The Molecular Formula and Proposed Structure
`of the Iron–Dextran Complex, IMFERON
`
`E. LONDON
`
`School of Pharmacy and Biomedical Sciences, St. Michael’s Building, White Swan Road,
`University of Portsmouth, Portsmouth, PO1 2DT, UK
`
`Received 11 July 2003; revised 9 February 2004; accepted 25 February 2004
`
`Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20093
`
`ABSTRACT: The first iron–dextran complex was discovered in 1953, when we attempted
`to synthesize an analog of ferritin, by substituting polysaccharide for its protein shell.
`This new complex soon became the most widely used parental therapy for hypochromic
`anemia in humans. No molecular formula has been proposed, but Cox has attributed an
`outline structure to it. The present article proposes a structure greatly different from the
`Cox model, by having a polynuclear b-ferric oxyhydroxide core, closely similar or identical
`to Akaganeite, chelated firmly by an encircling framework of dextran gluconic acid chains
`and surrounded by a removable outer sheath of colloidal dextran gluconic acid. The
`molecular weight of the iron–dextran core molecule, including its chelated framework,
`has been determined by gel filtration and analysis and its molecular formula (1.3)
`calculated. Also, these new data and existing electron photomicrographic, X-ray
`diffraction and crystallographic studies, have enabled a molecular weight, formula,
`and model structure to be proposed for its complex (2), which includes the outer sheath.
`The 480 iron atoms in both the core molecule and its sheathed complex are close to the
`number calculated from the core’s unit cell dimensions and volume. ß 2004 Wiley-Liss, Inc.
`and the American Pharmacists Association J Pharm Sci 93:1838–1846, 2004
`Keywords:
`iron–dextran complex; IMFERON; proposed structure
`
`INTRODUCTION
`
`The first synthesis of an iron–dextran complex was
`announced in a letter1 to the British Medical
`Journal by Fletcher & London (1954), wherein the
`medical need for such a product was described,
`together with an outline of its properties. In the
`same year a patent2 was assigned to London &
`Twigg, giving details of the preparation and proper-
`ties of the complex. Samples are still stable nearly
`50 years after manufacture. This iron–dextran
`complex called IMFERON (FisonTM), is specified
`in the British Pharmacopoeia (2000) and referred to
`forthwith as the complex or ‘‘Product A.’’
`
`Correspondence to: Eric London (Telephone: 02 392 551366;
`E-mail: londonye@aol.com)
`
`Journal of Pharmaceutical Sciences, Vol. 93, 1838–1846 (2004)
`ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association
`
`It regenerates hemoglobin quickly and effi-
`ciently in humans and piglets, and is well tolerated
`by both. It can be a life-saving treatment for
`mothers close to confinement with a low hemoglo-
`bin level.
`Essentially, all piglets in the UK and many
`elsewhere, receive their life store of iron from it
`within about 2 weeks of birth.
`in iron–dextran
`A resurgence of
`interest
`occurred in the 1990s following its widening use
`in hemodialysis and in imaging techniques. It has
`the ability during dialysis to effectively reduce
`blood makeup, which conserves both supply and
`expenditure.
`the discovery of
`About 40 years after
`IMFERON, Fison withdrew from the market,
`following an alleged difference on quality control
`with the FDA. in their American plant.
`The new prospective manufacturers such as
`Luitpold Pharmaceuticals, Inc., Shirley, NY, etc.,
`
`1838
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`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
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`

`STRUCTURE OF THE IRON–DEXTRAN COMPLEX, IMFERON
`
`1839
`
`have concentrated their efforts on securing control
`of molecular size, stability, reproducibility, and
`sterility of their iron–dextran complexes, as
`indicated by their publications,
`for example,
`Lawrence RJ, 1998.22
`The elucidation of factors controlling absorption
`at the intramuscular site and those influencing the
`antigenicity of the complexes should also be im-
`portant objectives.
`IMFERON may no longer appear under this
`trademark, as new legitimate synthesizers may
`well use their own for essentially the same
`product, or indeed market different iron–dextran
`complexes under various trademarks.
