PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 00/61191
`(51) International Patent Classification 7 :
`A61K 47/48, 49/00
`
`(43) International Publication Date:
`
`19 October 2000 (19.10.00)
`
`(11) International Publication Number:
`
`A2
`
`(21) International Application Number:
`
`PCT/US00/06047
`
`(22) International Filing Date:
`
`8 March 2000 (08.03.00)
`
`(81) Designated States: JP, European patent (AT, BE, CH, CY, DE,
`DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).
`
`(30) Priority Data:
`60/128,579
`
`9 April 1999 (09.04.99)
`
`US
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`(71) Applicant: ADVANCED MAGNETICS, INC. [US/US]; 61
`Mooney Street, Cambridge, MA 02138 (US).
`
`(72) Inventors: GROMAN, Ernest, V.; 80 Columbia Street, Brook(cid:173)
`line, MA 02146 (US). PAUL, Kenneth, G.; 1037 Washing(cid:173)
`ton Street, Holliston, MA 01746 (US). FRIGO, Timothy,
`B.; 14 Ripley Street, Waltham, MA 02454 (US). BEN(cid:173)
`GELE, Howard; 5 Historical Way, Canton, MA 02021 (US).
`LEWIS, Jerome, M.; 273 Upland Avenue, Newton, MA
`02461 (US).
`
`(74) Agents: SUNSTEIN, Bruce, D. et al.; Bromberg & Sunstein
`LLP, 125 Summer Street, Boston, MA 02110-1618 (US).
`
`(54) Title: HEAT STABLE COLLOIDAL IRON OXIDES COATED WITH REDUCED CARBO HYDRA TES AND CARBO HYDRA TE
`DERIVATIVES
`
`1600 . , - - - - - - - - - - - - - - - - - - - - - - -
`
`•
`
`1600
`
`fi! 1400
`a
`~ i 1300
`:I. s
`
`~- 1200
`
`1100
`
`1000 +---r---.----,----.--,---.....--"T"""-....... ----,,-~
`64
`62
`60
`88
`10
`78
`66
`74
`72
`76
`80
`[Bromoacetlc acid], mg/g
`
`(57) Abstract
`
`Compositions, methods of making the compositions, and methods of using the compositions are provided for an enhanced magnetic
`resonance imaging agent and a hematinic agent, the agents comprising carboxyalkylated reduced polysaccharides coated ultrasmaIJ
`superparamagnetic iron oxides. Methods of use of the carboxymethyl reduced dextran as a plasma extender are provided.
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 1
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`C6te d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 2
`
`

`

`WO 00/61191
`
`PCT/US00/06047
`
`HEAT ST ABLE COLLOIDAL IRON OXIDES COATED WITH REDUCED
`
`CARBOHYDRATES AND CARBOHYDRATE DERIVATIVES
`
`Technical Field
`
`5
`
`The field relates to compositions which are carboxymethyl reduced
`
`polysaccharides, and methods for use as plasma extenders and for coating iron oxide
`
`particles, and compositions comprised of superparamagnetic and non-superparamagnetic
`iron oxides coated with a reduced polysaccharide or derivatized reduced polysaccharide,
`and methods for use as MRI contrast agents and hematinics.
`
`10
`
`Background
`Since the invention of ~agnetic resonance imaging (MRI), a parallel technology
`of injectable chemicals called contrast agents has developed. Contrast agents play an
`
`important role in the practice of medicine in that they help produce more useful MRI
`
`images for diagnostic purposes. In particular, two classes of imaging agents have been
`
`15 developed and adopted in clinical practice. These are: low molecular weight
`gadolinium complexes such as Magnavist®; and colloidal iron oxides such as Feridex
`
`I.V.® and Combidex®. Neither of these two types of agents is ideal. Problems
`
`encountered with these agents are shown in Table 1, and include: expense of
`
`20
`
`components; inefficiency of synthesis; loss of coating during terminal sterilization
`(autoclaving); narrow range of organ uptake for purposes of imaging; toxic side-effects;
`restriction of use to either first pass or equilibrium dosing, and others that are described
`
`herein. Agents that overcome these problems, and that combine the properties of these
`
`two types of contrast agents, are highly desirable.
