808,751 B2
`
`Raloxifene HCI nano
`Raloxifene HCl
`
`Wmunumber
`
`IR spectra of raloxifene HCI commercial (initially higher absorbance) and post
`milling and washing particulate raloxifene HCI (initially lower absorbance).
`
`FIG. 14
`
`Page 13
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 13 0f 26
`
`US 8,808,751 B2
`
`-—Ralox HCI
`--~RAL HCL NaCl SM washe
`Ral HCl SM NaCl
`
`Page 14
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 14 0f 26
`
`US 8,808,751 B2
`
`Milled and washed sample
`//
`
`Absnrbance
`
`” ' 5466: ”
`Wavenumber
`
`FIG. 17
`
`Page 15
`
`

`
`U S. Patent
`
`Aug. 19, 2014
`
`Sheet 15 0f 26
`
`US 8,808,751 B2
`
`|G.18 "
`
`* iFlG.18‘D
`
`Page 16
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 16 0f 26
`
`US 8,808,751 B2
`
`MHZ
`
`HO
`
`OH
`
`H
`
`HO
`
`TRIS
`
`m
`0
`
`0 N
`
`m :1
`
`\\ [>0
`,8\
`CH3(CH2)1DCHZO
`ONE
`
`sodium dodecyl sulfate
`
`Mn = 25-30,000 g moi-1
`
`Plasdone 8-630
`
`FIG. 19
`
`FIG. 20A
`
`FIG. ZOB
`
`Page 17
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 17 0f 26
`
`US 8,808,751 B2
`
`4000
`
`3500 -
`
`3000 —
`
`| — Ralox H0!
`
`Z - Ralox Base
`3 "—- Ralox salt milled and washed
`4 —-~ Ralox Base salt milled
`
`Page 18
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 18 0f 26
`
`US 8,808,751 B2
`
`Abscrbanoa
`
`l
`
`Red:
`
`Raloxifene free base
`
`2. Green: Raloxifene HCL commercial
`
`3 Blue:
`
`Raloxifene free base and NaCl after ball mill
`
`I: I G
`
`4 Black: Raloxifene washed (once)
`
`Page 19
`
`

`
`US. Patent
`
`Aug. 19, 2014
`
`Sheet 19 0f 26
`
`US 8,808,751 B2
`
`1) Group A
`
`Order of Administration: Occasion 1 = Test Substance 1;
`Occasion 2 = Test Substance 2
`
`*Griseida Dec 1
`*we 0 cc 1
`*Ginger Dec 1
`mgr-“Champ Dec 1
`J‘Gh'unk Dec 1
`#050808 Occ 1
`
`FIG. 23A
`
`FIG. 23B
`
`Page 20
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 20 of 26
`
`US 8,808,751 B2
`
`Graphs of individual Concentration vs Time Data
`
`2) Group B
`Order of Administration: Occasion 1 = Test Substance 2; Occasion 2 =
`Test Substance 1
`
`._n_C4060G ocm
`_._.c306a5 Occ 2
`
`FIG. 23C
`
`-g-- CEOSOB OCG1
`._;._. Grizz Oct: 1
`_i—. C10608 0:: 1
`..»..-,_c2o6<2e occ1
`..,¢..c3osus O::c1
`
`..~,;... (220605 ()0: 2
`
`FIG. 23D
`
`Page 21
`
`Page 21
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 21 of 26
`
`US 8,808,751 B2
`
`Mean Concentration vs Time Data
`
`Raloxifene Plasma Concentration (nglmL)
`Test Substance 1
`Test Substance 2
`(commercial API
`(iceutica nanoparticles)