`We had found initially that alkaline treatment
`of the dextran was essential for good stability of the
`iron–dextran complex but only realized later that
`the reducing end groups of the dextran had largely
`been converted to carboxyl.3 This led to a study of
`the behavior of the complex in distilled water on a
`mixed-bed resin, showing that the main fraction
`passed readily through the column, while any
`uncomplexed carboxylated dextran was retained.4
`These important findings appeared as internal
`company reports by E. London, Head of Organic
`Research, 1951/1956, and were held unpublished
`until 1968, when a Fison patent5 revealed the
`carboxylation.
`Numerous clinical,6–8 veterinary,9,10 pharma-
`cological11/physiological,12 and several structural
`publications13–18 appeared pertaining to this
`complex during 1959–1972. Later references pri-
`marily concern the iron core,19–21 with a publica-
`tion in 199822 describing new developments of iron
`dextran products.
`However, none of these publications has given a
`molecular or structural formula for the complex or
`any of its components.
`This article has gone some way towards rectify-
`ing the situation by isolating a core molecule from
`the complex, determining its molar mass and
`molecular formula (1.3) and then proposing a che-
`mical structure (Fig. 2) for the core molecule; as
`well as a molecular formula (2) and model (Fig. 6)
`for the whole iron–dextran complex (‘‘Product A’’),
`comprising the core molecule wrapped in its
`colloidal sheath.
`
`MATERIALS AND METHODS
`
`Methodology for Gel Filtration23
`A column 1.2  50 cm of Sephacryl S-300 HR
`(allyldextran-N, N1-methylene bisacrylamide) was
`
`used, together with 0.05 M potassium phosphate
`buffer (pH7.4) as eluant, containing 6.8 g KH2PO4
`and 8 g NaOH per liter.This was fed from a header
`tank connected to the top of the column, a con-
`stant pressure (5–6 psi.) being supplied to it by
`an air pump with a controlled leak valve. The
`pressure line was transferred directly to the top of
`the column when the iron–dextran solution was
`being absorbed prior to development. Iron–dex-
`tran (1 mL) containing at least 2 mg/mL was
`usually applied, giving a clean separation of the
`core molecule from its sheath. One milliliter
`fractions per minute were collected automatically
`for analysis. The concentrations of other com-
`pounds applied to the column such as dextrans
`and their fluorescent derivatives, varied from 2–
`5 mg/mL, depending on the analytical procedure,
`molar absorptivity, or fluorescence.
`
`Iron Assay
`
`Gel filtration fractions were assayed spectro-
`scopically at 430 nm. Standard curves prepared
`from commercial samples of ‘‘Product A,’’ followed
`the Beer-Lambert law at least over the range of
`0.01–0.2 mg/mL Fe, even though 430 nm is not a
`true l max. The iron potency of ‘‘Product A’’ was
`based on an ammonium cerium (IV) sulphate/
`ferroin assay of
`the reduced test solution.
`(BritishPharmacopoeia, 2000).24
`
`Dextran/Dextran Gluconic Acid Assays
`The British Pharmacopoeia assay24 for dextran
`was found to work satisfactorily for dextran and
`dextran gluconic acid, after some important
`modifications in detail but not in principle. The
`assay involves reaction of an aqueous solution of
`the dextran or its derivative with a 2% Anthrone
`solution in concentrated sulphuric acid contain-
`ing 5% water. On heating a deep blue colour
`develops with a l max. of 625 nm.
`As the extent of the absorbance at 625 nm was
`sensitive to both time and temperature, we
`optimized these factors (to 16 min at 908C),
`following literature references25,26 and our own
`studies. The precision was further improved by
`keeping the Anthrone reagent at 0–58C or cooler
`and using it within 2 or 3 days. Also, dextran
`gluconic acid was used as its own internal
`standard, a fresh calibration curve being prepared
`for each series of assays. These were carried out in
`triplicate, giving a precision of 5% of the mean
`triple absorbance. The precision was improved to
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
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`1840
`
`LONDON
`
`3% if the reagent and test solution were sepa-
`rated by 1 mL of water to prevent premature
`interaction. This was achieved by dispensing 4 mL
`of Anthrone reagent at 0–58C into each test tube,
`followed carefully by 1 mL water and then 1 mL of
`test solution without mixing. Triplicate tubes and
`their contents were then shaken together and
`heated at 908C for 16 min in a waterbath, cooled in
`crushed ice and read at 625 nm.
