United States Patent [19]
`Lawrence et al.
`
`US005624668A
`Patent Number:
`Date of Patent:
`
`[11]
`
`[45]
`
`5,624,668
`Apr. 29, 1997
`
`[RON DEXTRAN FORMULATIONS
`
`OTHER PUBLICATIONS
`
`P. Carthew et al., Hepatology, vol. 13, No. 3, pp. 534-539
`(1991): Rapid Induction of Hepatic Fibrosis in the Gerbil
`After the Parenteral Administration of Iron-Dextran Com
`pleX.
`L.M. Fletcher, et al., Gastroenterology, vol. 97, pp.
`1011-1018 (1989): Effects of Iron Loading on Free Radical
`scavenging Enzymes and Lipid Peroxidation in Rat Liver.
`R.D. Hamstra et al., JAMA, vol. 243. No. 17, pp. 1726-1731
`(1980): Intravenous Iron Dextran in Clinical Medicine.
`P.A. Henderson et al., Blood, vol. 34, No. 3, pp. 357-375
`(1969): Characteristics of Iron Dextran Utilization in Man.
`T. O. Pitts et al., Nephron, vol. 22, pp. 316-321 (1978):
`Hemosiderosis Secondary to Chronic Parenteral Iron
`Therapy in Maintenance Hemodialysis Patients.
`L. R. Weintraub et al., British Journal of Haematology, vol.
`59. pp. 321-331 (1985): Pathogenesis of Hepatic Fibrosis in
`Experimental Iron Overload.
`Package Insert for INFeD (Iron Dextran Injection, USP).
`Package Insert for Imferon (Iron Dextran Injection, USP).
`1994 ASH Abstract Reproduction Form, 36th Annual Meet
`ing. Nashville, TN (1994): “Pharmacokinetics of Iron Dex
`tran In Iron De?cient Dialysis Patients: Evaluation and
`Comparison of Two Agents”. by D. Van Wyck et al.
`1994 ASH Abstract Reproduction Form, 36th Annual Meet
`ing, Nashville, TN (1994): “Iron Mobilization Early After
`Iron Dextran Infusion in Hemodialysis Patients: Evaluation
`and Comparison of 2 Agents”. by D. Van Wyck et al.
`
`Primary Examiner—Peter F. Kulkosky
`Attorney, Agent, or Firm-Morrison & Foerster LLP
`
`ABSTRACT
`[57]
`Ferric oxyhydroxide-dextran compositions for treating iron
`de?ciency having ellipsoidal particles with a preferred
`molecular Weight range of about 250,000 to 300,000 dal
`tons.
`
`28 Claims, 6 Drawing Sheets
`
`[54]
`
`[75]
`
`Inventors: Richard P. Lawrence, Baiting Hollow;
`Ralf A. Lange, Amagansett; Chin Wu,
`Shirley; Mary J. Helenek, Syosset, all
`of N.Y.
`
`[73]
`
`Assignee: Luitpold Pharmaceuticals, Inc.,
`Shirley, N.Y.
`
`[21]
`[22]
`[5 1]
`[52]
`[53]
`
`[5 6]
`
`Appl. No.: 536,984
`Filed:
`Sep. 29, 1995
`
`Int. Cl.6 .......................... .. A01N 59/16; A61K 33/26
`US. Cl. ....................................... .. 424/7817; 424/647
`Field of Search ................................... .. 424/421, 647.
`424/7817
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`Re. 24,642
`2,820,740
`2,885,393
`3,686,397
`3,697,502
`3,908,004
`4,101,435
`4,180,567
`4,452,773
`4,454,773
`4,505,726
`4,599,405
`4,659,697
`4,749,695
`4,788,281
`4,927,756
`5,102,652
`5,118,513
`5,248,492
`5,336,506
`5,354,350
`
`4/1959
`1/1958
`5/1959
`8/1972
`10/1972
`9/1975
`7/1978
`12/1979
`6/1984
`6/1984
`3/1985
`7/1986
`4/1987
`6/1988
`11/1988
`5/1990
`4/1992
`6/1992
`9/1993
`8/1994
`10/1994
`
`London at al. .
`London et al. .
`
`Herb .
`
`Muller .
`
`Christensen .
`Kitching .
`Hasegawa et a1. .
`Herb .
`Molday ................................. .. 436/526
`Brunner et al. .
`Takeuchi et al. .
`Muekker et al. .
`
`Tanaka .
`Schwengers .
`Tosoni et al. .
`Schwengers .
`Groman et al. ,
`Mehansho et al. .
`Groman et al. .
`Josephson et al. ,
`Moore .
`
`Pharmacosmos, Exh. 1010, p. 1
`
`