`Mean
`
`Mean (SD) Concentration vs Time Data All
`Animals
`
`FIG. 24A
`
`-9- Iceutica Nanopartides
`
`—-— Corrmerzial API
`
`FIG. 24B
`
`Page 22
`
`Page 22
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 22 of 26
`
`US 8,808,751 B2
`
`Mean Pharmacokinetic Data -— Test Substances 1 and 2
`
`Test Substance 1:
`
`Mean
`SD
`
`Cmax
`
`Tmaxa
`
`(ngImL)
`
`12.26
`5.47
`
`(h)
`
`1.00
`—--
`
`kelim
`
`(h")
`
`0.4191
`0.1714
`
`Test Substance 2:
`
`Mean
`
`SD
`
`7.69
`
`4.54
`
`1.50
`
`0.3050
`
`—--
`
`0.1576
`
`thalf
`
`(h)
`
`1.91
`0.77
`
`3.06
`
`2.12
`
`AUCO-1
`
`AUCO-inf
`
`(ng.hImL)
`
`(ng.h/mL)
`
`33.39
`20.54
`
`42.12 1
`21.82
`
`21.36
`
`16.79
`
`36.62
`
`23.45
`
`‘median
`
`Page 23
`
`Page 23
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 23 of 26
`
`US 8,808,751 B2
`
`Comparison of Cmax and AUCo_. Results: Test Substances 1 and 2
`
`
`- Pharmacokinetic Parameter
`Dog Name
`(Group)
`
`Test Substance
`
`Test Substance
`
`
`
`2
`
`1
`1
`
`
` 2
`
`
`
`
`
`Griselda (A)
`
`Ice (A)
`
`Ginger (A)
`
`Champ (A)
`
`Chuck (A)
`
`csosoe (A)
`
`C60606 (B)
`
`Grizz (B)
`
`C10606 (B)
`
`czosos (B)
`
`csoeoe (B)
`
`C40606 (B)
`
`FIG. 26A
`
`Page 24
`
`Page 24
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 24 of 26
`
`US 8,808,751 B2
`
`AU CO-t All Data
`
`*
`
`
`
`
`
`
`
`30.0
`
`25.0
`
`20.0
`
`
`
`AUCO-t(ng.hlmL) fa6OO
`
`0.0
`
`
`
`A
`
`A M Gtisexda (A)
`Ice (A)
`v-elem Ginger (A)
`—o—- Charrp (A)
`
`4 —o— Chuck (A)
`———I——- CSOBDB (A)
`-—|—- 060605 (B)
`
`Grizz (B)
`——-—~ C106UB (B)
`C20606 (B)
`CBOBO6 (B)
`C4060B (B)
`
`
`
`
`
`
`
`
`
`Test Substance
`
`FIG. 26B
`
`
`
`~ »~~,» Griselda (A)
`
`-—-)~€-- Ice (A)
`——-916-—— Ginger (A)
`
`—-o—- Champ (A)
`-0- Chuck (A)
`-—I——- C50606 (A)
`—-i-- C60606 (B)
`Grizz (B)
`
`
`
`----—-— C10606 (B)
`C20606 (B)
`C306D6 (B)
`040606 (B)
`
`6
`
`
`
`FIG. 26C
`
`Page 25
`
`Page 25
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 25 of 26
`
`US 8,808,751 B2
`
` .
`
`Efifinaanmfinafiwuuu
`60 minutes
`
`30 minutes
`
`45 minutes
`
`(~ 700 nm)
`
`(- 500 nm)
`
`(< 50 nm)
`
`FIG. 27
`
`
`
`Mag WD Spa
`100D0><5.3 mm 1.o 5.0 KVETD
`
`FIG. 28A
`
`FIG. 28B
`
`Page26
`
`Page 26
`
`

`
`U.S. Patent
`
`Aug. 19, 2014
`
`Sheet 26 of 26
`
`US 8,808,751 B2
`
`15.00
`
`10.00
`
`
`
`mgILdissolved
`
`-V-A—APlimi||ed in NaCl matrix
`
`—O—- API Raloxifene HCI
`—I— AP milled Lactose matrix
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`160
`
`180
`
`time (min)
`
`FIG. 29
`
`
`
`.
`..