`A blank correction should be made to the final
`absorbance by substituting 2 mL of water for the
`1 mL of water and 1 mL of test solution in the
`triplicate assay.
`Knowing the absorptivity of the test solution, its
`potency could be derived by reference to the cur-
`rent standard curve.
`
`Determination of the Molar Mass
`of the Core Molecule
`
`Gel filtration on a column of Sephacryl S-300
`HR was used (see methodology above), taking
`advantage of the relationship between elution
`volume (Ve), void volume (V0), packed bed volume
`(Vt) and molecular weight/size established by
`and Killander27/Ogston28/Granath33
`Laurent
`and summarized in the equations Kav ¼ Ve V0/
`Vt V0 and Kav¼ m log MW. Consequently,
`determination of the Ve values of a series of
`polysaccharides of known molar masses, allows
`their Kav values to be calculated and a plot to be
`drawn of Kav versus log MW. Then a related
`compound of unknown molar mass can be allo-
`cated one, simply by determining its Ve value on
`the same column, calculating its Kav, and using
`the log plot of the series.
`In practice, the elution volumes of a series of
`dextran fractions or their fluorescein isothiocya-
`nate (FITC) derivatives of known molecular
`weight (ex Sigma Chemical Company, Poole,
`Dorset, BH12 4QH, England) were determined,
`using the same Sephacryl S-300 HR column and
`packing at approximately 148C. The FITC deriva-
`tives, where available, enabled the elution to be
`followed visually and measured quantitatively
`using their l max at 490 nm. For colourless
`dextran fractions, such as dextran MW.74,000
`(ex Sigma), the Anthrone assay was used on
`multiple fractions collected at timed intervals
`(usually 1 min), as neither a flow-through refract-
`ometer or pulsed amperometric detector were
`available at the time. Plotting the cumulative
`elution volume versus absorbance for each frac-
`tion, gave a series of curves, the peak value of each
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
`
`the
`representing the elution volume (Ve) of
`particular solute. The use of blue dextran (MW
`2106) gave the void volume (V0) and the pack
`dimensions gave (Vt).
`Added precision could sometimes be given to a
`peak value by differentiating the equation for the
`curve in question (of absorptivity versus cumula-
`tive eluant volume) and obtaining the x value
`where dy/dx¼ 0. This is the (Ve) value at peak
`absorptivity used to calculate Kav above.
`
`Progressive Release of Iron from the Iron–
`Dextran Complex (‘‘Product A’’): Expt.544/1
`
`The reduction of the iron–complex with thiogly-
`collic acid (or hydroxylamine) resulted in the
`release of ferric ions as ferrous, giving a deep red
`color with a,a0-dipyridyl [Fe2þ(dipyridyl)3]. The
`extent and rate of this loss of ferric ions was
`revealed as follows: 1 mL of ‘‘Product A’’ contain-
`ing 50 mg Fe was diluted with deionised water to
`100 mL. To 50 mL of this 0.5 mg/mL Fe solution
`was added 46 mg of thioglycollic acid (equivalent
`to 25 mg Fe), retaining the remaining 50 mL for
`Expt. 544/2. The solution of complexþ thioglycol-
`oglycollic acid was diluted with water to 0.1 mg/
`mL Fe and 5 mL of this solution was added to1 mL
`of 20 mg/mL a,a0-dipyridyl in 0.5 N HCl. After
`further dilution with water to 0.01 mg/mL Fe, the
`absorptivity at 522 nm [l max for Fe2þ(a,a0-
`dipyridyl)3] and the time were recorded. These
`readings were repeated at hourly intervals until
`the absorptivity plateaued.
`
`Expt.544/2
`
`The above experiment was repeated using only
`half the thioglycollic acid.
`
`RESULTS AND DISCUSSION
`
`Initially (1953), our search for the structure of the
`iron–dextran complex (‘‘Product A’’) began with
`the use of mixed bed ion exchange incorporating
`both strong anionic and cationic resins (formerly
`‘‘Biodeminrolit’’) (see Introduction). In the current
`work, Amberlite 400 and 402 (Rohm & Haas Co.,
`Philadelphia, PA) were at first used to purify the
`commercial iron–dextran complex (‘‘Product A’’)
`from any uncomplexed dextran gluconic acid,
`possibly remaining from the biosynthetic process.
