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`
`US005399363A
`Patent Number:
`Date of Patent:
`
`5,399,363
`Mar. 21, 1995
`
`United States Patent [191
`Liversidge et al.
`
`[11]
`[45]
`
`[54]
`
`[75]
`
`[73]
`
`[21]
`[22]
`
`[63]
`
`[51]
`[52]
`[53]
`[56]
`
`SURFACE MODIFIED ANTICANCER
`NANOPARTICLES
`
`Inventors: Gary G. Liversidge; Elaine
`Liversidge, both of West Chester;
`Framed P. Sarpotdar, Malvern, all of
`Pa.
`
`Assignee: Eastman Kodak Company,
`Rochester, NY.
`
`Appl. No.: 908,125
`
`Filed:
`
`Jul. 1, 1992
`
`Related US. Application Data
`Continuation-impart of Ser. No. 647,105, Jan. 25, 1991,
`Pat. No. 5,145,684.
`
`Int. Cl.6 . . . . .
`
`. . . . . . . . . . . . . . .. A61K 9/_ 14
`
`US. Cl. ......................... .. 424/490; 424/489
`Field of Search ............... .. 424/490, 487; 514/ 352
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`2,671,750 3/ 1954 Macek ............................... .. 514/ 179
`3,881,020 4/1975 Nakamura et al. .
`514/619
`
`4,107,288 8/1978 Oppenheim . . . . . . . . .
`
`. . . . .. 424/22
`
`4,225,581 9/1980 Kreuter et al.
`4,269,821 5/ 1981 Kreuter et a1.
`4,540,602 9/ 1985 Motoyarna .... ..
`
`.. 424/88
`424/489
`427/213.31
`
`4,826,689 5/1989 Violanto . . . . . .
`
`. . . .. 424/489
`
`4,851,421 7/1989 Iwasaki et a1. ..
`5,049,322 9/1991 Devissaguet
`
`514/352
`424/490
`
`5,091,188 2/ 1992 Haynes . . . . . . . .
`
`. . . .. 424/450
`
`424/487
`5,118,525 6/1992 Fessi
`5,124,338 6/ 1992 King .................................. .. 514/352
`
`European Pat. Off. .
`European Pat. Off. .
`European Pat. Off. .
`France .
`Germany .
`Japan .
`United Kingdom .
`United Kingdom .
`WIPO .
`
`FOREIGN PATENT DOCUMENTS
`262560 9/1987
`411629 2/1991
`499299 1/1992
`2118987 4/1972
`3772837 7/1987
`2282330 11/1990
`2185397 7/1987
`2200048 ‘ 7/1988
`8400294 7/1983
`9115193 6/1989
`9015593 6/1990
`9203380 8/1990
`9106292 11/1990
`OTHER PUBLICATIONS
`Lachman et al., “The Theory and Practice of Industrial
`Pharmacy”, Chapter 2 (1986).
`Remington’s Pharmaceutical Sciences, 17th Edition,
`Chapter 20, Schott, H., “Colloidal Dispersions”.
`Goodman and Gilman, “The Pharmacological Basis of
`Therapeutics”, Eighth Edition, pp. 68-69.
`Primary Examiner—Thurman K. Page
`Assistant Examiner-William E. Benston, Jr.
`Attorney, Agent, or Finn-Arthur H. Rosenstein
`[57]
`ABSTRACT
`Dispersible particles consisting essentially of a crystal
`line anticancer agent having a surface modi?er ad
`sorbed on the surface thereof in an amount sufficient to
`maintain an effective average particle size of less than
`about 1000 nm. Anticancer compositions comprising
`the particles exhibit reduced toxicity and/or enhanced
`efficacy, and can be administered by IV bolus injection.
`
`17 Claims, N0 Drawings
`
`CIPLA EXHIBIT 1005
`Page 1 of 10
`
`

`

`1
`
`SURFACE MODIFIED ANTICANCER
`NAN OPARTICLES
`
`5,599,363
`
`CROSS REFERENCED TO RELATED
`APPLICATION
`This application is a continuation-in-part of US. pa
`tent application Ser. No. 647,105, ?led Jan. 25, 1991,
`now U.S. Pat. No. 5,145,684, the disclosure of which is
`hereby incorporated by reference in its entirety.