`

`US. Patent
`
`Apr. 29, 1997
`
`Sheet 1 of 6
`
`5,624,668
`
`_ _
`
`oodw
`OOdN
`
`p
`
`p h
`
`8552
`
`oosvm _
`QQNN
`006m
`
`00
`
`Pharmacosmos, Exh. 1010, p. 2
`
`

`

`SU
`
`a
`
`6
`
`8
`
`P..88
`
`m25:in\w8.8
`
`
`
`z<Exmo287:8.9
`
`9Wm8.Ar._8.:p‘A.8Fzo=<m<mmE..8.2
`WIlz<mhxmo..w29:..8.3
`
`30$:on.
`
`amv28mm..8.mNzo=<m<ammm_.
`
`.Eommzzoo..8.9
`
`6.6.,N0EMmmSzSM,8.88.88.88+...“8.88.8
`
`8....
`
`8a
`
`Pharmacosmos, Exh. 1010, p. 3
`
`Pharmacosmos, Exh. 1010, p. 3
`
`
`

`

`US. Patent
`
`Apr. 29, 1997
`
`Sheet 3 of 6
`
`5,624,668
`
`00.0n
`
`00.0w
`
`00.0w
`
`MMFDZHE
`
`00.09
`
`00.¢N
`
`00.NN
`
`00.0w
`
`760.O2
`
`00.0w
`
`00.3...
`
`00.Nn
`
`00.00
`
`8.8.
`
`00.0w
`
`00.¢N
`
`00.NN
`
`00.0w
`
`00.09
`
`AW
`
`00.0—
`
`00.0F00.NF006;
`
`00.0
`
`00.0
`
`00..—.
`
`00.N
`
`00.0
`
`m.9...
`
`Pharmacosmos, Exh. 1010, p. 4
`
`Pharmacosmos, Exh. 1010, p. 4
`
`

`

`US. Patent
`
`Apr. 29, 1997
`
`Sheet 4 0f 6
`
`5,624,668
`
`_ _ _ _ _ _ _ _ b _ _ _ _ _ P _ _ _ _ _ _ _ p P _ r _ _ _
`
`
`
`
`
`
`
`
`
`
`
`
`
`006m oodm 006w 006w OQNN 00.0w oo.w—
`
`lllllllllTl
`I
`o
`o.
`co
`N
`
`Pharmacosmos, Exh. 1010, p. 5
`
`

`

`U S Patent
`
`Apr. 29, 1997
`
`Sheet 5 of 6
`
`5,624,668
`
`FIG, 5
`
`Pharmacosmos, Exh. 1010, p. 6
`
`

`

`US. Patent
`
`Apr. 29, 1997
`
`Sheet 6 0f 6
`
`5,624,668
`
`Pharmacosmos, Exh. 1010, p. 7
`
`