`However, gel filtration on Sephadex or Sephacryl
`columns made it possible to visualize the devel-
`
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`STRUCTURE OF THE IRON–DEXTRAN COMPLEX, IMFERON
`
`1841
`
`The molar mass estimate (MW) of this isolated
`core molecule was determined as described under
`Materials and Methods, yielding the data sum-
`marized in Table 1.
`The key relationship, Kav ¼ Ve V0/Vt V0
`allowed the conversion of elution volumes (Ve) to
`Kav values, representing the fractions of the gel
`volume available to the solutes used.
`Plotting Kav versus log MW from Table 1 gave a
`sigmoidal curve linear over a wide range, the
`linear section having an equation y¼ 3.6749x þ
`5.6101, where R2¼ 0.9855 (y ¼ log MW and x¼
`Kav).
`This technique enabled a molar mass estimate
`to be made for any related gel filtration fraction,
`given its elution volume and gave a molar mass
`estimate for the iron–dextran core molecule of
`105,000 Da, replacing the 73,000 assigned by Cox
`et al.18
`Having now determined the molar mass esti-
`mate of the core molecule and the molecular
`formulae of both the core molecule and the original
`complex, the molar mass estimate of the complex
`could be calculated, providing we knew the ratios
`of iron to dextran gluconic acid in each. So using
`the same assays for iron and dextran gluconic acid
`as had been used to obtain the elution profiles in
`Figure 1 (see Materials and Methods), these ratios
`were found to be 1:1.7 and 1:4 for the core and
`complex, respectively. Knowing the ratios, the
`molar mass of the core and the fact that the iron is
`present as b-FeO.OH,17,21 the molecular formulae
`of both the core (1.3), the original complex (2), and
`the molar mass of the complex were calculated as
`shown below.
`
`Calc. of Mol. Formula of Core Molecule
`
`Let the molecular formula of the core molecule be
`FeO:OH:2H2O
`ð
`
`Þa DxCOOHð
`Þb
`ð1Þ
`where a and b are uknown integers.
`This formula must also account for the molar
`mass estimate of 105,000 Da determined above.
`This gives the equation:
`Þa þ ð5056 Þb ¼ MW ¼ 105;000
`55:84 þ 69
`ð
`ð1:1Þ
`iron to
`Having already found the ratio of
`dextran gluconic acid in the core molecule to be
`1:1.7, we obtain 55.84a:5056b¼ 1:1.7. giving the
`equation:
`
`55:84a  1:7 ¼ 5056b
`
`ð1:2Þ
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
`
`Figure 1. Separation of iron–dextran core molecule
`from its sheath (DxCOOH ) in ‘‘Product A.’’ [Color figure
`can be seen in the online version of this article, available
`on the website, www.interscience.wiley.com.]
`
`opment and elution. Sephacryl S-300 HR also
`showed good stability, flow rate, high resolution,
`and covered the dextran molar mass range of
`2000–400,000 Da, and so was used in subsequent
`work.
`The complex was resolved into two fractions,
`one containing iron and dextran gluconic acid,
`the other dextran gluconic acid (abbreviated to
`dextran acid or DxCOOH; this consists of 31 es-
`sentially 1:6 linked anhydroglucose units, the re-
`ducing end group of the final unit having been
`oxidized to carboxyl, MW 5056) in a clean separa-
`tion. A typical graph of this separation is shown in
`Figure 1.
`Attempts to repeat the gel filtration of the iron–
`dextran acid fraction to see if more could be
`removed, always resulted in polymodal separa-
`tions. This led to the conclusion that before its
`removal
`from the column,
`the uncomplexed
`DxCOOH had probably been acting as a protective
`sheath around what it is proposed to call the ‘‘core
`molecule,’’
`the sheath preventing interaction
`between the core molecule and the dextran
`derivative column (see Materials and Methods).
`This was partially confirmed by comparing the
`stabilities of the eluted core molecule and the
`original complex, after each had been diluted
`(1:900) with water and allowed to stand. The core
`precipitated ferric oxyhydroxide after 35 days,
`while the complex survived 63 days. The use of a
`nondextran derivative column in place of Sepha-
`cryl, for example, ‘‘Toya Pearl,’’ may well allow
`repeat gel filtration without degradation and
`support our hypothesis of column interaction.