`
`5
`
`25
`
`5
`
`2
`It is another advantageous feature of this invention
`that anticancer compositions are provided exhibiting
`improved ef?cacy.
`Yet another advantageous feature of this invention is
`that compositions are provided featuring poorly soluble
`anticancer agents that can be administered by IV bolus
`injection.
`Still another advantageous feature of this invention is
`that compositions are provided containing poorly solu
`ble anticancer agents exhibiting prolonged circulation
`in the blood pool after IV bolus injection.
`Other advantageous features will become readily
`apparent upon reference to the following descriptions
`of preferred embodiments.
`DESCRIPTION OF PREFERRED
`EMBODIMENTS
`This invention is based partly on the discovery that
`surface modi?ed anticancer nanoparticles exhibit re
`duced toxicity and/or enhanced ef?cacy. While the
`invention is described herein primarily in connection
`with its preferred class of drugs, i.e., anticancer agents
`including immunosuppressive agents, it is also useful in
`conjunction with poorly water soluble drugs, particu
`larly those with low therapeutic indices, from other
`classes of drug substances.
`The particles of this invention comprise an anticancer
`agent. The anticancer agent is present in one or more
`discrete crystalline phases. The crystalline phase differs
`from an amorphous, i.e., non-crystalline phase which
`results from conventional solvent precipitation tech
`niques for the preparation of particles in the submicron
`size range, such as described in US. Pat. No. 4,826,689.
`The invention can be practiced with a wide variety of
`anticancer agents. However, the anticancer agent must
`be poorly soluble and dispersible in at least one liquid
`medium. By “poorly soluble”, it is meant that the drug
`substance has a solubility in the liquid dispersion me
`dium, e.g., water, of less than about 10 mg/ml, and
`preferably, of less than 1 mg/ml at processing tempera
`ture, e.g., room temperature. The preferred liquid dis
`persion medium is water. However, the invention can
`be practiced with other liquid media in which the anti
`cancer agent is dispersible including, for example, aque
`ous salt solutions, safflower oil, and solvents such as
`ethanol, t-butanol, hexane, and glycol. The pH of the
`aqueous dispersion media can be adjusted by techniques
`known in the art.
`The anticancer agent preferably is selected from al
`kylating agents, antimetabolites, natural products, hor
`mones and antagonists, and miscellaneous agents, such
`as radiosensitizers.
`Examples of alkylating agents include alkylating
`agents having the bis-(2-chloroethyl)-amine group such
`as, for example, chlormethine, chlorambucile, melpha
`lan, uramustine, mannomustine, extramustinephoshate,
`mechlore-thaminoxide, cyclophosphamide, ifosfamide,
`and trifosfamide;
`alkylating agents having a substituted aziridine group
`such as, for example, tretamine, thiotepa, triaziquone
`and mitomycine;
`alkylating agents of the alkyl sulfonate type, such as,
`for example, busulfan, piposulfan, and piposulfam;
`alkylating N-alkyl-N-nitrosourea derivatives, such as,
`for example, carmustine, lomustine, semustine, or strep
`tozotocine; and alkylating agents of the mitobronitole,
`dacarbazine and procarbazine type.
`
`BACKGROUND OF THE INVENTION
`1. Field of Invention
`This invention relates to anticancer agents in the form
`of particles, to anticancer compositions comprising the
`particles, and to methods employing the particles.
`2. Description of the Prior Art
`The therapeutic index is a measure of how selective a
`drug is at producing its desired effects and can be de
`?ned as the ratio of the median lethal dose to the median
`effective dose, i.e., (LD50/ED50) (see Goodman and
`Gilman, The Pharmacological Basis of Therapeutics,
`Eight Edition, p. 68-69). Virtually all anticancer agents
`have a low therapeutic index, e.g., less than about 1.0.
`Increasing the therapeutic index, e.g., by reducing tox
`icity or enhancing ef?cacy would provide more latitude
`to physicians in their duty of administering anticancer
`drugs to patients in need thereof. Consequently, meth
`ods to reduce toxicity and/or enhance ef?cacy of anti
`cancer drugs and thus increase the therapeutic indices
`of such drugs would be of great value in the treatment
`of cancers.