`A
`.
`.
`.
`V Den
`ag WD span
`smmax 5,4 mm 1.0 5.0 kVETD
`
`.34; w
`£2
`‘Q '
`Mag WD Spol HV Del
`oooux 5.4 mm 1.0 5.0 KVETD
`
`FIG. 30A
`
`FIG. 30B
`
`Page 27
`
`Page 27
`
`

`
`US 8,808,751 B2
`
`1
`METHODS FOR THE PREPARATION OF
`BIOLOGICALLY ACTIVE COMPOUNDS IN
`NANOPARTICULATE FORM
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods for the prepara-
`tion of biologically active compounds in nanoparticulate
`form. The invention also relates to biologically active com-
`pounds in nanoparticulate form produced by said methods, to
`compositions comprising such compounds, to medicaments
`produced using said biologically active compounds in nano-
`particulate form and/or compositions, and to methods of
`treatment of an animal, including man, using a therapeuti-
`cally effective amount of said biologically active compounds
`administered by way of said medicaments.
`
`BACKGROUND
`
`Poorbioavailability is a significant problem encountered in
`the development of therapeutic compositions, particularly
`those compounds containing a biologically active compound
`that is poorly soluble in water at physiological pH. An active
`compound’s bioavailability is the degree to which the active
`compound becomes available to the target tissue in the body
`after systemic administration through, for example, oral or
`intravenous means. Many factors affect bioavailability,
`including the form of dosage and the solubility and dissolu-
`tion rate of the active compound.
`Poorly and slowly water-soluble compounds tend to be
`eliminated from the gastrointestinal
`tract before being
`absorbed into the circulation. In addition, poorly soluble
`active agents tend to be disfavored or even unsafe for intra-
`venous administration due to the risk of particles of agent
`blocking blood flow through capillaries.
`It is known that the rate of dissolution of a particulate drug
`will
`increase with increasing surface area. One way of
`increasing surface area is decreasing particle size. Conse-
`quently, methods ofmaking finely divided or sized drugs have
`been studied with a view to controlling the size and size range
`of drug particles for pharmaceutical compositions.
`For example, dry milling techniques have been used to
`reduce particle size and hence influence drug absorption.
`However, in conventional dry milling the limit of fineness is
`reached generally in the region of about 100 microns (100,
`000 nm), at which point material cakes on the milling cham-
`ber and prevents any further diminution ofparticle size. Alter-
`natively, wet grinding may be employed to reduce particle
`size, but flocculation restricts the lower particle size limit to
`approximately 10 microns (10,000 nm). The wet milling pro-
`cess, however, is prone to contamination, thereby leading to a
`bias in the pharmaceutical art against wet milling. Another
`alternative milling technique, commercial airj et milling, has
`provided particles ranging in average size from as low as
`about 1 to about 50 microns (1,000-50,000 nm).
`There are several approaches currently used to formulate
`poorly soluble active agents. One approach is to prepare the
`active agent as a soluble salt. Where this approach cannot be
`employed, alternate (usually physical) approaches are
`employed to improve the solubility of the active agent. Alter-
`nate approaches generally subject the active agent to physical
`conditions that change the agent’s physical and or chemical
`properties to improve its solubility. These include process
`technologies such as micro-ionisation, modification of crys-
`tal or polymorphic structure, development of oil based solu-
`tions, use of co-solvents, surface stabilizers or complexing
`agents, micro-emulsions, super critical fluid and production
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`of solid dispersions or solutions. More than one of these
`processes may be used in combination to improve formula-
`tion of a particular therapeutic compound.
`These techniques for preparing such pharmaceutical com-
`positions tend to be complex. By way of example, a principal
`technical difliculty encountered with emulsion polymeriza-
`tion is the removal of contaminants, such as unreacted mono-
`mers or initiators (which may have undesirable levels of
`toxicity), at the end of the manufacturing process.