`

`5,624,668
`
`1
`IRON DEXTRAN FORMULATIONS
`
`FIELD OF THE INVENTION
`
`The present invention relates to improved iron dextran
`formulations for the treatment of iron de?ciency, and to
`methods for preparing such formulations.
`
`BACKGROUND OF THE INVENTION
`
`The intravenous or intramuscular injection of sterile solu
`tions of an iron dextran complex is clinically indicated for
`the treatment of patients with documented iron de?ciency in
`whom oral administration is unsatisfactory or impossible.
`Iron dextran is absorbed from the injection site after
`intramuscular injection, for example. into the capillaries and
`the lymphatic system Circulating iron dextran is cleared
`from the plasma by cells of the reticuloendothelial system,
`which split the complex into its components of iron and
`dextran. IMFERON®. for example. a product previously
`marketed by Fisons Pharmaceuticals. is released to the blood
`after uptake by the phagocytic activity of macrophages. See
`Henderson. et al., Blood 34:357-375 (1969). The iron
`immediately is bound to available protein moieties to form
`hemosiderin or ferritin. the physiological forms of iron or, to
`a lesser extent. to transferrin. This iron. which is subject to
`physiological control, replenishes the iron component of
`hemoglobin and other depleted iron stores.
`The major bene?t of the clinical use of iron dextran is that,
`due to its large molecular weight (i.e.. greater than 70,000
`daltons). the iron dextran complex is not excreted by the
`kidneys. Therefore almost the entire dose of iron dextran
`remains bioavailable as the iron dextran is metabolized in
`the liver. The major portion of an intramuscular injection of
`iron dextran is absorbed within 72 hours. Most of the
`remaining iron is absorbed over the ensuing 3 to 4 weeks.
`Iron dextran for parenteral administration currently is
`marketed by Steris Pharmaceuticals, Inc. under the brand
`name lNFeD®. As formulated. this product is a dark brown
`and slightly viscous sterile liquid complex of ferric
`oxyhydroxide. beta-FeO(OH). and is a low molecular
`weight dextran derivative in approximately 0.9% weight per
`volume sodium chloride for intravenous or intramuscular
`use. It contains the equivalent of 50 mg of elemental iron (as
`an iron dextran complex) per ml. Sodium chloride may be
`added for tonicity. The pH of the solution is between 5.2 and
`6.5.
`Under electron microscopy, IMFERON® has been shown
`to have an inner electron-dense FeO(OH) core with a
`diameter of approximately 3 nm and an outer moldable
`plastic dextran shell with a diameter of approximately 13
`nm. Almost all of the iron. about 98-99% is present as a
`stable ferric-dextran complex. The remaining iron represents
`a very weak ferrous complex.
`The dextran component of conventional iron dextran
`products is a polyglucose that either is metabolized or
`excreted Negligible amounts of iron are lost via the urinary
`or alimentary pathways after administration of iron dextran.
`Staining from inadvertent deposition of iron dextran in
`subcutaneous and cutaneous tissues usually resolves or fades
`within several weeks or months. Various studies have
`reported that the half life of iron dextran in iron de?cient
`subjects ranges from 5 to more than 20 hours. Notably. these
`half-life values do not represent clearance of iron from the
`body because iron is not readily eliminated from the body.
`See. for example. the package inserts for IMFERON® and
`INFeD®. or Hamstra, et al. JAMA 24321726-1731 (1980).
`
`15
`
`25
`
`35
`
`45
`
`55
`
`65
`
`2
`U.S. Pat. No. 2,820,740 and its reissue RE 24,642 to
`London et al. describe colloidal injectable iron preparations