`In addition, poorly water-soluble drugs, such as
`poorly water-soluble anticancer agents, are not readily
`injectable via an intravenous (IV) bolus injection. The
`creation of injectable forms of poorly soluble drugs
`represents a formidable problem. It would be highly
`desirable to be able to provide poorly soluble drugs,
`such as poorly soluble anticancer agents, in an IV bolus
`injectable form.
`SUMMARY OF THE INVENTION
`We have discovered that anticancer compositions
`comprising anticancer agents in the form of surface
`modi?ed nanoparticles exhibit reduced toxicity and/or
`enhanced efficacy.
`More particularly, in accordance with this invention,
`there are provided particles consisting essentially of a
`crystalline anticancer agent having a surface modi?er
`adsorbed on the surface thereof in an amount su?icient
`to maintain an effective average particle size of less than
`about 1000 nm.
`This invention further provides an anticancer compo
`sition comprising the above-described particles.
`In another embodiment of the invention, there is
`provided a method of treating a mammal comprising
`administering to the mammal the above-described anti
`cancer composition.
`In yet another embodiment of the invention, there is
`provided a method of enhancing the ef?cacy and/or
`reducing the toxicity of an anticancer agent which in
`cludes the step of administering the agent in the form of
`the above-described particles.
`65
`It is an advantageous feature of this invention that
`anticancer compositions are provided exhibiting re
`duced toxicity.
`
`55
`
`60
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`CIPLA EXHIBIT 1005
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`5,399,363
`3
`Examples of antimetabolites include folic acid ana
`logs, such as, for example, methotrexate;
`pyrimidine analogs such as, for example, ?uorouracil,
`?oxuridine, tegafur, cytarabine, idoxuridine, and
`?ucytosine; and
`purine derivatives such as, for example, mercaptopu
`rine, thioguanine, azathioprine, tiamiprine, vidarabine,
`pentostatin, and puromycine.
`Examples of natural products include vinca alkaloids,
`such as, for example, vinblastine and vincristine;
`epipodophylotoxins, such as, for example, etoposide
`and teniposide;
`antibiotics, such as, for example, adriamycine, dauno
`mycine, doctinomycin, daunorubicin, doxorubicin, mi
`thrarnycin, bleomycin, and mitomycin;
`enzymes, such as, for example, L-asparaginase;
`biological response modi?ers, such as, for example,
`a-interferon;
`camptothecin;
`M01; and
`retinoids, such as retinoic acid.
`Examples of hormones and antagonists include adre
`nocorticosteroids, such as, for example, prednisone;
`progestins, such as, for example, hydroxyprogester
`one caproate, medroxyprogesterone acetate and meges
`trol acetate;
`estrogens, such as, for example, diethylstilbestrol and
`ethinyl estradiol;
`antiestrogens, such as, for example, tamoxifen;
`androgens, such as, for example, testosterone propio
`nate and ?uoxymesterone;
`antiandrogens, such as, for example, ?utamide;
`and gonadotropin-releasing hormone analogs, such
`as, for example leuprolide.
`Examples of miscellaneous agents include radiosen
`sitizers, such as, for example, 1,2,4-benzotriazin-3-amine
`1,4-dioxide (SR 4889) and 1,2,4-benzotriazine-7-amine
`1,4-dioxide (WIN 59075);
`platinum coordination complexes such as cisplatin
`and carboplatin;
`anthracenediones, such as, for example, mitoxan
`trone;
`substituted ureas, such as, for example, hydroxyurea;
`and adrenocortical suppressants, such as, for exam
`ple, mitotane and aminoglutethimide.
`In addition, the anticancer agent can be an immuno
`suppressive drug, such as, for example, cyclosporine,
`azathioprine, sulfasalazine, methoxsalen and thalido
`mide.
`The anticancer agents useful in the practice of this
`invention are known compounds and/or can be pre
`pared by techniques known in the art.
`The anticancer agent can be used alone or in combi
`nation with one or more anticancer agents.
`The particles of this invention contain an anticancer
`55
`agent as described above having a surface modi?er
`adsorbed on the surface thereof. Useful surface modi?
