(12)
`
`United States Patent
`Groman et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,599,498 B1
`Jul. 29, 2003
`
`US006599498B1
`
`(54) HEAT STABLE COLLOIDAL IRON OXIDES
`COATED WITH REDUCED
`CARBOHYDRATES AND CARBOHDRATE
`
`6,165,378 A 12/2000 Maruno et a1. ........ .. 252/6253
`
`FOREIGN PATENT DOCUMENTS
`
`........... .. B03C/1/00
`12/1986
`0 230 768 B1
`......... .. C08B/11/12
`10/1991
`0 450 092
`....... .. G01N/33/543
`7/1991
`W0 91 09678
`........ .. A61K/49/00
`4/1996
`W0 96 09840
`....... .. A61K/31/715
`6/2000
`W0 00 30657
`OTHER PUBLICATIONS
`
`DERIVATIVES
`
`EP
`EP
`W0
`(75) Inventors: Ernest V. Groman, Brookline, MA
`W0
`(Us); Kenneth G_ Paul, Holliston, M A
`(Us); Timothy B_ Frigo, Waltham, MA W0
`(US); Howard Bengele, Canton, MA
`(US); Jerome M. Lewis, Newton, MA
`(Us)
`
`(73) Assigneej Advanced Magnetics, Inc,’ Cambridge,
`MA (Us)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) APPL NO; 09/521,264
`
`(22) Filed?
`
`Mar- 8! 2000
`
`Related US. Application Data
`(60) Provisional application No. 60/128,579, ?led on Apr. 9,
`1999'
`(51) Int. Cl.7 ........................ .. A61B 5/055; A61K 9/16;
`A61K 9/50; A61K 31/70; A61K 31/715;
`A01N 43/04
`(52) US. Cl. ................... .. 424/9.34; 424/9.35; 424/493;
`514/54; 514/59
`_
`(58) Field of Search ............................... .. 424/9.3, 9.32,
`424/935, 493; 514/54, 59
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,770,183 A
`4,827,945 A
`
`9/1988 Groman et a1. ........... .. 128/654
`5/1989 Groman et a1. ........... .. 128/653
`
`. . . . .. 424/9
`5,055,288 A 10/1991 Lewis et a1. . . . . . . . . .
`424/9
`5,102,652 A
`4/1992 Groman et a1. .... ..
`424/9
`5,128,121 A * 7/1992 Berg etal. ......... ..
`.... .. 424/9
`5,160,726 A 11/1992 Josephson et a1.
`5,204,457 A
`4/1993 Maruno et a1. ........... .. 536/101
`5,262,176 A 11/1993 Palmacci et a1. ............ .. 424/9
`5,985,245 A * 11/1999 Golman et a1. .......... .. 424/9.36
`
`Grimm, Jan et a1. “Characterization of Ultrasmall, Paramag
`netic Magnetite Particles as Superparamagnetic Contrast
`Agents in MRI”, Database Chemabs [Online]: Invest.
`RadioL, 2000, vol. 35(9), pp. 553—556.
`Voorhees, A.B. et al., Proc. Soc. Exp. Biol. Med. 1951,
`76:254.
`Squire, J .R. et al., in “Dextran, Its Properties and Use in
`Medicine” Charles C Thomas, Spring?eld, IL, 1955.
`Hanna, C.H. et al., Am. J. Physiol. 1957, 191:615.
`Briseid, G. et al., Acta Pharmcol. Et toxicaL, 1980,
`47:119—126.
`Kitchen, R.,Proc. Sugar Process. Res. Conf, 1983, 232—47.
`Jue, C. K. et al., J. Biochem. Biophys. Methods, 1985,
`1110945
`Hedin, H' et all” Int Arch Allergy and ImmlmOL)
`1997:113:358—359.
`Kumar K., J. Liq. Chromatogr Relat. TehcnoL, 1997, 20,
`3351—3364.
`HasegaWa et a1. Japan J. Appl. Phys., 1998, Part
`II37(3A)I1029
`.
`*
`.
`cited by examiner
`
`Primary Examiner—Sreeni Padmanabhan
`(74) Attorney, Agent, or Firm—Bromberg & Sunstein LLP
`(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 hema
`tinic agent, the agents comprising carboxyalkylated reduced
`polysaccharides coated ultrasmall superparamagnetic iron
`oxides. Methods of use of the carboxymethyl reduced dex
`tran as a plasma extender are provided.
`
`26 Claims, 12 Drawing Sheets
`
`Pharmacosmos, Exh. 1016, p. 1
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 1 0f 12
`
`US 6,599,498 B1
`
`
`
`:59 55385
`
`Q8 8.9 82 8.8 8% 8.8 8mm 83
`
`F .6E
`
`Pharmacosmos, Exh. 1016, p. 2
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 2 0f 12
`
`US 6,599,498 B1
`
`com
`
`
`
`A129 GEDEE
`
`m ._@E
`
`Pharmacosmos, Exh. 1016, p. 3
`
`