`Another method of providing reduced particle size is the
`formation of pharmaceutical drug microcapsules, which
`techniques include micronizing, polymerisation and co-dis-
`persion. However, these techniques suffer from a number of
`disadvantages including at least the inability to produce suf-
`ficiently small particles such as those obtained by milling, and
`the presence of co-solvents and/or contaminants such as toxic
`monomers which are diflicult to remove, leading to expensive
`manufacturing processes.
`Over the last decade, intense scientific investigation has
`been carried out to improving the solubility of active agents
`by converting the agents to ultra fine powders by methods
`such as milling and grinding. These techniques may be used
`to increase the dissolution rate of a particulate solid by
`increasing the overall surface area and decreasing the average
`particle size.
`U.S. Pat. No. 6,634,576 discloses examples ofwet-milling
`a solid substrate, such as a pharmaceutically active com-
`pound, to produce a “synergetic co-mixture”.
`lntemational Patent Application PCT/AU2005/001977
`(Nanoparticle Composition(s) and Method for Synthesis
`Thereof) describes, inter alia, a method comprising the step of
`contacting a precursor compound with a co-reactant under
`mechanochemical synthesis conditions wherein a solid-state
`chemical reaction between the precursor compound and the
`co-reactant produces therapeutically active nanoparticles dis-
`persed in a carrier matrix. Mechanochemical synthesis, as
`discussed in lntemational Patent Application PCT/AU2005/
`001977, refers to the use of mechanical energy to activate,
`initiate or promote a chemical reaction, a crystal structure
`transformation or a phase change in a material or a mixture of
`materials, for example by agitating a reaction mixture in the
`presence of a milling media to transfer mechanical energy to
`the reaction mixture, and includes without limitation “mecha-
`nochemical activation”, “mechanochemical processing”,
`“reactive milling”, and related processes.
`The present invention provides methods for the preparation
`of biologically active compounds in nanoparticulate form,
`which ameliorate some of the problems attendant with prior
`technologies, or provides an alternative thereto.
`As an example of the need for such novel compounds and
`methods
`for synthesizing them, consider osteoporosis.
`Osteoporosis describes a group of diseases which arises from
`diverse etiologies, but which are characterized by the net loss
`ofbone mass per unit volume. The consequence ofthis loss of
`bone mass and resulting bone fracture is the failure of the
`skeleton to provide adequate support for the body. One of the
`most common types of osteoporosis is associated with meno-
`pause. Most women lose from about 20% to about 60% ofthe
`bone mass in the trabecular compartment of the bone within
`3 to 6 years after the cessation of menses. This rapid loss is
`generally associated with an increase of bone resorption and
`formation. However, the resorptive cycle is more dominant
`and the result is a net loss of bone mass. Osteoporosis is a
`common and serious disease among postmenopausal women.
`The most generally accepted method for the treatment of
`postmenopausal osteoporosis
`is
`estrogen replacement
`therapy. Although therapy is generally successful, patient
`
`Page 28
`
`Page 28
`
`

`
`US 8,808,751 B2
`
`3
`compliance with the therapy is low, primarily because estro-
`gen treatment frequently produces undesirable side effects.
`An additional method of treatment would be the administra-
`
`tion of a bisphosphonate compound, such as, for example,
`FosamaxTM (Merck & Co., Inc.).
`Throughout premenopausal time, most women have less
`incidence of cardiovascular disease than men ofthe same age.
`Following menopause, however, the rate of cardiovascular
`disease in women slowly increases to match the rate seen in
`men. This loss of protection has been linked to the loss of
`estrogen and, in particular, to the loss of estrogen’s ability to
`regulate the levels of serum lipids. The nature of estrogen’s
`ability to regulate serum lipids is not well understood, but
`evidence to date indicates that estrogen can up regulate the
`low density lipid (LDL) receptors in the liver to remove
`excess cholesterol. Additionally, estrogen appears to have
`some effect on the biosynthesis of cholesterol, and other
`beneficial effects on cardiovascular health.