`ers are believed to include those which physically ad
`here to the surface of the anticancer agent but do not
`chemically bond to the anticancer agent.
`Suitable surface modi?ers can preferably be selected
`from known organic and inorganic pharmaceutical ex
`cipients. Such excipients include various polymers, low
`molecular weight oligomers, natural products and sur
`factants. Preferred surface modi?ers include nonionic
`65
`and anionic surfactants. Representative examples of
`excipients include gelatin, casein, lecithin (phospha
`tides), gum acacia, cholesterol, tragacanth, stearic acid,
`
`4
`benzalkonium chloride, calcium stearate, glyceryl mon
`ostearate, cetostearyl alcohol, cetomacrogol emulsify
`ing wax, sorbitan esters, polyoxyethylene alkyl ethers,
`e.g., macrogol ethers such as cetomacrogol 1000, poly
`oxyethylene castor oil derivatives, polyoxyethylene
`sorbitan fatty acid esters, e.g., the commercially avail
`able Tweens TM , polyethylene glycols, polyoxyethyl
`ene stearates, colloidol silicon dioxide, phosphates, so
`dium dodecylsulfate, carboxymethylcellulose calcium,
`carboxymethylcellulose sodium, methylcellulose, hy
`droxyethylcellulose, hydroxypropylcellulose, hydroxy
`propylmethylcellulose phthalate, noncrystalline cellu
`lose, magnesium aluminum silicate, triethanolamine,
`polyvinyl alcohol (PVA), and polyvinylpyrrolidone
`(PVP). Most of these excipients are described in detail
`in the Handbook of Pharmaceutical Excipients, published
`jointly by the American Pharmaceutical Association
`and The Pharmaceutical Society of Great Britain, the
`Pharmaceutical Press, 1986. The surface modi?ers are
`commercially available and/ or can be prepared by tech
`niques known in the art. Two or more surface modi?ers
`can be used in combination.
`Particularly preferred surface modi?ers include poly
`vinylpyrrolidone, tyloxapol, polaxomers, such as Pluro
`nic TM F68, F108 and F127, which are block copoly
`mers of ethylene oxide and propylene oxide available
`from BASF, and poloxamines, such as Tetronic TM 908
`(T908), which is a tetrafunctional block copolymer
`derived from sequential addition of ethylene oxide and
`propylene oxide to ethylenediamine available from
`BASF, dextran, lecithin, Aerosol OT TM (AOT),
`which is a dioctyl ester of sodium sulfosuccinic acid,
`available from American Cyanamid, Duponol TM P,
`which is a sodium lauryl sulfate, available from DuPont,
`Triton'I'M X-200, which is an alkyl aryl polyether
`sulfonate, available from Rohm and Haas, Tween 20,
`40, 60 and 80, which are polyoxyethylene sorbitan fatty
`acid esters, available from ICI Speciality Chemicals,
`Span 20, 40, 60 and 80, which are sorbitan esters of fatty
`acids, Arlacel 20, 40, 60 and 80, which are sorbitan
`esters of fatty acids, available from Hercules, Inc., Car
`bowax TM 3550 and 934, which are polyethylene gly
`cols available from Union Carbide, Crodesta TM F-l10,
`which is a mixture of sucrose stearate and sucrose dis
`tearate, available from Croda Inc., Crodesta SL-40,
`which is available from Croda, Inc., hexyldecyl tri
`methyl ammonium chloride (CTAC), bovine serum
`albumin and SA90HCO, which is C1gH37 CH1 (CON
`(CH3) CH2 (CHOH)4CH2OH)2. Surface modi?ers
`which have been found to be particularly useful include
`polyvinylpyrrolidone, Pluronic F-108, polyvinyl alco
`hol and gum acacia.
`The surface modi?er is adsorbed on the surface of the
`anticancer agent in an amount suf?cient to maintain an
`effective average particle size of less than about 1000
`nm. The surface modi?er does not chemically react
`with the anticancer agent or itself. Furthermore, the
`individually adsorbed molecules of the surface modi?er
`are essentially free of intermolecular crosslinkages.
`As used herein, particle size refers to a number aver
`age particle size as measured by conventional particle
`size measuring techniques well known to those skilled
`in the art, such as sedimentation ?eld ?ow fractionation,
`photon correlation spectroscopy, or disk centrifugation.