`
`U.S. Patent
`
`Jul. 29,2003
`
`Sheet 3 0f 12
`
`US 6,599,498 B1
`
`
`
`QmE A23 uzmumcEe?
`
`- - _ - n n - n -
`
`E on 3 mm 3 mm mm 3 Nu cm W . mu _ n_
`
`- can?
`
`I 8.3.
`
`1 came
`
`- cc:
`
`
`
`- 82 9.56 9.2.1.502
`
`Pharmacosmos, Exh. 1016, p. 4
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 4 0f 12
`
`US 6,599,498 B1
`
`
`
`
`
`
`
`wowowowm $.04 wmww wQOO
`
`2m 2m Q2 Q2 cm? 8 8 0m
`
`
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`
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`Q CE W. $2 Em _H_ 5 Em @
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`
`Pharmacosmos, Exh. 1016, p. 5
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`21f05LI.66_.nS
`
`US 6,599,498 B1
`
`mmmuH.500
`
`
`
`Svomomfi._.O._
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`....--4.----Iw.m.5z_._\§3.3.31-----mz:mm<m-S:
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`
`m._®_n_
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`
`Pharmacosmos, Exh. 1016, p. 6
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 6 0f 12
`
`US 6,599,498 B1
`
`HG, 6A
`
`Pharmacosmos, Exh. 1016, p. 7
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 7 0f 12
`
`US 6,599,498 B1
`
`FIG. 6B
`
`Pharmacosmos, Exh. 1016, p. 8
`
`

`
`U S Patent
`
`Jul. 29, 2003
`
`Sheet 8 0f 12
`
`US 6,599,498 B1
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`Pharmacosmos, Exh. 1016, p. 9
`
`

`
`U S Patent
`
`Jul. 29, 2003
`
`Sheet 9 0f 12
`
`US 6,599,498 B1
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`PEG. 7
`
`Pharmacosmos, Exh. 1016, p. 10
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 10 0f 12
`
`US 6,599,498 B1
`
`FIG. 8A
`
`Pharmacosmos, Exh. 1016, p. 11
`
`

`
`U.S. Patent
`
`Jul. 29, 2003
`
`Sheet 11 0f 12
`
`US 6,599,498 B1
`
`FEG.
`
`1
`
`Pharmacosmos, Exh. 1016, p. 12
`
`

`
`U.S. Patent
`
`Jul. 29,2003
`
`Sheet 12 0f 12
`
`US 6,599,498 B1
`
`BLOOD CLEARANCE IN HUMANS AT 4 mg/kg
`
`160
`
`(ug/mL)
`
`140
`120 E
`\
`100
`\
`
`80
`
`6O
`
`CONCENTRATION
`
`0
`
`20
`
`40
`TIME (HOURS)
`FIG. 9A
`
`60
`
`BLOOD CLEARANCE IN HUMANS AT 4 mg/kg
`
`160
`
`(ug/mL)
`
`CONCENTRATION
`
`O
`
`T
`
`0.0 OI1
`
`\
`\
`\
`\
`M
`0.2 0.3 OI5 ‘IIO 4IO BIO 24I.O 48LO 72I.O16I8.O
`TIME (HOURS)
`FIG. 9B
`
`|
`
`l
`
`Pharmacosmos, Exh. 1016, p. 13
`
`