`
`It has been reported in the literature that serum lipid levels
`in postmenopausal women having estrogen replacement
`therapy return to concentrations found in the premenopausal
`state. Thus, estrogen would appear to be a reasonable treat-
`ment for this condition. However, the side effects of estrogen
`replacement therapy are not acceptable to many women, thus
`limiting the use of this therapy. An ideal therapy for this
`condition would be an agent which regulates serum lipid
`levels in a manner analogous to estrogen, but which is devoid
`of the side effects and risks associated with estrogen therapy.
`A number of structurally unrelated compounds are capable
`of interacting with the estrogen receptor and producing
`unique in vivo profiles. Compounds with in vivo profiles
`typical of a “pure” antagonist (for example, ICI 164,384) or of
`a relatively “pure” agonist (for example, 17[3-estradiol) rep-
`resent opposite ends of a spectrum in this classification.
`Between these two extremes lie the SERMs (“selective estro-
`gen receptor modulator”), characterized by clinical and/or
`preclinical selectivity as full or partial agonists in certain
`desired tissues (for example, bone), and antagonists or mini-
`mal agonists in reproductive tissues. Within this pharmaco-
`logic class, individual SERMs may be further differentiated
`based on profiles of activity in reproductive tissues.
`Raloxifene, a second generation SERM, displays poten-
`tially useful selectivity in uterine tissue with apparent advan-
`tages over triphenylethylene-based estrogen receptor ligands.
`As such, raloxifene appears to be well-suited at least for the
`treatment of postmenopausal
`complications,
`including
`osteoporosis and cardiovascular disease. It is anticipated that,
`as further advances are made in the pharmacology and
`molecular biology of estrogen receptor active agents, further
`subclassifications of SERMs may evolve in the future along
`with an increased understanding of the therapeutic utility of
`these novel classes of estrogemc compounds.
`The advancement of raloxifene has been hampered by its
`physical characteristics, particularly low solubility, which
`affects bioavailability. Accordingly, any improvement in the
`physical characteristics of raloxifene would potentially offer
`more beneficial therapies. In particular, it would be a signifi-
`cant contribution to the art to provide forms of raloxifene
`which have increased solubility, methods of preparation of
`such forms, pharmaceutical formulations comprising such
`forms, and methods of use of such formulations.
`Although the background to the present invention is dis-
`cussed in the context of improving the bioavailability of com-
`pounds that are poorly or slowly water soluble, the applica-
`tions ofthe methods ofthe present invention are not limited to
`such, as is evident from the following description of the
`invention.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`
`Further, although the background to the present invention
`is largely discussed in the context of improving the bioavail-
`ability of therapeutic or pharmaceutical compounds,
`the
`applications of the methods of the present invention are
`clearly not limited to such. For example, as is evident from the
`following description, applications of the methods of the
`present invention include but are not limited to: veterinary
`therapeutic applications and agricultural chemical applica-
`tions, such as pesticide and herbicide applications.
`
`SUMMARY OF THE INVENTION
`
`The present invention is directed to the unexpected discov-
`ery that biologically active compounds in nanoparticulate
`form can be produced by dry milling solid biologically active
`compound together with a millable grinding compound, such
`that the resulting nanoparticulate biologically active com-
`pound dispersed in milled grinding compound resists
`reagglomeration.
`Thus, in one aspect, the present invention comprises a
`method for producing a biologically active compound in
`nanoparticulate form, the method comprising the step of:
`dry milling a mixture of a solid biologically active com-
`pound and a millable grinding compound, in a mill com-
`prising a plurality of milling bodies, to produce a solid
`dispersion or solution comprising nanoparticulate bio-
`logically active compound dispersed in at least partially
`milled grinding compound.
`The term millable means that the grinding compound is
`capable of being physically degraded under the dry milling
`conditions ofthe method ofthe invention. In one embodiment
`
`of the invention, the milled grinding compound is of a com-
`parable particle size to the nanoparticulate biologically active
`compound.