`By “an effective average particle size of less than about
`1000 nm” it is meant that at least 90% of the particles
`have a number average particle size of less than about
`1000 nm when measured by the above-noted tech
`
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`5
`niques. In particularly preferred embodiments of the
`The mechanical means applied to reduce the particle
`size of the anticancer agent conveniently can take the
`invention, the effective average particle size is less than
`form of a dispersion mill. Suitable dispersion mills in
`about 400 nm. In some embodiments of the invention,
`clude a ball mill, an attritor mill, a vibratory mill, a
`the effective average particle size is less than about 300
`planetary mill, media mills such as a sand mill and a
`nm. With reference to the effective average particle
`bead mill. A media mill is preferred due to the relatively
`size, it is preferred that at least 95% and, more prefera
`shorter milling time required to provide the intended
`bly, at least 99% of the particles have a particle size of
`result, i.e., the desired reduction in particle size. For
`less than the effective average, e. g., 1000 nm. In particu
`media milling, the apparent viscosity of the premix
`larly preferred embodiments, essentially all of the parti
`preferably is from about 100 to about 1000 centipoise.
`cles have a size less than 1000 nm.
`For ball milling, the apparent viscosity of the premix
`Motoyama et al, US. Pat. No. 4,540,602 disclose that
`preferably is from about 1 up to about 100 centipoise.
`a solid drug can be pulverized in an aqueous solution of
`Such ranges tend to afford an optimal balance between
`a water-soluble high molecular substance, and that as a
`efficient particle fragmentation and media erosion.
`result of such wet grinding, the drug is formed into
`The grinding media for the particle size reduction
`?nely divided particles ranging from 0.5 pm or less to 5
`step can be selected from rigid media preferably spheri
`pm in diameter. However, there is no suggestion that
`cal or particulate in form having an average size less
`particles having an average particle size of less than
`than about 3 mm and, more preferably, less than about
`about 1 pm can be obtained. Attempts to reproduce the
`1 mm. Such media desirably can provide the particles of
`wet grinding procedures described by Motoyama et a1
`the invention with shorter processing times and impart
`resulted in particles having an average particle size of
`less wear to the milling equipment. The selection of
`much greater than 1 nm.
`material for the grinding media is not believed to be
`The particles of this invention can be prepared by a
`critical. However, zirconium oxide, such as 95% ZrO
`method comprising the steps of dispersing an anticancer
`stabilized with magnesia, zirconium silicate, and glass
`agent in a liquid dispersion medium and applying me
`grinding media provide particles having levels of con
`chanical means in the presence of grinding media to
`tamination which are believed to be acceptable for the
`reduce the particle size of the anticancer agent to an
`preparation of pharmaceutical compositions. Further,
`effective average particle size of less than about 1000
`other media, such as stainless steel, titania, alumina, and
`nm. The particles can be reduced in size in the presence
`95% ZrO stabilized with yttrium, are expected to be
`of a surface modi?er. Alternatively, the particles can be
`useful. Preferred media have a density greater than
`contacted with a surface modi?er after attrition.
`about 2.5 g/cm3.
`A general procedure for preparing the particles of
`The attrition time can vary widely and depends pri
`this invention is set forth below. The anticancer agent
`marily upon the particular mechanical means and pro
`selected is obtained commercially and/or prepared by
`cessing conditions selected. For ball mills, processing
`techniques known in the art in a conventional coarse
`times of up to ?ve days or longer may be required. On
`35
`form. It is preferred, but not essential, that the particle
`the other hand, processing times of less than 1 day (resi
`size of the coarse anticancer agent selected be less than
`dence times of one minute up to several hours) have
`about 100 pm as determined by sieve analysis. If the
`provided the desired results using a high shear media
`coarse particle size of the anticancer agent is greater
`mill.
`than about 100 pm, then it is preferred that the particles
`The particles must be reduced in size at a temperature
`of the anticancer agent be reduced in size to less than
`which does not signi?cantly degrade the anticancer
`100 pm using a conventional milling method such as
`agent. Processing temperatures of less than about
`airjet or fragmentation milling.