`
`Summary
`
`25
`
`An embodiment of the invention is a method of providing an iron oxide complex
`
`for administration to a mammal subject, the method comprising: producing a reduced
`polysaccharide iron oxide complex, and sterilizing the complex by autoclaving. In
`general, the reduced polysaccharide is a reduced polymer of glucose. An example of a
`
`reduced polymer of glucose is a reduced dextran. The reduced polysaccharide is
`
`1
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`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 3
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`PCT/US00/06047
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`Table 1. Comparison of ideal properties of MRI contrast agents with properties
`
`of low molecular weight gadolinium based contrast agents and colloidal iron oxides.
`
`Properties of an ideal
`contrast agent
`Low production costs:
`efficient synthesis
`Autoclavable without
`excipients
`Tl agent
`T2 agent
`Non toxic
`Imaging vascular
`compartment at early phase
`(as a bolus administration)
`and at a late stage
`(equilibrium phase)
`Multiple administration in
`single examination
`Image of multiple target
`organs
`Bolus injection
`Low volume of injection
`Iron source for anemia
`
`5
`
`10
`
`15
`
`20
`
`25
`
`low molecular weight
`gadolinium
`Yes
`
`colloidal iron
`oxides
`No
`
`Yes
`
`Yes
`No
`Yes
`No
`
`No
`
`Yes
`
`Yes
`No
`No
`
`No
`
`Sometimes
`Yes
`No
`No
`
`No
`
`Sometimes
`
`No
`No
`Yes
`
`produced through reaction of a polysaccharide with a reagent selected from the group
`
`consisting of a borohydride salt or hydrogen in the presence of a hydrogenation catalyst.
`
`In a further aspect of the method, the iron oxide is superparamagnetic.
`
`Another preferred embodiment of the invention is a method of providing an iron oxide
`
`30 complex for administration to a mammalian subject, the method comprising: producing
`
`a derivatized reduced polysaccharide iron oxide complex, and sterilizing the complex by
`
`autoclaving. According to this method, producing the complex can include derivatizing
`
`a reduced polysaccharide by carboxyalkylation, for example, wherein the
`
`carboxyalkylation is a carboxymethylation. Further according to this method, the
`
`35
`
`reduced polysaccharide can be a reduced dextran. The derivatized, reduced
`
`polysaccharide can be isolated as the sodium salt and does not contain an infrared
`absorption peak in the region of 1650-1800 cm· 1
`• In one aspect of the method, producing
`
`2
`
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`Pharmacosmos A/S v. American Regent, Inc.
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`WO 00/61191
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`PCT/US00/06047
`
`the derivatized reduced polysaccharide is achieved at a temperature of less than
`
`approximately 50°C. In another aspect of the method, producing the derivatized reduced
`
`polysaccharide is achieved at a temperature of less than approximately 40°C. In a
`
`further aspect of the method, the iron oxide is superparamagnetic.
`
`5
`
`In yet another embodiment, the invention provides a method of formulating an
`
`iron oxide complex coated with a reduced polysaccharide. This composition is for
`
`pharmacological use and the composition has decreased toxicity in comparison to an
`
`analogous iron oxide complex coated with native polysaccharide. The method of
`
`formulating such an iron oxide complex comprises: producing a reduced polysaccharide
`
`10
`
`iron oxide complex, and sterilizing the complex by autoclaving .. The formulation
`
`provides polysaccharide which was produced by reacting the polysaccharide with one of
`
`a reducing agent selected from the group consisting of a borohydride salt or hydrogen in
`
`the presence of an hydrogenation catalyst. The reduced polysaccharide iron oxide
`
`complex having such decreased toxicity. In a further aspect of the method, the iron
`
`15 oxide is superparamagnetic.
`
`In yet another embodiment, the invention provides a method of formulating an
`
`iron oxide complex coated with a reduced derivatized polysaccharide. This composition
`
`is for pharmacological use and the composition has decreased toxicity in comparison to
`
`an analogous iron oxide complex coated with native derivatized polysaccharide. The
`
`20 method of formulating such an iron oxide complex comprises: producing a reduced
`
`derivatized polysaccharide iron oxide complex; and sterilizing the complex by
`
`autoclaving. According to this method, producing the complex can include derivatizing
`
`a reduced polysaccharide by carboxyalkylation, for example, wherein the
`
`carboxyalkylation is a carboxymethylation. Further according to this method, the
`
`25 . reduced polysaccharide can be a reduced dextran. The derivatized, reduced
`
`polysaccharide can be isolated as the sodium salt and does not contain an infrared
`absorption peak in the region of 1650-1800 cm· 1
`• In one aspect of the method, producing
`
`the derivatized reduced polysaccharide is achieved at a temperature of less than
`
`approximately 50°C. In another aspect of the method, producing the derivatized reduced
`
`3
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`Pharmacosmos A/S v. American Regent, Inc.