`suitable for parenteral injection formed of a nonionic ferric
`hydroxide, partially depolymerized dextran complex. Cur
`rent commercial iron dextran products, based on these two
`prior patents do not have su?icient purity (see FIGS. 1 and
`2) and needed thermal stability (see FIGS. 3 and 4) to
`safeguard safety and sterility concerns. Also, these commer
`cial products have a relatively short plasma residence time
`which could cause a potential risk of iron overload in
`speci?c organs. See, Carthew. R. E. . et al. Hepatology 13 (3)
`:534-538 (1991); Pitts, T. 0., et al. Nephron 22:316 (1978);
`Weintraub, L. R., et al. Brit. J. Hematology 59:321 (1985);
`and Fletcher, L. M., et al., Gastroenterology 97:1011 (1989).
`Similarly, U.S. Pat. No. 2,885.393 to Herb also discloses
`iron dextran complexes. The most suitable range in molecu
`lar weight of the partially depolymerized dextran for injec
`tion was found to be 30,000 to 80,000 daltons or lower. A
`subsequent patent to Herb, U.S. Pat. No. 4,180,567, dis
`closes other iron preparations and methods for making and
`administering such preparations; however, the method dis
`closed does not teach the heating of iron dextran complexes
`above 100° C.
`Other methods for the production of iron dextran com
`plexes have been described, for example, in U.S. Pat. No.
`4,599,405 to Muller et al. regarding iron (IlI) hydroxy/
`dextran complexes that are produced using an alkali
`carbonate, ammonium carbonate or a carbonate of an
`organic base added to an acid solution containing a partially
`depolymerized dextran and an iron (111) salt. Thereafter, an
`alkali metal hydroxide or ammonium hydroxide is added.
`The suspension so formed is then converted into a solution
`by heating, and the solution worked up in a known manner.
`Alternatively, ferric chloride and dextran can be reacted in
`aqueous solution in the presence of citric acid as disclosed
`in U.S. Pat. No. 3,697,502 or by treating reactive trivalent
`iron with a complex-forming agent consisting of sorbitol,
`gluconic acid and certain oligosaccharides, in particular
`proportions and amounts as taught in U.S. Pat No. 3,686,
`397.
`U.S. Pat. No. 4,749,695 and its divisional, U.S. Pat. No.
`4,927,756. both to Schwengers, disclose a water-soluble iron
`dextran and a process for its manufacture. As disclosed, the
`dextran utilized has an average molar mass of from 2,000 to
`4,000 daltons. Another alternative includes the complex
`ation of ferric hydroxide with hexonic acid derivatives of
`dextran as in U.S. Pat. No. 4,788,281 to Tosoni.
`U.S. Pat. No. 3,908,004 to Kitching discloses the prepa
`ration of iron compositions to treat iron-de?ciency anemia.
`Methods of formulating these compositions include the
`heating of an aqueous alkaline solution of a polysaccharide
`with a water soluble inorganic iron compound such as ferric
`oxychloride. The presence of the alkali is said to be neces
`sary to bring about the formation of the complex. However,
`the alkaline conditions also cause some degradation of the
`polysaccharide and the low molecular-weight species so
`formed produce iron compounds which are responsible for
`undesirable effects.
`U.S. Pat. No. 4,659,697 to Tanaka discloses a process for
`producing an organoiron (II) compound-containing antiane
`mic composition which through the cultivation of a yeast in
`a saccharide-containing nutrient medium, such as grape
`juice, in the presence of an iron compound to form a cultured
`broth comprising an organoiron(lI) compound, alcohol and
`water and removing the alcohol from the cultured broth to an
`extent that the resulting cultured broth has an alcohol
`
`Pharmacosmos, Exh. 1010, p. 8
`
`