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`Table 1. Data Required for MW Determination
`
`Sample Ref.
`
`Blue Dx IX/63
`FITCa Dx IX/70
`Dx IX/35
`FTIC Dx IX/41
`Iron–Dx IX/31
`
`MW
`2 106
`50,700
`74,000
`145,000
`(105,200)
`
`V0
`
`22
`
`aFluorescein derivative label (ex Sigma plc.)
`bEach Ve value is an average of a number of expts.
`
`b
`
`Ve
`
`—
`28.15
`27.25
`25.05
`26.2
`
`Kav
`
`—
`0.24
`0.21
`0.12
`0.16
`
`Log MW
`
`—
`4.7050
`4.8692
`5.1614
`(5.013)
`
`where a and b are the same unknown integers as
`in eq 2.
`Solving eqs 1.1 and 1.2 for their common roots
`a and b, we find
`a¼ 477:8 and b ¼ 8:87
`Therefore, the molecular formula of the core
`molecule is:
`Þ478 Dx:COOHð
`
`ð
`FeO:OH:2H2O
`MW ¼ 105; 000 Da ðby expt:Þ
`*This agrees with the dihydrate of FeOOH
`being present, as required by the proposed struc-
`tural formula, Figure 2. **MW 5056, based on an
`intrinsic viscosity of (0.05) for the original dextran,
`with the addition of a carboxyl end group to the
`terminal anhydroglucose unit. ***Further details
`can be obtained from the author.
`
`ð1:3Þ
`
`Þ9
`
`Calc. of the Mol. Formula of the Complex
`(‘‘Product A’’)
`Fe :ðDxCOOHÞ in Core Mol: ¼ 1:1:7 by wt
`
`Fe :ðDxCOOHÞ in the Complex
`¼ 1:4:0 by wt: No: of ðDxCOOHÞ in Core is 9:
`
`No: of ðDxCOOHÞ in the Complex is 9x ð4:0=1:7Þ
`¼ 21:2
`
`
`
`Therefore Molecular Formula of Complex is:
`ð
`Þ478ðDxCOOHÞ9
`ðDxCOOHÞ12
`FeOOH:2H2O
`ð2Þ
`
`covalent core þ colloidal sheath
`MW ¼ 166; 000 Da ðby calc:Þ
`As the molar mass estimate of the Core Molec-
`ule (1.3) was 105,000 Da, the MW calculated for
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
`
`the Complex (2) from its molecular formula¼
`166,000 Da.
`This is supported by the zone centrifugation
`data (>156,000 Da.) of Ricketts et al.14
`Our earlier work using iron exchange (see
`Introduction) had established that this main frac-
`tion eluted by gel filtration had a smaller molar
`mass than the original complex and is named here
`the ‘‘core molecule.’’
`To accommodate the new molecular features of
`the iron–dextran complex (‘‘Product A’’), that is,
`two new molecular formulae and their correspond-
`ing molar mass estimates, it is proposed that a
`ligand structure is adopted for the polynuclear
`b-ferric oxyhydroxide core molecule. This was
`inspired by Muller’s30 1967 article on iron hydro-
`xide complexes.
`This proposal seals the core between two
`DxCOOH ligands, the remaining seven ligands
`being wrapped probably in random helices (an ex-
`pandable coil formation for the ligands would be
`typical of dextran chains of MWffi 2000–10,00032)
`around the core and attached to it by covalent links at
`their carboxyl ends and hydrogen bonds at the other.
`
`Figure 2. Proposed iron dextran core molecule.
`[Color figure can be seen in the online version of this
`article, available on the website, www.interscience.
`wiley.com.]
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`STRUCTURE OF THE IRON–DEXTRAN COMPLEX, IMFERON
`
`1843
`
`Figure 2 shows this proposal, the initial and terminal
`dextran gluconic acid ligands appearing in italics.
`The dimensions of the metal shadowed electron
`photomicrographs of either the core molecule or
`complex do not support the extended radial
`dextran acid chains proposed by Cox,18 although
`at that time the separate existence of a core
`molecule was not recognized. It was thought to be
`the iron dextran complex (‘‘Product A’’). The com-
`plex is now considered to combine both the core
`molecule and its associated dextran acid sheath.