`
`US 6,599,498 B1
`
`1
`HEAT STABLE COLLOIDAL IRON OXIDES
`COATED WITH REDUCED
`CARBOHYDRATES AND CARBOHDRATE
`DERIVATIVES
`
`RELATED APPLICATIONS
`This application claims the bene?t of Provisional Appli
`cation No. 60/128,579, ?led in the United States Patent and
`Trademark Of?ce on Apr. 9, 1999, and Which is hereby
`incorporated by reference herein.
`TECHNICAL FIELD
`The ?eld relates to compositions Which are carboxym
`ethyl 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.
`
`BACKGROUND
`Since the invention of magnetic 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 devel
`oped and adopted in clinical practice. These are: loW
`molecular Weight gadolinium complexes such as Mag
`navist®; and colloidal iron oxides. Neither of these tWo
`types of agents is ideal. Problems encountered With these
`agents are shoWn in Table 1, and include: expense of
`components; inef?ciency of synthesis; loss of coating if
`sterilized by autoclaving; narrow range of organ uptake for
`purposes of imaging; side-effects; restriction of use to either
`?rst 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.
`
`1O
`
`15
`
`25
`
`35
`
`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 molecular Weight
`gadolinium
`
`colloidal
`iron oxides
`
`45
`
`LoW production costs:
`ef?cient synthesis
`Autoclavable Without
`excipients
`T1 agent
`T2 agent
`Non toxic at vast excess
`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
`
`Yes
`
`Yes
`
`Yes
`No
`Yes
`No
`
`No
`
`Yes
`
`Yes
`No
`No
`
`No
`
`No
`
`Sometimes
`Yes
`No
`No
`
`No
`
`Sometimes
`
`No
`No
`Yes
`
`55
`
`SUMMARY
`An embodiment of the invention is a method of providing
`an iron oxide complex for administration to a mammal
`
`65
`
`2
`subject, the method comprising: producing a reduced
`polysaccharide iron oxide complex, and steriliZing the com
`plex 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 produced through reaction of a polysac
`charide With a reagent selected from the group consisting of
`a borohydride salt or hydrogen in the presence of a hydro
`genation 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 complex for administra
`tion to a mammalian subject, the method comprising: pro
`ducing 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
`caboxyalkylation, for example, Wherein the carboxyalkyla
`tion is a carboxymethylation. The term “derivatiZing” and
`related terms (eg derivatives, derivatiZed, derivatiZation,
`etc) refer to the conventional sense of functionaliZation at
`the reactive sites of the composition. 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 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 polysaccharide is
`achieved at a temperature of less than approximately 40° C.
`In a further aspect of the method, the iron oxide is super
`paramagnetic.
`In yet another embodiment, the invention provides a
`method of formulating an iron oxide complex coated With a
`reduced polysaccharide. This composition is for pharmaco
`logical 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 iron oxide complex, and steriliZing the com
`plex by autoclaving. The formulation provides polysaccha
`ride 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 has such decreased toxicity. In a further
`aspect of the method, the iron 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 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 carboxyalky
`lation is a carboxymethylation. Further according to this
`method, the reduced polysaccharide can be a reduced dex
`tran. The derivatiZed, reduced polysaccharide can be iso
`lated 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
`
`Pharmacosmos, Exh. 1016, p. 14
`
`