`

`3
`content of less than about 1% by volume, and an antianemic
`composition produced thereby. The antianernic composition
`was said to be very stable, with excellent absorbability into
`a living body and incorporation of iron into hemoglobin.
`Iron dextran complexes also have application as imaging
`agents. For example, dextran/magnetite is disclosed as a
`particulate solution speci?cally noted to be stabilized by
`polymeric dextran. (See Hasegawa et al.. U.S. Pat. No.
`4.l01,435. Several others have used dextrans of various
`molecular weights as ingredients in the synthesis of mag
`netic colloids or particles. (See Hasegawa et al., U.S. Pat.
`No. 4.101.435; Molday, US. Pat. No. 4,454,773; and
`Schroder, US. Pat. No. 4.505.726. The resulting complexes
`of dextran and iron oxide have varying sizes and structures,
`but all have molecular weights of at least about 500,000
`daltons.
`The incorporation of high molecular weight dextran into
`magnetic particles or colloids may, however, cause some
`patients to experience adverse reactions to the dextran,
`particularly when such complexes are administered as
`parenteral magnetic resonance contrast agents. These
`adverse reactions may also be due in part to problems of
`high molecular weight polymers such as dextran dissociat
`ing from the metal oxide colloid upon prolonged storage or
`under high temperatures,'thereby leaving the metal oxide
`free to aggregate.
`Despite the variety of iron dextran formulations described
`in the prior act. current iron de?ciency products are based on
`technology that has not satisfactorily resolved stability and
`purity concerns. What is needed in the therapeutic ?eld of
`iron supplementation. is an improved next-generation iron
`dextran product with enhanced purity and thermal stability,
`as well as prolonged plasma residence time to minimize
`possible iron overload complications without compromising
`the e?icacy of iron dextran therapy.
`SUMMARY OF THE INVENTION
`These and other objects are achieved by the iron dextran
`product prepared according to this invention. It has excellent
`attributes and thermal stability but also has prolonged
`plasma residence time to minimize possible iron overload
`problem without compromising the e?icacy of iron dextran.
`It is an object of the present invention to provide methods
`for synthesizing iron dextran compositions useful in the
`treatment of iron de?ciency. Associated compositions also
`are disclosed. Such compositions include aqueous colloidal
`suspensions or solutions of a ferric oxyhydroxide-dextran
`complex, having an average molecular weight of about
`100,000 to 600,000 daltons and a substantially uniform size
`distribution. Physiologically acceptable carriers for these
`compositions also are contemplated. The administration of
`such compositions to humans and other mammals for the
`treatment of iron de?ciency or, in the case of non-human
`mammals. for medicinal as well as investigational purposes
`also are described.
`In a preferred embodiment of the present invention, the
`molecular weight range of the iron dextran compositions are
`about 150.000 to 350,000 daltons, and more particularly
`preferred are compositions with a molecular weight range of
`about 250.000 to 300.000 daltons.
`It is a further object of the present invention to provide
`iron dextran compositions having a beta-FeO(OH) core. A
`further object of the invention is to provide ellipsoidal
`iron-dextran particles with a length in the range of about 25
`to 45 nanometers, more preferably about 31.5 to 36.5
`nanometers, and a width of about 3.5 to 5.5 nanometers,
`more preferably about 4 to 5 nanometers.
`
`4
`It is a further object of the present invention to provide
`methods for synthesizing iron-dextran compositions as
`described above. The process of the present invention
`involves the initial production of iron-dextran particles by
`conventional methods. Applicants, however, have discov
`ered that superior particles may be produced by the follow
`ing process. Generally, as discussed in greater detail below,
`iron-dextran particles are puri?ed by conventional tech
`niques to remove various impurities, in particular, chloride
`iron, but also including any toxic by-products. uncomplexed
`dextran and, generally, any component of the initial iron
`dextran reaction mixture which would not be appropriate or
`permitted to be administered to patients in an approvable
`composition.
`
`BRIEF DESCRIPTION OF THE DRAWING
`FIGURES
`
`FIG. 1 shows a HPGPC chromatograrn of an iron dextran
`formulation according to the present invention demonstrat
`ing its uniform molecular weight distribution.
`FIG. 2 shows the HPGPC chromatogram of two commer
`cial preparations of iron dextran demonstrating a signi?cant
`heterogeneity relative to the formulations in FIG. 1.
`FIG. 3 shows the HPGPC chromatogram of an iron
`dextran formulation according to the present invention
`assessed over a period of seven days, demonstrating the
`stability of formulations.
`FIG. 4 shows the HPGPC chromatograrn of a commercial
`iron dextran formulation assessed over a period of seven
`days, demonstrating a signi?cant instability relative to the
`formulation of FIG. 3. At a magni?cation of 140,000 times.
`FIG. 5 shows that an electron photomicrograph of iron
`dextran particles according to the present invention at a
`magni?cation of 140,000><.
`FIG. 6 shows electron photomicrograph of particles sold
`under the brand name INFeD® at a magni?cation of 140,
`000x.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`The present inventors have found that iron dextran for
`mulations prepared according to the following speci?cations
`are surprisingly more temperature stable and/or exhibit a
`much greater degree of homogeneity than is evidenced by or
`would have been expected from iron dextran formulations of
`the prior art such as IMFERON® and INFED®. The
`improved methods and compositions disclosed for the
`preparation of these iron dextran formulations achieve uni
`form molecular weight distribution. Safety, reliability and
`quality of iron dextran injectable and infusible products can
`be signi?cantly improved over previous products. Our prod
`uct now in development is called DEEG'ERRUM®. DEX
`FERRUM® is a pharmaceuticaJly-equivalent iron dextran
`characterized by a higher mean molecular weight (266,
`608i1.4% daltons).
`In the following discussion and examples, certain calcu
`lations as set forth below are required to determine the
`amounts of active and inactive ingredients:
`The amount of iron dextran is based on its iron (Fe
`content. The amount in mg/ml is calculated by dividing the
`desired iron concentration in mg/ml of elemental iron by the
`powder’s % w/w iron content divided by 100. This amount
`is then multiplied by the batch size in liters for the amount
`required in grams for that batch size. This value is then
`corrected for its moisture content.
`
`5,624,668
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`Pharmacosmos, Exh. 1010, p. 9
`
`