`However, Figure 2, while quantitatively
`accounting for the iron–dextran hydrated poly-
`nuclear ferric oxyhydroxide core with its dextran
`gluconic acid framework and newly determined
`molar mass, nevertheless does not indicate the
`intricate 3D nature of the core, which is shown
`essentially as a ferric oxyhydroxide polymer for
`purposes of clarity.
`The core detail was revealed first by the work of
`Towe19 and extended by Kilcoyne and Lawrence,21
`the latter using Mossbauer spectroscopy and
`Rietveld refinement of their X-ray powder diffrac-
`tion data. These techniques were used earlier by
`Buckwald and Post,31 on powdered crystallites of
`Akaganeite obtained from an iron–nickel meteor-
`ite. The iron–dextran cores and the natural
`Akaganeite were shown to be essentially identical
`by Kilcoyne and Lawrernce, although Towe had
`indicated their great similarity.
`Kilcoyne and Lawrence concluded that the core
`consisted of b-FeOOH with a monoclinic unit cell,
`space group I2/m, containing two nonequivalent
`Fe3þ sites in a distorted octahedral environment.
`Their data gave a projected crystal structure for an
`iron–dextran core down the b-axis (Fig. 4), which
`could be regarded as a ball and stick equivalent of
`
`the space-filled unit cell of natural Akaganeite
`(Fig. 5), based on Buchwald and Post’s article.
`This article gave unit cell dimensions for
`Akaganeite of 10.6  3.0339  10.513 A˚ containing
`8 Fe, 16 O, and 2 Cl atoms (Fig. 5), giving a core
`density of 0.0422 Fe atoms/nm3.
`These data are close to those of Kilcoyne and
`Lawrence.21
`A 3.5-nm diameter spherical core with this
`structure would contain 532 Fe atoms and so
`approximates to our formula (1.3), which contains
`478. Rutherford17,18 had proposed a 3–4 nm core
`from electron photomicrographs.
`The chlorine atoms shown in the unit cell
`(Fig. 5), are usually considered essential for the
`formation of this polynuclear ferric oxyhydroxide
`crystal cell structure, and their complete or partial
`removal after synthesis would leave channels in
`the structure that could facilitate the ready
`transport of ferrous ions. In fact, Kilcoyne and
`Lawrence only claimed an occupancy of 0.91 atoms
`of Cl per unit cell, so the average degree of
`substitution at any one Cl site must be less than
`50%, and some channels must already exist in the
`structure.
`Such a possibility for an ion transport mechan-
`ism would again be reminiscent of that considered
`to operate in Ferritin.
`As each unit cell of the core contains 8 Fe atoms,
`then the 478 Fe atoms found in the molecular
`formula calculation of the Core Molecule (1.3),
`should probably be 480, equivalent to 60 unit cells
`in the core.
`Further study has shown that, as with Ferritin,
`the complex loses its iron atoms progressively
`by reduction with thioglycollic acid, and the
`(DxCOOH)12 sheath seemed to remain intact
`
`Figure 3.
`Iron release by reduction. [Color figure can be seen in the online version of
`this article, available on the website, www.interscience.wiley.com.]
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
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`1844
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`Figure 4.
`Iron–dextran core. A projection of the
`crystal structure of the iron–dextran core down the
`b-axis as determined by Rietveld refinement. [ß 1999.
`Reprinted with permission. S.H. Kilcoyne, J.L.
`Lawrence, Z. Kristallogr 214: 668.21] [Color figure can
`be seen in the online version of this article, available on
`the website, www.interscience.wiley.com.]
`
`during their loss, as there was little change in the
`viscosity of the reaction medium, but this work
`needs extending.
`The progressive loss of ferric ions after reduc-
`tion to ferrous is illustrated in Figure 3.
`These curves show that reduction, as expected,
`proceeds more rapidly at the higher thioglycollic
`acid to iron concentration (544/1) of 1 mol equiva-
`lent of each and its extent is roughly proportional
`to the amount of reductant used, that is, 0.5 mol
`equivalent of thioglycollic acid to iron (544/2),
`liberates 53% of the ferrous ions freed by 1 mol
`equivalent (544/1).
`About 42 and 80% of the theoretical amount of
`iron available to be freed were accounted for in
`Figure 3, 544/2 and 544/1, respectively.