`30°40" C. are ordinarily preferred. If desired, the pro
`The coarse anticancer agent selected can then be
`cessing equipment can be cooled with conventional
`added to a liquid medium in which it is essentially insol
`cooling equipment. The method is conveniently carried
`uble to form a premix. The concentration of the antican
`out under conditions of ambient temperature and at
`cer agent in the liquid medium can vary from about
`processing pressures which are safe and effective for the
`01-60% and preferably is from 5—30% (w/w). It is
`milling process. For example, ambient processing pres
`preferred, but not essential, that the surface modi?er be
`sures are typical of ball mills, attritor mills and vibratory
`present in the premix. The concentration of the surface
`mills. Processing pressures up to about 20 psi (1.4
`kg/cmz) are typical of media milling.
`modi?er can vary from about 0.1 to about 90% and
`preferably is l—75%, more preferably 20-60%, by
`The surface modi?er, if it was not present in the
`weight based on the total combined weight of the drug
`premix, must be added to the dispersion after attrition in
`substance and surface modi?er. The apparent viscosity
`an amount as described for the premix above. Thereaf
`of the premix suspension is preferably less than about
`ter, the dispersion can be mixed, e.g., by shaking vigor
`1000 centipoise.
`ously. Optionally, the dispersion can be subjected to a
`The premix can be used directly by subjecting it to
`sonication step, e.g., using an ultrasonic power supply.
`For example, the dispersion can be subjected to ultra
`mechanical means to reduce the average particle size in
`the dispersion to less than 1000 nm. It is preferred that
`sonic energy having a frequency of 20-80 kHz for a
`the premix be used directly when a ball mill is used for
`time of about 1 to 120 seconds.
`attrition. Alternatively, the anticancer agent and, op
`The relative amount of the anticancer agent and sur
`tionally, the surface modi?er, can be dispersed in the
`face modi?er can vary widely and the optimal amount
`liquid medium using suitable agitation, e.g., a roller mill
`of the surface modi?er can depend, for example, upon
`the particular anticancer agent and surface modi?er
`or a Cowles type mixer, until a homogeneous dispersion
`is observed in which there are no large agglomerates
`selected, the critical micelle concentration of the sur
`visible to the naked eye. It is preferred that the premix
`face modi?er if it forms micelles, the surface area of the
`anticancer agent, etc. The surface modi?er preferably is
`be subjected to such a premilling dispersion step when a
`recirculating media mill is used for attrition.
`present in an amount of about 01-10 mg per square
`
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`meter surface area of the anticancer agent. The surface
`modi?er can be present in an amount of 01-90%, pref
`erably 05-80% and more preferably l—60% by weight
`based on the total weight of the dry particle.
`A simple screening process has been developed
`whereby compatible surface modi?ers and anticancer
`agents can be selected which provide stable dispersions
`of the desired particles. First, coarse particles of an
`anticancer agent are dispersed in a liquid in which the
`anticancer agent is essentially insoluble, e.g., water at
`2% (w/v) and milled for 120 hours in a roller mill under
`the following milling conditions:
`Grinding vessel: 8 oz. (250 ml) glass jar
`Available volume of grinding vessel: 250 ml
`Media volume: 120 ml
`Media type: 1.0 mm pre-cleaned zirconium oxide
`beads (distributed by Zircoa, Inc.)
`Milling time: 120 hours
`Slurry volume: 60 ml
`RPM: 92
`Room Temperature
`The slurry is separated from the milling media by
`conventional means, e.g., by pouring the slurry out of
`the vessel, or by using a pipette. The separated slurry is
`then divided into aliquots and surface modi?ers are
`added at a concentration of between 2 and 50% by
`weight based on the total combined weight of the anti
`cancer agent and surface modi?er. The dispersions are
`then sonicated (1 minute, 20 kHz) or vortexed using a
`multitubed vortexer for one minute, to disperse agglom
`erates and subjected to particle size analysis, e.g., by
`photon correlation spectroscopy (PCS) and/or by ex
`amination under an optical microscope (IOOOXmagni?