`
`US 6,599,498 B1
`
`3
`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 super
`paramagnetic.
`Another embodiment of the invention provides a reduced
`derivatiZed polysaccharide iron oxide complex With T1 and
`T2 relaxation properties to alloW contrast agent signal
`enhancement With T1 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 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: injec
`tion 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 super
`paramagnetic. 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 compris
`ing: injection of an autoclaved reduced derivatiZed polysac
`charide 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.
`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 compo
`sition 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 super
`paramagnetic.
`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 polysaccha
`ride. 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 carboxym
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`ethyl dextran. A further aspect of this embodiment of the
`invention is that the level of derivatiZation of the reduced
`dextran is at least 750 pmole but less than 1500 pmole 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 tem
`perature 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 1210 C.
`for a time sufficient to steriliZe the complex. In a further
`aspect of the embodiment, the iron oxide is superparamag
`netic.
`An embodiment of the invention provides a reduced
`derivatiZed polysaccharide iron oxide complex, such com
`plex 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 super
`paramagnetic.
`A preferred embodiment of the invention is a method of
`formulating for pharmacological use a reduced polysaccha
`ride 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.
`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; com
`prising: 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. Producing the composition can include deriva
`tiZing a reduced polysaccharide by carboxyalkylation, for
`example, Wherein the carboxyalkylation is a carboxymethy
`lation. 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 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 polysaccharide is achieved at a temperature of less
`than approximately 40° C.
`
`Pharmacosmos, Exh. 1016, p. 15
`
`

`
`US 6,599,498 B1
`
`5
`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 com
`prises 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 polysac
`charide 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 deriva
`tiZation 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 carboxym
`ethylated dextran having decreased toxicity.
`Another embodiment of the invention is an improved
`method of administering to a mammalian subject a polysac
`charide composition of the type Wherein the composition
`includes dextran in a manner that the composition provides
`reduced toxicity, Wherein the improvement comprises uti
`liZing 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 carboxymethy
`lated polysaccharide in formulation of the composition.
`An embodiment of the invention provides a method of use
`of reduced derivatiZed dextrans as blood expanders.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shoWs a Fourier transform infrared (FTIR) spec
`trographic analysis of carboxymethyl reduced dextran
`(CMRD) sodium salt obtained With Example 5.
`FIG. 2 shoWs an FTIR spectrographic analysis of sodium
`salt CMRD coated ultrasmall superparamagnetic iron oxide
`(USPIO; see US. Pat. No. 5,055,288) obtained in Example
`31.
`FIG. 3 is a graph that shoWs the amount of carboxymethyl
`groups (micromoles) 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 graph is plotted from the data of Table 2.
`FIG. 4 shoWs pharmacokinetics of CMRD coated USPIO
`in the blood of three male rats folloWing intravenous admin
`istration 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.
`FIG. 5 shoWs the graph used to determine a half-life (67
`minutes) of CMRD coated USPIO in rat blood. The data of
`FIG. 4 Were used to generate the graph in FIG. 5. The
`half-life range of 61 to 75 minutes Was Within the 95%
`con?dence level.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`FIG. 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 ?gure
`illustrates enhanced visualiZation of the heart and surround
`ing arteries and veins caused by administration of CMRD
`coated USPIO. Imaging Was performed using a General
`Electric 2 Tesla magnetic resonance imager.
`
`FIG. 7 shoWs MRI images of a pig, pre-administration (A)
`and post-administration (B) of contrast agent, anterior por
`tion 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
`?gure illustrates enhanced visualiZation of the heart and
`surrounding arteries and veins caused by administration of
`CMRD coated USPIO. Imaging Was performed using a
`Siemans 1.5T Magnatom Vision magnetic resonance imager.
`
`FIG. 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.
`
`FIG. 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/T 2 relaxation to determine the blood concentration of
`the CMRD coated USPIO. The graph shoWs CMRD coated
`USPIO 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 characteristics, as shoWn herein.
`Surprisingly, the development and synthesis of prepara
`tions 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 T10. 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 alde
`hyde form of the polysaccharide (structure 2). Structure 2
`represents less than 0.01% of the equilibrium mixture
`(Brucker, G. (1974) Organic Chemistry: Amino Acids, Pep
`tides and Carb0hydrates., Tankonykiado Press, Budapest, 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 equilib
`rium betWeen structures 1 and 2 alloWs complete
`
`Pharmacosmos, Exh. 1016, p. 16
`
`