`
`Reduction Data
`Chart 544/1
`Elapsed Time Hrs
`1.08
`2.83
`3.75
`4.67
`21
`
`Chart 554/2
`Elapsed Time Hrs
`1.25
`2.75
`3.83
`4.75
`21.08
`
`A 522 nm
`0.32
`0.59
`0.76
`0.93
`1.08
`
`A 522 nm
`0.12
`0.29
`0.4
`0.49
`0.58
`
`Further study is needed to explore the possibi-
`lity of replacing the lost iron atoms or adding more
`
`JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 7, JULY 2004
`
`Figure 5. Akaganeite unit cell. Chlorine is usually
`considered essential for the buildup of this structure
`during the formation of polynuclear b-FeOOH. Its
`removal leaves space for core access. [Color figure can
`be seen in the online version of this article, available on
`the website, www.interscience.wiley.com.]
`
`than were originally present, as can be done with
`Ferritin. If it is confirmed that the iron–dextran
`complex is essentially intact after removal of some
`or all of the iron in a reversible process, then its
`modus operandi as a synthetic iron store in man
`would seem to be similar to that of Ferritin.
`However, metal shadowing of the complex
`revealed an electron translucent sheath around
`the core (but not around the core freed from
`uncomplexed dextran acid), giving an overall size
`for the complete complex of 11.5  7.5 nm. If one
`assumes a symmetrical even thickness sheath
`around the core, the complex could be represented
`by Figure 6, agreeing with the lozenge shape of
`some electron photomicrographs17 and Schnei-
`der’s29 b-Fe.O.OH models. This suggests a par-
`tially filled cavity of ca 9.5 5.5 nm may exist in
`the ‘‘Product A’’ complex. If correct, this would
`allow iron–dextran complexes like Dexferrum22
`with its higher MW to exist within a similar
`structure, by enlarging the core and if necessary
`the sheath.
`Ferritin is known32 to have a cavity that is
`normally incompletely filled.
`While it is recognized that electron photomicro-
`graphs may not allow precise measurement, they
`
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`Petitioner Ex. 1058 - Page 7
`
`

`

`STRUCTURE OF THE IRON–DEXTRAN COMPLEX, IMFERON
`
`1845
`
`Figure 6. Proposed cross section of iron–dextran complex, Imferon. (11.5 7.5 nm).
`[Color figure can be seen in the online version of this article, available on the website,
`www.interscience.wiley.com.]
`
`will usually give a sufficiently correct order of
`magnitude to justify a model of the type proposed
`in Figure 6, which considerably aids the visualiza-
`tion of this somewhat unusual structure.
`Finally, it is possible that the use of the core
`molecule shown as the central feature of Figure 6, in
`place of the whole iron–dextran complex normally
`used, would sharpen the data obtained using such
`techniques as X-ray diffraction and Mossbauer
`spectroscopy and make them easier to interpret.
`In particular, the broad overlapping Bragg
`peaks referred to in the Kilcoyne and Lawrence21
`article might be further resolved, and could
`simplify the determination of the space group
`using X-ray diffraction, and possibly remove any
`residual uncertainty from the very close similarity
`already established by Kilcoyne and Lawrence
`between the iron–dextran core structure and that
`of natural Akaganeite.
`By so excluding the sheath around the core, less
`than 43% of the total complex polysaccharide would
`be available to interfere with these sensitive inves-
`tigations, and none of it would be present as a purely
`polysaccharide barrier at the outer surface of the
`complex, as it has been in previous investigations on
`iron–dextran cores. (The author could supply such
`material given reasonable notice.)
`
`ACKNOWLEDGMENTS
`
`I would like to thank Prof. G. Blunden for my Hon
`Research Fellowship and help in many ways,
`including reviewing this article. I also thank
`Dr. Brian Carpenter and Dr. Paul Cox for valuable
`manuscript critiques and the latter for Figure 5 and
`related data. Finally, thanks are due to Professor
`
`to
`R.C. Hider for some challenging concepts,
`Hannah and Stephen Chambers, Jon and Jim
`Murphy, and Peter London for computer support,
`and Dr. P. Woodruff of the Brit.Technology Group
`for a most welcome Seed Corn Grant, and my wife
`for patient encouragement.
`
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