`cation). If a stable dispersion is observed, then the pro
`cess for preparing the particular anticancer agent sur
`face modi?er combination can be optimized in accor
`dance with the teachings above. By stable it is meant
`that the dispersion exhibits no flocculation or particle
`agglomeration visible to the naked eye and, preferably,
`when viewed under the optical microscope at 1000>< , at
`40
`least 15 minutes, and preferably, at least two days or
`longer after preparation. In addition, preferred particles
`exhibit no ?occulation or agglomeration when dis
`persed in 0.1N HCl and/or phosphate buffered saline,
`pH 7.4 (PBS) or rat plasma.
`The resulting dispersion is stable and consists of the
`liquid dispersion medium and the above-described parti
`cles. The dispersion of surface modi?ed anticancer
`agent nanoparticles can be spray coated onto sugar
`spheres or onto a pharmaceutical excipient in a ?uid
`bed spray coater by techniques well known in the art.
`Anticancer pharmaceutical compositions according
`to this invention include the particles described above
`and a pharmaceutically acceptable carrier therefor.
`Suitable pharmaceutically acceptable carriers are well
`known to those skilled in the art. These include non
`toxic physiologically acceptable carriers, adjuvants or
`vehicles for parenteral injection, for oral administration
`in solid or liquid form, for rectal administration, nasal
`administration, intramuscular administration, subcuta
`neous administration, and the like.
`A method of treating a mammal in accordance with
`this invention comprises the step of administering to the
`mammal in need of treatment an effective amount of the
`above-described anticancer composition. The selected
`dosage level of the anticancer agent for treatment is
`effective to obtain a desired therapeutic response for a
`particular composition and method of administration.
`
`8
`The selected dosage can be readily determined by one
`skilled in the art and depends upon the particular anti
`cancer agent, the desired therapeutic effect, the route of
`administration, the desired duration of treatment and
`other factors.
`It is a particularly advantageous feature that the anti
`cancer compositions of this invention exhibit reduced
`toxicity and/or enhanced ef?cacy as illustrated in the
`examples that follow. Further, the particles of this in
`vention exhibit prolonged circulation in the blood pool.
`Moreover, anticancer agents which heretofore could
`not be administered by injection, when prepared as
`nanoparticles and formulated in anticancer composi
`tions according to this invention, can be effectively
`administered by injection, e.g., by an intravenous bolus
`injection.
`The following examples further illustrate the inven
`tion.
`EXAMPLES l-4 Nanoparticulate Piposulfan
`EXAMPLE 1
`Piposulfan (purchased from Eastman Kodak) was
`milled in a mixture of 0.33% polyoxyethylene sorbitan
`monooleate, Tween 80, (ICI Americas, Inc., Wilming
`ton, Del.) and 0.67% sorbitan monooleate, Span 80,
`(ICI) using 1 mm zirconium oxide beads for about 96
`hours to produce particles approximately 240 nm in
`diameter. The ?nal piposulfan concentration in the sus
`pension was 10 mg/mL. The particles were stable to
`?occulation/ aggregation in rat plasma.
`Milling Conditions: A coarse suspension of piposul
`fan was prepared by adding 300 mg of the drug to a 4
`oz. (120 mL) amber bottle which was previously ?lled
`with 60 mL of 1 mm precleaned zirconium oxide beads
`(Zircoa Inc., Solon, Ohio) and 30 mL of 1% Tween
`80/Span 80 (l to 2 ratio) solution. The surfactant solu
`tion was prepared by accurately weighing 333 mg of
`Tween 80 and 667 mg of Span 80 in a volumetric ?ask
`followed by addition of sterile water for injection to
`dissolve/ disperse the surfactants. Sufficient quantity of
`water was added to make the ?nal volume 100 mL.
`Zirconium oxide beads were cleaned by ?rst rinsing in
`1N sulfuric acid followed by several rinses with deion
`ized water. The media was dried in a vacuum oven at
`about 100° C. overnight.
`The sealed primary container was loaded into a sec
`ondary padded aluminium containment can to ensure a
`tight ?t. It was milled on a roller mill (US Stoneware,
`Mawah, NJ.) at 144 RPM for about 96 hours. At the
`end of the milling time the slurry was separated from
`the media and particle size was measured using a PCS
`device. Stability of these particles to rat plasma was
`assessed by optical microscopy at IOOOXmagni?catiOn.