`

`5,624,668
`
`15
`
`25
`
`5
`In general. a suitable iron 1]] salt, such as ferric chloride,
`is neutralized with a suitable alkali to which a modi?ed
`dextran is added either before. concomitantly or after neu
`tralization to produce an iron dextran complex with a
`molecular weight in the range of about 100,000 to about
`600.000 daltons. The resulting solution is puri?ed of excess
`dextran, salts. toxic impurities, etc., such as are identi?ed in
`Table 2 by any suitable method to produce an iron dextran
`aqueous concentrate or powder with an elemental iron
`concentration of between about 5% to about 50%. Puri?ed
`iron dextran powder or concentrate is then used in the
`preparation of a ?nal solution made of the foregoing iron
`dextran composition, with an elemental iron content of
`about 25 to about 100 mg/ml.
`We have observed that in solution. dextran is not tightly
`bound to the iron core, and complexes formed of aggregates
`in which. e.g.. two cores might be bound to the same dextran
`molecule. can be observed. The dextran serves to stabilize
`the core. but the puri?cation process associated with the
`initial preparation of iron dextran particules in which. e.g.,
`chloride iron is removed, also tends to remove some of the
`dextran.
`To a ?nal solution made of the foregoing iron-dextran
`composition. an appropriate amount of oxidized dextran is
`added to provide a desired ?nal ratio of the content of iron
`to dextran in the ?nal iron dextran composition in a range
`from about 1:2 to 1:5. but preferably about 1:4 as described
`in greater detail below. The iron-dextran and oxidized dex
`tran mixture is heated and reacted for an appropriate length
`of time with a suitable alkali. Generally, an appropriate
`length of time is not less than about one hour. The actual
`amount of time required to complete the reaction is depen
`dent on the amounts and ratios of starting materials. Deter
`mination of the end point may be measured by the absence
`of dextran enhancement of the LAL endotoxins test. We
`35
`have determined that oxidized dextran enhances the LAL gel
`clot method for assessing endotoxins, whereas reacted
`material. prepared according to our disclosure, demonstrates
`no such enhancement. Thus. in our manufacturing
`procedure, the reaction end point is determined by this
`technique to be complete when the amount of unreacted
`dextran does not exceed about 0.05 percent. After cooling
`and dilution to a ?nal volume, the pH of the solution is
`adjusted to a physiologically acceptable pH range. This
`adjusted solution is then aseptically ?lled and/or terminally
`sterilized for administration, such as by injection.
`We believe that the reaction of the iron dextran complex
`with an oxidized dextran under alkaline conditions converts
`the terminal unit of oxidized dextran from fS-Gluconolactone
`to sodium gluconate. The resulting solution contains dextran
`that is both bound and unbound to the iron complex where
`the molecular weight distributions of the bound and
`unbound dextrans are in equilibrium. Without wishing to be
`bound by any particular mechanism of action. we believe
`that the oxidized dextran at this stage of processing of iron
`dextran compositions minimizes or substantially eliminates
`aggregate complexes in which two iron cores might be
`bound to the same dextran molecule. Moreover. oxidized
`dextran has a terminal carboxyl group and has superior
`chelating abilities.
`The amount of oxidized dextran required to produce the
`desired product meeting its desired nonvolatile residue is
`calculated by subtracting the calculated # mg/ml iron dex
`tran (dry weight) from the theoretical total weight based on
`the nonvolatile residue of the desired product. That is. for a
`nonvolatile residue of 28-43 % w/v. the theoretical total
`weight would range from 280 to 430 mg/ml. The value