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`PCT/US00/06047
`
`polysaccharide is achieved at a temperature of less than approximately 40°C. In a
`further aspect of the method, the iron oxide is superparamagnetic.
`Another embodiment of the invention provides a reduced derivatized
`polysaccharide iron oxide complex with Tl and T2 relaxation properties to allow
`5 contrast agent signal enhancement with Tl sequences and signal diminishment with T2
`sequences. A further aspect of the embodiment is that the reduced derivatized
`polysaccharide iron oxide can be administered multiple times for sequential imaging in a
`single examination. Yet another aspect of the agent is that it can be used to image
`multiple organ systems including the vascular system, liver, spleen, bone marrow, and
`
`10
`
`15
`
`20
`
`25
`
`lymph nodes.
`Another embodiment of the invention provides a reduced polysaccharide iron
`oxide complex for use as an intravenous iron supplement.
`Another embodiment of the invention provides a reduced derivatized
`polysaccharide iron oxide complex for use as an intravenous iron supplement.
`In yet a further embodiment, the invention provides an improved method of
`administering to a mammalian subject an autoclaved reduced polysaccharide iron oxide
`complex. The improved method of administration comprising: injection of an
`autoclaved reduced polysaccharide iron oxide complex in a volume of 15 ml or less. In
`another aspect of the embodiment the injected volume is injected as a bolus. In a
`further aspect of the method, the iron oxide is superparamagnetic. In a further aspect of
`the embodiment the injected volume provides improved image quality.
`In yet a further embodiment, the invention provides an improved method of
`administering to a mammalian subject an autoclaved derivatized reduced
`polysaccharide iron oxide complex. The improved method of administration
`comprising: injection of an autoclaved reduced derivatized polysaccharide iron oxide
`complex in a volume of 15 ml or less. In another aspect of the embodiment the
`injected volume is injected as a bolus. In a further aspect of the method, the iron oxide
`is superparamagnetic. In a further aspect of the embodiment the injected volume
`provides improved image quality.
`
`4
`
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`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 6
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`

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`WO 00/61191
`
`PCT/US00/06047
`
`An embodiment of the invention provides an improved method of administering
`to a mammalian subject a reduced polysaccharide iron complex in a manner that the
`composition provides reduced toxicity, wherein the improvement comprises utilizing a
`reduced polysaccharide in formulation of the composition. In a further aspect of the
`embodiment, the iron oxide is superparamagnetic.
`An embodiment of the invention provides an improved method of administering
`to a mammalian subject a reduced derivatized polysaccharide iron complex in a manner
`that the composition provides reduced toxicity, wherein the improvement comprises
`utilizing a reduced derivatized polysaccharide in formulation of the composition. In a
`further aspect of the embodiment, the iron oxide is superparamagnetic.
`An embodiment of the invention provides a reduced polysaccharide iron oxide
`complex, wherein the reduced polysaccharide is derivatized, for example, the reduced
`derivatized polysaccharide is a carboxyalkyl polysaccharide. The carboxyalkyl is
`selected from the group consisting of carboxymethyl, carboxyethyl and carboxypropyl.
`Further, the reduced polysaccharide can be a reduced dextran, for example, the reduced
`dextran can be a reduced carboxymethyl dextran. A further aspect of this embodiment
`of the invention is that the level of derivatization of the reduced dextran is at least 750
`µmole but less than 1500 µmole of carboxyl groups per gram of polysaccharide wherein
`said composition has reduced toxicity relative to composition with respect to lower
`levels of derivatization.
`An embodiment of the invention provides a reduced polysaccharide iron oxide
`complex, such complex being stable at a temperature of at least approximately 100°C.