`The ?nal pH of the formulation was 6.
`Control A (unmilled), a coarse suspension containing
`40 mg of bulk piposulfan was dispersed in water in the
`presence of 3% ethanol and 1% Tween 80. This suspen
`sion could not be injected IV.
`Example 1 was evaluated for ef?cacy studies in fe
`male mice (avg. wt. 22 g) which were implanted with
`early stage Mammary Adenocarcinoma #l6/C on day
`0. The formulation was injected starting from day l for
`several days. The antitumor activity was assessed by
`monitoring tumor weight and comparing it to the con
`trol animals. The results were as follows:
`
`45
`
`55
`
`65
`
`CIPLA EXHIBIT 1005
`Page 5 of 10
`
`

`

`Treatment
`
`Control
`
`Example 1
`(243 nm)
`
`Control A
`
`9
`
`5,399,363
`
`Rome *
`of Adm'
`
`Tmal
`Dose (mg/kg)
`
`% wt‘ T/C L°gl9
`LOSS % Cell K111
`
`10
`EXAMPLE 7
`Example 5 was repeated except that Tetronic 908 was
`replaced by gum acacia. The ?nal particle size was 298
`
`+5-5 — —
`
`5 nm.
`
`—-
`
`Iv
`
`Iv
`5c
`
`--
`
`356
`
`85
`300
`
`‘5'5
`
`o
`i
`'0 18
`_ 10.8
`0
`
`2-75
`
`1'0
`21
`
`Nanocamptothecin formulations were evaluated for
`ef?cacy in two murine tumor models, i.e., Mammary
`Adenocarcinoma #16/ C and Pancreatic Ductal Adeno
`carcinoma #03. The antitumor activity was assessed by
`10 momtormg tumor weight from experimental and con
`trol animals.
`1. Ef?cacy Studies in Pancreatic Ductal
`Adenocarcinoma #03:
`
`‘Administration - IV Intravenous; SC Subcutaneous
`Example I could be injected directly as 10 mg/ml suspension. There was no acute
`toxicity after injection of 78 mg/kg single dose.
`
`T/C=Tumor weight in treated animals divided by the
`tumor weight of the control animals, reported as per
`cent value. Lower value indicates better ef?cacy, 0%
`suggests cures. < 10% is considered highly active, 10 to
`42% is moderately active formulation. >42% is consid
`ered inactive.
`Log Kill=(T—C)/3.32 (T d), where T is the time in
`days for the median tumor to reach 1000 mg mass in
`treated animals, C is the time in days for the median
`tumor to reach 1000 mg in control animals and Td is the
`tumor volume doubling time in days. Cures (tumor free
`animals) are excluded from (T —C) calculations.
`Example 1 demonstrates that a composition of this
`invention exhibited reduced toxicity and enhanced ef?
`cacy compared to a prior art composition and could be
`administered by IV bolus injection.
`
`20
`
`30
`
`EXAMPLES '2-4
`The milling procedure described in Example 1 was
`repeated except that the ratio of Tween 80 to Span 80
`was 2:1. The resulting average particle size was 297 nm.
`The milling procedure described in Example 1 was
`repeated except that the ratio of Tween 80 to Span 80
`was 1:1. The resulting average particle size was 380 nm.
`The milling procedure described in Example 1 was
`repeated except that the surface modi?er was a 1:1 ratio
`of Tween 60 and Span 60. The resulting average parti»
`cle size was 301 nm.
`Stable pipsulfan nanoparticles were also prepared
`using bovine serum albumin as the surface modi?er.
`EXAMPLES 5-7 Nanoparticulate Camptothecin
`EXAMPLE 5
`Approximately 60 mL of precleaned zirconium oxide
`beads (1 mm) were placed in a 120 mL wide mouth
`round amber bottle. To it was added 0.35 g of Tetronic
`908 (BASF) followed by 0.35 g of Camptothecin
`(Sigma Chemicals, 95% pure). To the above mixture, 35
`mL of water for injection (Abbott) was added. The
`bottle was sealed and mounted on a roller mill. Milling
`was effected by rotating the bottle at 100 RPM for 7
`days.
`At the end of milling, an aliquot (100 PL) was
`checked for particle