`
`6
`obtained is then corrected for the oxidized dextran’s loss on
`drying by dividing this value by (l-(loss on drying/100)).
`This amount is then multiplied by the batch volume in liters
`for the amount of grams for that batch size.
`The amount of alkali (such as sodium hydroxide) is
`dependent on the amount of oxidized dextran since it reacts
`with the alkali to form a carboxylic acid. The reaction is 1: 1.
`To determine the appropriate amount of alkali (such as
`NaOH) in grams, the molecular weight of the alkali is
`multiplied by the number of grams of oxidized dextran
`required for the desired product which is then divided by the
`average molecular weight of the oxidized dextran.
`A maximum limit for the hydrochloric acid used to adjust
`pH is calculated using the desired product’s upper limit for
`chloride content. The amount of chloride supplied by the
`starting materials (iron dextran and oxidized dextran) is
`calculated, then the maximum amount of hydrochloric acid
`added is determined by subtracting the total amount of
`chloride supplied from the starting materials from the
`desired product’s upper limit for chloride content, then
`multiplying the value obtained by the batch size in liters,
`divide this value by the atomic weight of chloride (35.5) and
`then divide by the normality of the hydrochloric acid solu
`tion to be used for the ?nal value.
`The low molecular weight carbohydrates of the invention
`must be oxidized in order to avoid problems in lack of
`uniformity and with the presence of endotoxins. Such car
`bohydrates preferably have a molecular Weight in the range
`of about 2,000 to 15,000 daltons, most preferably around
`6,000 to 7,000 daltons. The preferred concentrations of the
`carbohydrates of the invention which effectively impart
`stabilization to the carrier phase of the metal oxide compo
`sition are in the range of about 0.001M to about 2M, most
`preferably about 0.05M to about 0.5M, but optimal concen
`trations can be determined by those skilled in the art
`according to conventional techniques.
`Some preferred low molecular weight stabilizing agents
`include. but are not limited to, mannitol, sorbitol, glycerol,
`inositol, dextran 1 (Pharmacia Inc., Piscataway, NJ.) and
`ascorbate. Other useful agents include dextrins, celluloses,
`hydroxyethylstarches, heparins, starches, dextran sulfates,
`carboxylmethylated dextran and carboxymethyl cellulose.
`In the case of dextran 1, which has a molecular weight of
`about\1,000 daltons, the same compound can both stabilize
`the colloid or particulate suspension against unwanted
`physical changes and block possible adverse reactions. The
`simultaneous injection of dextran 1 and a complex of
`dextran and the magnetic iron oxide decreases adverse
`reactions to high molecular weight dextran alone.
`Preferred methods of manufacture of iron dextran solu
`tions involve the neutralization of ferric chloride solution
`with an alkaline solution of dextran. The mixture is heated,
`then cooled to room temperature and clari?ed by centrifu
`gation. The resulting solution is then concentrated to the
`desired iron content by dialysis against running water. The
`iron dextran is composed of a beta-FeO(OH) core formed by
`the neutralization of an acidic ferric chloride/dextran solu
`tion with alkaline sodium bicarbonate. The by-products of
`this reaction are sodium chloride and carbon dioxide. During
`neutralization. the modi?ed dextran is absorbed (complexes)
`to the iron core’s surface where the dextran’s hydroxyl
`groups provide the “OH” needed for stabilization of the
`core’s beta-FeO(OH) structure.
`
`50
`
`55
`
`65
`
`EXAMPLES
`
`Experimental studies describing the use of low molecular
`Weight carbohydrates as stabilizing agents for metal oxide
`
`Pharmacosmos, Exh. 1010, p. 10
`
`