`In a preferred embodiment, such complex is stable at a temperature of approximately
`121 °C. In an even more preferred aspect of the reduced polysaccharide iron oxide
`complex, such complex is stable at a temperature of at least 121 °C for a time sufficient
`to sterilize the complex. In a further aspect of the embodiment, the iron oxide is
`
`5
`
`10
`
`15
`
`20
`
`25
`
`superparamagnetic.
`An embodiment of the invention provides a reduced derivatized polysaccharide
`iron oxide complex, such complex being stable at a temperature of at least
`approximately 100°C. In a preferred embodiment, such complex is stable at a
`
`30
`
`5
`
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`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 7
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`WO 00/61191
`
`PCT/US00/06047
`
`temperature of approximately 121 °C. In an even more preferred aspect of the reduced
`polysaccharide iron oxide complex, such complex is stable at a temperature of at least
`121 °C for a time sufficient to sterilize the complex. In a further aspect of the
`
`5
`
`15
`
`embodiment, the iron oxide is superparamagnetic.
`A preferred embodiment of the invention is a method of formulating for
`pharmacological use a reduced polysaccharide iron oxide complex having increased pH
`stability in comparison to the corresponding native dextran iron oxide, the method
`comprising: providing dextran; and reacting the dextran with a borohydride salt or
`hydrogen in the presence of an hydrogenation catalyst, reacting the reduced dextran
`10 with iron salts to provide a formulation having a stable pH.
`A preferred embodiment of the invention is a method of formulating for
`pharmacological use a reduced derivatized polysaccharide iron oxide complex having
`increased pH stability in comparison to the corresponding native dextran iron oxide, the
`method comprising: providing dextran; and reacting the dextran with a borohydride salt
`or hydrogen in the presence of an hydrogenation catalyst, reacting the reduced dextran
`with iron salts to provide a formulation having a stable pH.
`In another embodiment, the invention provides a method of formulating a
`reduced derivatized dextran composition for pharmacological use wherein the
`composition has decreased toxicity in comparison to native dextran, comprising:
`20 producing a reduced derivatized polysaccharide; and sterilizing the product by
`autoclaving. According to this method, the reduced polysaccharide is obtained by
`reacting the native polysaccharide with one of several reducing agents selected from the
`group consisting of a borohydride salt, or hydrogen in the presence of a hydrogenation
`catalyst. In a preferred aspect of the embodiment the polysaccharide is dextran.
`25 Producing the composition can include derivatizing a reduced polysaccharide by
`carboxyalkylation, for example, wherein the carboxyalkylation is a carboxymethylation.
`Further according to this method, the reduced polysaccharide can be a reduced dextran.
`The derivatized, reduced polysaccharide can be isolated as the sodium salt and does not
`contain an infrared absorption peak in the region of 1650-1800 cm· 1
`• In one aspect of the
`30 method, producing the derivatized reduced polysaccharide is achieved at a temperature
`
`6
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 8
`
`

`

`WO 00/61191
`
`PCT/US00/06047
`
`of less than approximately 50°C. In another aspect of the method, producing the
`derivatized reduced polysaccharide is achieved at a temperature of less than
`approximately 40°C.
`An embodiment of the invention provides an improved method of administering
`to a mammalian subject a reduced derivatized polysaccharide in a manner that the
`composition provides reduced toxicity, wherein the improvement comprises utilizing a
`reduced polysaccharide in formulation of the composition.
`An embodiment of the invention provides a reduced polysaccharide, wherein the
`reduced polysaccharide is derivatized, for example, the reduced derivatized
`polysaccharide is a carboxyalkyl polysaccharide. The carboxyalkyl is selected from the
`group consisting of carboxymethyl, carboxyethyl and carboxypropyl. Further, the
`reduced polysaccharide can be a reduced dextran. A further aspect of this embodiment
`of the invention is that the level of derivatization of the reduced dextran is at least 750
`micromolar of carboxyl groups per gram of polysaccharide wherein said composition
`has reduced toxicity relative to composition with lower levels of derivatization.
`Another embodiment of the invention is a method of formulating a dextran
`composition for pharmacological use and having decreased toxicity in comparison to
`native dextran, the method comprising: providing dextran; and reacting the provided
`dextran with a borohydride salt or hydrogen in the presence of an hydrogenation catalyst
`followed by carboxymethylation, the reduced carboxymethylated dextran having
`decreased toxicity.