`Without wishing to be bound by theory, it is believed that
`the physical degradation of the millable grinding compound
`affords the advantage of the invention by acting as a more
`effective diluent than grinding compounds of a larger particle
`size.
`
`In a highly preferred form, the grinding compound is
`harder than the biologically active compound, and is thus
`capable of physically degrading such under the dry milling
`conditions of the invention. Again without wishing to be
`bound by theory, under these circumstances it is believed that
`the millable grinding compound affords the advantage of the
`present invention through a second route, with the smaller
`particles of grinding compound produced under the dry mill-
`ing conditions enabling the production of smaller particles of
`biologically active compound.
`The solid dispersion or solution may then be separated
`from the milling bodies and removed from the mill.
`In a preferred aspect, the grinding compound is separated
`from the dispersion or solution. In one aspect, where the
`grinding compound is not fully milled, the unmilled grinding
`compound is separated from the nanoparticulate biologically
`active compound. In a further aspect, at least a portion of the
`milled grinding compound is separated from the nanoparticu-
`late biologically active compound.
`The milling bodies are essentially resistant to fracture and
`erosion in the dry milling process.
`The quantity of the grinding compound relative to the
`quantity of biologically active compound in nanoparticulate
`form, and the extent of physical degradation of the grinding
`compound, is sufiicient to inhibit reagglomeration of the bio-
`logically active compound in nanoparticulate form. The
`
`Page 29
`
`Page 29
`
`

`
`US 8,808,751 B2
`
`5
`grinding compound is not chemically reactive with the phar-
`maceutical compound under the milling conditions of the
`invention.
`
`In additional aspects, the present invention also relates to
`biologically active compounds in nanoparticulate form pro-
`duced by said methods, to compositions comprising said
`compounds, to medicaments produced using said biologi-
`cally active compounds in nanoparticulate form and/or said
`compositions, and to methods of treatment of an animal,
`including man, using a therapeutically effective amount of
`said biologically active compounds administered by way of
`said medicaments.
`
`Medicaments of the invention may comprise only the bio-
`logically active compounds in nanoparticulate form or, more
`preferably, the biologically active compounds in nanoparticu-
`late form may be combined with one or more pharmaceuti-
`cally acceptable carriers, as well as any desired excipients or
`other like agents commonly used in the preparation of medi-
`caments.
`
`While the method of the present invention has particular
`application in the preparation of poorly water-soluble bio-
`logically active compounds in nanoparticulate form,
`the
`scope of the invention is not limited thereto. For example, the
`method of the present invention enables production of highly
`water-soluble biologically active compounds in nanoparticu-
`late form. Such compounds may exhibit advantages over
`conventional compounds by way of, for example, more rapid
`therapeutic action or lower dose. In contrast, wet grinding
`techniques utilizing water (or other comparably polar sol-
`vents) are incapable of being applied to such compounds, as
`the particles dissolve appreciably in the solvent.
`As will be described subsequently, selection of an appro-
`priate grinding compound affords particular highly advanta-
`geous applications of the method of the present invention.
`Some grinding compounds appropriate for use in the inven-
`tion are readily separable from the biologically active com-
`pound in nanoparticulate form by methods not dependent on
`particle size (such methods being inappropriate due to the
`degradation of the grinding compound). For example, select-
`ing an appropriate grinding compound that also possesses
`solubility properties different from the biologically active
`compound in nanoparticulate form allows separation of the
`two by relatively straightforward selective dissolution tech-
`niques. Examples of such grinding compounds are provided
`in the detailed description of the invention. Thus, a particu-
`larly advantageous application ofthe method ofthe invention
`is the use of a water-soluble salt as a grinding compound in
`conjunction with a poorly water-soluble biologically active
`compound.