`
`US 6,599,498 B1
`
`7
`conversion, When treated With sodium borohydride, to the
`linear polyol (structure 3).
`
`Scheme 1:
`
`OH
`
`OH
`
`0
`
`o
`
`OH
`
`OH
`
`0
`
`o
`
`OH
`
`o
`
`0 OH
`
`OH
`
`OH —
`
`OH
`
`0
`
`OH
`
`OH H
`
`OH
`
`1
`
`0
`
`OH
`
`OH
`
`OH
`
`NaBH
`CH0 _4,
`
`OH
`
`0
`
`OH
`
`OH H
`
`OH
`
`2
`
`OH
`
`OH
`
`OH
`
`20
`
`25
`
`30
`
`8
`Several solutions to the problem of imparting resistance to
`heat stress have been described. Palmacci et al., US. Pat.
`No. 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 pro
`cess 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. Pat. No. 4,827,945, and Lewis et al., US. Pat. No.
`5,055,288, both patents hereby incorporated herein by
`reference, use citrate to prevent clumping of the particles
`When the coating dissociates. Groman et al., US. Pat. No.
`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., US. Pat. No. 5,160,726, hereby incor
`porated herein by reference, avoids heat stress on the coating
`by using ?lter steriliZation rather than heat to steriliZe the
`colloid. Filter steriliZation is expensive since both the ster
`iliZation process and container closure must be performed in
`a germ free environment. Additionally, ?lter steriliZing has
`a higher rate of failure than the process of autoclaving,
`Which re?ects the inability to obtain an environment for the
`?ltration step that is entirely germ free.
`Maruno et al., US. Pat. No. 5,204,457, describes a
`carboxymethyl-dextran coated particle With improved sta
`bility 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 1, 37(3A):1029—1032, 1998)
`describes carboxymethyl dextran coated iron particles With
`thermal stability at 80° C., but does not teach use of a
`carboxymethyl reduced dextran coated particle, nor of ter
`minal 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
`T1 relaxation, and the second knoWn as spin-lattice or T2
`relaxation. T1 relaxation generally results in a brightening of
`the image caused by an increase in signal. T1 processes are
`most useful in imaging of the vascular system. T2 relaxation
`generally results in a darkening of the image caused by a
`decrease in signal. T2 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
`T1 and T2 properties; hoWever, either T1 or T2 relaxation
`can characteriZe the dominant relaxation property of a
`particular contrast agent. LoW molecular Weight gadolinium
`based contrast agents are T1 agents, and have primary
`application in the imaging of vascular related medical prob
`lems such as stroke and aneurysms and the brain. Iron oxide
`based colloidal contrast agents are T2 agents, and have
`primary application in imaging tumors of the liver and
`lymph nodes (prostate and breast cancer). An agent possess
`ing both T1 and T2 properties Would be desirable. Using
`such an agent Would (I) provide a single drug for all
`applications, and simplify the inventory of the pharmacy, (ii)
`simplify imaging in the MRI suite, and (iii) improve patient
`care by permitting simultaneous examination of multiple
`medical problems in a single patient during a single
`examination, rather than requiring use of either a T1 or a T2
`contrast agent.
`
`35
`
`40
`
`45
`
`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 Mass.) 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 superpara
`magnetic iron oxide (USPIO) Which has completed Phase III
`clinical trials for both liver imaging and Phase III 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 combin
`ing the ingredients, heating, and purifying by ultra?ltration.
`HoWever, the yield of dextran added to the particles in the
`reaction is inef?cient. Pharmaceutical grade dextran is the
`most expensive component of the Combidex® synthesis. A
`50
`more ef?cient use of dextran in the synthesis of Combidex®
`is desirable to loWer production costs.
`Terminal steriliZation (autoclaving) is a preferred method
`of steriliZing drugs for injection. HoWever, many superpara
`magnetic iron oxide colloids that are used as MRI contrast
`agents are synthesiZed With polymer coatings and coverings
`that in?uence the biodistribution and elimination of these
`colloids. Upon exposure to the heat for the duration of the
`autoclaving process, the polymer coating can become dis
`sociated from the iron oxide cores. The functional conse
`quences of polymer dissociation from the iron oxide are
`physical changes in the material, such as clumping, biodis
`tribution changes (changes in plasma half life), and changes
`in toxicity pro?le (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.