`Another embodiment of the invention is an improved method of administering to
`a mammalian subject a polysaccharide composition of the type wherein the composition
`includes dextran in a manner that the composition provides reduced toxicity, wherein
`the improvement comprises utilizing reduced carboxymethylated dextran in lieu of
`dextran in the formulation. In another aspect, an embodiment of the invention is an
`improved method of administering to a mammalian subject a polysaccharide in a
`manner that the composition provides reduced toxicity, wherein the improvement
`comprises utilizing a reduced carboxymethylated polysaccharide in formulation of the
`composition.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`7
`
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`Pharmacosmos A/S v. American Regent, Inc.
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`

`

`WO 00/61191
`
`PCT/US00/06047
`
`An embodiment of the invention provides a method of use of reduced
`
`derivatized dextrans as blood expanders.
`
`Brief Description of the Drawings
`
`Figure 1 shows a Fourier transform infrared (FTIR) spectrographic analysis of
`
`5
`
`carboxyrnethyl reduced dextran (CMRD) sodium salt obtained with Example 5.
`
`Figure 2 shows an FTIR spectrographic analysis of sodium salt CMRD coated
`
`ultrasmall superparamagentic iron oxide (USPIO; see U.S. Patent 5,055,288) obtained
`
`in Example 31.
`Figure 3 is a graph that shows the amount of carboxyrnethyl groups (micromoles)
`
`10
`
`per gram of product, on the ordinate, as a function of the amount of bromoacetic acid
`mg/gram used in reactions with reduced dextran starting material, on the abscissa. The
`
`15
`
`20
`
`25
`
`graph is plotted from the data of Table 2.
`Figure 4 shows pharrnacokinetics of CMRD coated USPIO in the blood of three
`male rats following intravenous administration of 2.2 mg of iron per kg body weight.
`Samples (0.25 ml) of blood were collected at the times indicated on the abcissa, and
`
`relaxation times were measured on a Brucker Minispec spectrometer.
`Figure 5 shows the graph used to determine a half-life (67 minutes) of CMRD
`
`coated USPIO in rat blood. The data of Figure 4 were used to generate the graph in
`Figure 5. The half-life range of 61 to 75 minutes was within the 95% confidence level.
`Figure 6 shows MRis of a rat, pre-administration (A) and post-administration (B)
`
`of contrast agents, anterior portion at top. CMRD coated USPIO (5 mg of iron per kg
`
`body weight) was administered into the femoral vein prior to taking the post
`administration contrast image. The figure illustrates enhanced visualization of the heart
`and surrounding arteries and veins caused by administration of CMRD coated USPIO.
`Imaging was performed using a General Electric 2 Tesla magnetic resonance imager.
`
`Figure 7 shows MRI images of a pig, pre-administration (A) and post(cid:173)
`
`administration (B) of contrast agent, anterior portion at top. CMRD coated USPIO
`(Example 31; 4 mg of iron per kg body weight) was administered into the femoral vein
`prior to taking the post administration contrast image. The figure illustrates enhanced
`
`30
`
`visualization of the heart and surrounding arteries and veins caused by administration of
`
`8
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`PCT/US00/06047
`
`CMRD coated USPIO. Imaging was performed using a Siemans 1.5T Magnatom
`
`Vision magnetic resonance imager.
`Figure 8 shows MRI images of the anterior portion of a normal human subject,
`pre-administration (A) and post-administration (B) of contrast imaging agent. CMRD
`coated USPIO (4 mg of iron per kg body weight) was administered as a bolus into a
`vein in the arm prior to taking the post contrast image. Imaging was performed 15 to 30
`minutes after administration of contrast agent. The image illustrates enhanced
`visualization of the heart and surrounding arteries and veins.
`Figure 9 shows the blood clearance kinetics in humans of imaging agent. CMRD
`coated USPIO ( 4 mg of iron per kg body weight), was administered as a bolus into a
`vein in the arm prior to taking blood samples. Samples were analyzed for 1/f2
`relaxation to determine the blood concentration of the CMRD coated USPIO. The
`graph shows CMRD coated US PIO concentration (ordinate) as a function of time
`
`(abscissa).