`Again, as will be described subsequently, a highly advan-
`tageous aspect ofthe present invention is that certain grinding
`compounds appropriate for use in the method ofthe invention
`are also appropriate for use in a medicament. The present
`invention encompasses methods for the production of a medi-
`cament incorporating both the biologically active compound
`in nanoparticulate form and at least a portion of the grinding
`compound, medicaments so produced, and methods of treat-
`ment of an animal, including man, using a therapeutically
`effective amount of said biologically active compounds by
`way of said medicaments.
`Analogously, as will be described subsequently, a highly
`advantageous aspect of the present invention is that certain
`grinding compounds appropriate for use in the method of the
`invention are also appropriate for use in a carrier for an
`agricultural chemical, such as a pesticide or a herbicide. The
`present invention encompasses methods for the production of
`an agricultural chemical composition incorporating both the
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`biologically active compound in nanoparticulate form and at
`least a portion of the grinding compound, and agricultural
`chemical compositions so produced.
`The agricultural chemical compound may include only the
`biologically active compound in nanoparticulate form
`together with the milled grinding compound or, more prefer-
`ably, the biologically active compounds in nanoparticulate
`form and milled grinding compound may be combined with
`one or more pharmaceutically acceptable carriers, as well as
`any desired excipients or other like agents commonly used in
`the preparation of medicaments.
`Analogously, the agricultural chemical composition may
`include only the biologically active compound in nanopar-
`ticulate form together with the milled grinding compound or,
`more preferably, the biologically active compounds in nano-
`particulate form and milled grinding compound may be com-
`bined with one or more carriers, as well as any desired excipi-
`ents or other like agents commonly used in the preparation of
`agricultural chemical compositions.
`In one particular form of the invention, the grinding com-
`pound is both appropriate for use in a medicament and readily
`separable from the biologically active compound in nanopar-
`ticulate form by methods not dependent on particle size. Such
`grinding compounds are described in the following detailed
`description of the invention. Such grinding compounds are
`highly advantageous in that they afford significant flexibility
`in the extent to which the grinding compound may be incor-
`porated with the biologically active compound in nanopar-
`ticulate form into a medicament.
`
`In one aspect, the invention provides novel formulations of
`raloxifene. Raloxifene is [6-hydroxy-2-(4-hydroxyphenyl)
`benzol[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl-,
`and is also known as 6-hydroxy-2-(4-hydrophenyl)-3-[4-(2-
`piperidinoethoxy)-benzoyl]benzo- [b] -thiophene.
`Other
`names for raloxifene may also be found in the literature. The
`structural formula for raloxifene is illustrated below:
`
` HO
`
`This invention provides raloxifene, or a pharmaceutically
`acceptable salt or solvate thereof, in particulate form having a
`mean particle size ofbetween about 10 nm and about 500 nm.
`The invention further provides methods for producing said
`particulate raloxifene, pharmaceutically acceptable salt or
`solvate thereof.
`
`The invention also provides pharmaceutical compositions
`comprising or formulated using the said particulate ralox-
`ifene, or pharmaceutically acceptable salt or solvate thereof.
`The present invention further provides the use of the said
`particulate raloxifene, or pharmaceutically acceptable salt or
`solvate thereof, in the manufacture of a pharmaceutical com-
`position for alleviating human pathologies,
`including
`osteoporosis,
`serum lipid
`lowering,
`and
`inhibiting
`endometriosis, uterine fibrosis, and breast cancer.
`The present invention further provides the use of such
`compositions comprising or formulated using the said ralox-
`ifene, or pharmaceutically acceptable salt or solvate thereof,
`
`Page 30
`
`Page 30
`
`

`
`US 8,808,751 B2
`
`7
`for alleviating human pathologies, including osteoporosis,
`serum lipid lowering, and inhibiting endometriosis, uterine
`fibrosis, and breast cancer.
`In one aspect, then, the invention provides a method for
`producing a composition comprising nanoparticles of a bio-
`logically active compound, the method comprising the step
`of:
`
`5
`
`dry milling a solid biologically active compou