`

`5,624,668
`
`7
`compositions prepared according to the present invention
`are presented below. These examples are to be considered as
`illustrative of the present invention rather than lirnitative of
`its scope in any way.
`The preferred dextran formulation for the production of
`iron dextran formulations according to the present invention
`are prepared by fermentation of sucrose using Ieuconostoc
`mesenteroides bacteria (NRRL B-512 (F)). The crude dex
`tran is precipitated, hydrolyzed. and fractionated by conven
`tional means. The dextran fraction is oxidized with an
`oxidizing agent under alkaline conditions, then puri?ed.
`Studies on the structure of the iron dextran complex report
`that it is composed of a beta-FeO(OH) core complexed with
`low molecular weight dextrans ranging from 3,500 to 7,500
`daltons. The oxidized dextran used in this invention is the
`dextran which is depolymerized to an average molecular
`weight ranging from 3.500 to 7,500 daltons. The dextran’s
`terminal unit. D-glucose. is then oxidized to gluconolactone.
`During the manufacturing process described in this inven
`tion the oxidized deXtIan’s terminal unit, gluconolactone, is
`converted to D-glucuronic acid via alkaline hydrolysis.
`The oxidized dextran used to produce iron dextran prod
`ucts according to the present invention has the following
`physical properties as set forth in Table 1:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`TABLE 1
`
`Parameter
`
`Tolerance
`
`Description
`odor
`Loss on Drying (w/w %)
`Sodium chloride content
`(w/W %)
`Nitrogenous Impurities
`Bromide content
`Alcohol and Related
`Impurities
`Relative Viscosity of a 10
`% sol
`Average Molecular Weight
`Phosphate (w/w %)
`Reducing Sugars (w/w %)
`Pyrogen Test
`
`White, amorphous powder
`Odorless
`Not more than 5.0%
`Not more than 2.0%
`
`Not more than 0.015%
`Less than 5 ppm
`Less than 0.05% w/w
`
`Less than 4.0 centistokes
`
`Between 3,000 and 7,000
`Not more than 0.28%
`Not more than 7.0%
`Passes test
`
`The characteristics and physical properties of the pre
`ferred iron dextran powder used to produce iron dextran
`formulations of the present invention are as follows in Table
`2. This composition is commercially available from Labo
`ratorien Hausmann AG in Switzerland, and U.S. Pat. No.
`4.599.405, discussed above. is relevant to the preparation of
`such compositions. U.S. Pat. No. 3,697,502 also is relevant.
`
`TABLE 2
`
`Parameter
`
`Tolerance
`
`Description
`Identi?cation
`Loss on Drying (w/w %)
`Sodium chloride content
`
`Brown, amorphous powder
`Complies
`Not more than 10.0%
`Not more than 6.0%
`
`Dextran content
`Iron Content
`Bromide content
`Alcohol and Related
`Impurities
`pH of a 5% Solution
`Molecular Weight
`Determination by GPC
`
`Between 29.0 and 36.0%
`Between 28.0 and 35.0%
`Less than 5 ppm
`Less than 0.05% w/w
`
`5.2 to 6.5
`
`Mw
`Mn
`
`Between 255,000-520,000
`Between 200,000-365,000
`
`TABLE 2-continued
`
`Parameter
`
`Tolerance
`
`MWJMl1
`Arsenic
`Lead
`Copper
`Zinc
`Bacterial Endotoxins
`
`Not more than 1.7
`Not more than 2 ppm
`Not more than 100 ppm
`Not more than 100 ppm
`Not more than 100 ppm
`Passes test
`
`EXAMPLE 1
`Preparation of Iron dextran Compositions
`In a 200 liter steam-jacket reaction vessel, 114 liter of hot
`(70° C. —90° C.) water was added. Next, 30.0 kg of iron
`dextran, satisfying the parameters described above, along
`with 28.3 kg oxidized dextran, also satisfying the parameters
`discussed above. The mixture was diluted up to 175 liters.
`Next. 185 g of NaOH was added and mixed with the iron
`dextran mixture. The vessel was sealed and then heated to a
`range of 110° C.—1l5° C. using a steam jacket for three
`hours. The vessel was then cooled to approximately 25° C.
`and vented during the cooling process. The pH was tested
`and adjusted to the range of 5.7-6.0.
`The reaction solution was pre?ltered through a 1.0 micron
`membrane into a holding vessel. Next, the ?ltered solution
`was passed through a 0.2 micron ?lter into sterilized receiv
`ing vessels. and depyrogenated vials were ?lled and stop
`pered with aliquots of the sterilized solution.
`
`EXAMPLE 2
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Evaluation of Process Results to Determine Molecular
`Weight Using HP-GPC
`The molecular Weight of the iron dextran complex of
`Example 1 was determined by gel permeation chromatog
`raphy in a'HP-GPC system equipped with a differential
`refractometer as the detector and an integrator with a GPC
`program for molecular weight calculations. The HP-GPC
`column was packed with porous particles of polyacrylic acid
`containing pore sizes up to 1000 angstroms. The pores act as
`sieves where smaller molecules permeate through in the
`pacln'ng’s pores while the larger molecules are excluded
`from the packing and are eluted by the more mobile phase.
`Thus, macromolecules elute from the columns, from largest
`to smallest.
`FIGS. 1-4 show comparisons between the iron dextran
`formulations of the present invention and two commercial
`preparations. These ?gures present data generated by a
`refractive index detector. This detector measures the con
`centration of the iron dextran, dextran and other molecules
`and the integrator’s GPC program interprets the data and
`calculates the relative: weight average molecular weight
`(MW), number average molecular weight (Mn) and polydis
`persity index (MW/Mn) of the sample. The reported values
`are based on polyethyleneglycol (PEG) and polyethylenox
`ide (PEO) standards used for calibration of the instrument,
`and are considered relative molecular weights which should
`be within 5% of the actual values.
`Ellipsoidal particles of the present invention are shown in
`FIG. 5. This shows DE)G1ERRUM® at a magni?cation of
`about 140.000X. In comparison. FIG. 6 shows particles sold
`under the name INFeD®. The unique conformation and
`
`Pharmacosmos, Exh. 1010, p. 11
`
`

`

`5,624,668
`
`9
`consistency of the DEXFERRUM® particles. as compared
`with another iron dextran supplement product. is evident
`from the foregoing ?gures and comparative electron photo
`micrographs. This information is consistent with the litera
`ture analyses of prior art iron-dextran complexes as re?ected
`in the paper by Cog. et al. from J. Pharm. Pharmac
`24:513-517 (1972).
`
`10
`
`TABLE 4
`
`Comparison of Transferrin Levels
`Transfe