`
`Detailed Description of Specific Embodiments
`Table 1 summarizes the characteristics of two classes of MRI contrast agents that
`have been previously described, and shows a comparison of their characteristics to
`those of an ideal contrast agent. Agents of the invention embody the ideal
`
`5
`
`10
`
`15
`
`20
`
`characteristics, as shown herein.
`Surprisingly, the development and synthesis of preparations of ultrasmall
`superparamagnetic iron oxide (USPIOs) coated with polysaccharide reduced dextrans
`and derivatives of reduced dextrans, such as the agents with the desirable properties as
`shown herein, are derived from a change in the chemical nature of one constituent,
`dextran TlO. This change involved reduction of the terminal aldehyde group to an
`alcohol of the polysaccharide used in its synthesis to an alcohol (Scheme 1 ). Scheme 1
`illustrates the chemical change in a polysaccharide such as dextran upon treatment with
`sodium borohydride. The hemiacetal form of the polysaccharide (structure 1) is in
`equilibrium with the aldehyde form of the polysaccharide (structure 2). Structure 2
`represents less than 0.01 % of the equilibrium mixture (Brucker, G. (1974) Organic
`30 Chemistry: Amino Acids, Peptides and Carbohydrates., Tankonykiado Press, Budapest,
`
`25
`
`9
`
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`WO 00/61191
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`
`p. 991 ). Treatment of structure 2 with sodium borohydride results in its irreversible
`conversion to the linear polyol form of the polysaccharide (structure 3). The dynamic
`equilibrium between structures 1 and 2 allows complete conversion, when treated with
`
`sodium borohydride, to the linear polyol (structure 3).
`
`5
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`Scheme 1:
`
`0 OH
`0
`OH
`OH
`OH~~
`O 0
`O O
`OH
`OH
`
`OH
`
`n
`
`O
`
`OH
`0
`CHO
`OH
`OH
`OH~~
`0
`0
`0
`0
`OH
`OH
`
`OH
`
`n
`
`0
`
`0
`
`OH
`O O
`OH
`
`NaBH4 - OH~~
`
`0
`
`OH
`OH
`
`OH
`
`O 0
`OH
`
`n
`
`OH
`
`2
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`3
`
`Dextran coated superparamagnetic iron oxide particles have particular interest as
`magnetic resonance imaging (MRI) contrast agents because of their ability to enhance
`images of the liver and lymph. Feridex I.V.® (Advanced Magnetics, Inc., Cambridge
`10 MA) is a dextran coated superparamagnetic iron oxide MRI contrast agent, and
`approved for use in humans. Combidex® (Advanced Magnetics, Inc.) is a dextran
`coated ultrasmall superparamagnetic iron oxide (USPIO) which has completed Phase ill
`clinical trials for both liver imaging and Phase ill trials for lymph imaging. Combidex®
`has a smaller mean diameter (20 nm) than Feridex I.V.® (60 nm), which gives it a
`different biodistribution in humans. Combidex® is made by addition of base to a
`solution of dextran, ferric chloride and ferrous chloride. The synthetic process
`comprises combining the ingredients, heating, and purifying by ultrafiltration.
`However, the yield of dextran added to the particles in the reaction is inefficient.
`Pharmaceutical grade dextran is the most expensive component of the Combidex®
`synthesis. A more efficient use of dextran in the synthesis of Combidex® is desirable to
`
`15
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`20
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`lower production costs.
`Terminal sterilization (autoclaving) is a preferred method of sterilizing drugs for
`injection. However, many superparamagnetic iron oxide colloids that are used as MRI
`contrast agents are synthesized with polymer coatings and coverings that influence the
`biodistribution and elimination of these colloids. Upon exposure to the heat for the
`duration of the autoclaving process. the polymer coating can become dissociated from
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`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 12
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`WO00/61191
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`PCT /US00/0604 7
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`15
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`the iron oxide cores. The functional consequences of polymer dissociation from the
`iron oxide are physical changes in the material, such as clumping, biodistribution
`changes (changes in plasma half life), and changes in toxicity profile (potential
`increases in adverse events). For example, a substantial decrease in the pH of the
`solution can be detected following autoclaving of iron dextran particles, and the pH
`continues to fall upon further storage.
`Several solutions to the problem of imparting resistance to heat stress have been
`described. Palmacci et al., U.S. Patent 5,262,176, hereby incorporated herein by
`reference, used crosslinked dextran to stabilize the covering on the iron oxide particles
`prior to autoclaving. The crosslinking process uses noxious agents such as
`epichlorohydrin and epibromohydrin, which must be removed from the colloid after the
`crosslinking reaction.
`Methods of preventing clumping of the colloid induced by heat stress that have
`no effect on coating dissociation have also been described. These methods generally
`include the use of excipients during the autoclaving process. Groman et al., U.S. Patent
`4,827,945, and Lewis et al., U.S. Patent 5,055,288, both patents hereby incorporated
`herein by reference, use citrate to prevent clumping of the particles when the coating
`dissociates. However, the use of citrate in high concentrations in combination with heat
`can cause toxicity. Groman et al., U.S. Patent 5,102,652, hereby incorporated herein by
`reference, uses low molecular weight carbohydrates such as mannitol to prevent
`clumping during autoclaving. These excipients increase the cost and complexity of
`manufacturing the product, yet do not solve the problem of dissociation of the polymer
`from the iron particle.
`Josephson et al., U.S. Patent 5,160,726, hereby incorporated herein by reference,
`avoids heat stress on the coating by using filter sterilization rather than heat to sterilize
`the colloid. Filter sterilization is expensive since both the sterilization process and
`container closure must be performed in a germ free environment. Additionally, filter
`sterilizing has a higher rate of failure than the process of autoclaving, which reflects the
`inability to obtain an environment for the filtration step that is entirely germ free.
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`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1017 - Page 13
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`WO 00/61191
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`PCT /US00/06047
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`Maruno et al., U.S. Patent 5,204,457, describes a carboxymethyl-dextran coated
`
`particle with improved stability up to 80°C for an extended period but does not teach
`
`use of terminal sterilization by autoclaving. Hasegawa et al. (Japan J. Appl. Phys., Part
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`1, 37(3A): 1029-1032, 1998) describes carboxymethyl dextran coated iron particles with
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`thermal stability at 80°C, but does not teach use of a carboxymethyl reduced dextran
`
`coated particle, nor of terminal sterilization by autoclaving.
`
`Magnetic resonance imaging agents act by affecting the normal relaxation times,
`
`principally on the protons of water. There are two types of relaxation, one known as
`
`spin-spin or Tl relaxation, and the second known as spin-lattice or T2 relaxation. Tl
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`relaxation generally results in a brightening of the image caused by an increase in
`
`signal. T 1 processes are most useful in imaging of the vascular system. T2 relaxation
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`generally results in a darkening of the image caused by a decrease in signal. T2
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`processes are most useful in imaging of organs such as the liver, spleen, or lymph nodes
`
`that contain lesions such as tumors. All contrast agents have both Tl and T2 properties;
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`however, either Tl or T2 relaxation can characterize the dominant relaxation property
`
`of a particular contrast agent. Low molecular weight gadolinium based contrast agents
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`are T 1 agents, and have primary application in the imaging of vascular related medical
`
`problems such as stroke and aneurysms and the brain. Iron oxide based colloidal
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`contrast agents are T2 agents, and have primary application in imaging tumors of the
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`liver and lymph nodes (prostate and breast cancer). An agent possessing both Tl and
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`T2 properties would be desirable. Using such an agent would (I) provide a single drug
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`for all applications, and simplify the inventory of the pharmacy, (ii) simplify imaging in
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`the MRI suite, and (iii) improve patient care by permitting simultaneous examination of
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`multiple medical problems in a single patient during a single examination, rather than
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`requiring use of either a Tl or a T2 contrast agent.
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`A dextran can elicit a sometimes fatal anaphylactic response when administered
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`intravenously (i.v.) in man (Briseid, G. et al., Acta Phanncol. et Toxicol., 1980, 47: 119-
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`126; Hedin, H. et al., Int. Arch. Allergy and Immunol., 1997:113:358-359). Related
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`adverse reactions have been observed also on administration of magnetic dextran coated
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`iron oxide colloids. Non-magnetic dextran coated iron oxide colloids that have utility
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`Petitioner Ex. 1017 - Page 14
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`PCT/US00/06047
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`as hematinics, particularly as an adjunct to erythropoietin treatment