';,', ,,;"' f ~
`~+,k;,;!':\S :_'f:.
`, ::; : ' 1/.",--,- ,1,/; < ~v:-c;
`\>'f~~;;:;1~,~\"~,:,
`,'1:;',.;;'
`
`,~
`
`Structure / Histotoxicity Relationship
`of Parenteral Iron Preparations
`
`P. Geisser, M. Baer, and E. Schaub
`
`Summary
`Commercial iron preparations with different chemical
`structures and stabilities which are indicated for paren(cid:173)
`teral application were analyzed. After intravenous appli(cid:173)
`cation in mice, toxie effeets were screened by histological
`examination of liver," kidney, adrenal, lung and spleen.
`The various iron complexes were classified into fOllr
`gro ups according to their physicochemica/ properties (mo(cid:173)
`leclllar mass, kinetic and thermodynamic stability). It IVas
`fOllnd that the toxic effects can be forecasted by the eh~m­
`icaI properties. The results clear/y show that r:ot all lron
`preparations tested can be recommended for Intrav~nm~s
`application. After injection, the id~al iron preparatlOn !S
`deposited in the reticlllo-endothelzal. syst.em, and ~lOt zn
`the parenchyma of the liver, nor maznly In the penportal
`area. FlIrthermore, i/s renal elimination rate should be
`below J % of the dase, and there should be practically no
`iran detectable in the tubuli. The moleclllar mass of an
`oplimal product is between 30 000 and 100000 Dalions,
`and the preparation does not contain any slow/y degr~d­
`able biopo/ymers, so that the incidence af allerglc Side
`errecls is reduced to a minimum. Iran preparatlons con(cid:173)
`sEsting on/y of weak iran comp/exes, whi~h liberale iran
`ions slochaslically, sholl/d nol be used for lIltravenOllS ap(cid:173)
`pficalion.
`
`Zusammenfassung
`Struktur / Hislotoxizitats-Beziehung von parenteralen Ei(cid:173)
`senpraparaten
`
`Handelsubliche, zur parentemlen Anwendung empfohlene
`Eisenkomplexe mit verschiedenen chemischen Strukturen
`lind unterschiedlichen Slabilitiiten wurden analysiert und
`nach intravenoser Gabe an Miillse hist%gisch auf ihre
`toxisehen Wirkungen auf Leber, Niere, Nebenniere,
`Lunge und Mi/z untersllcht. Die Eisenkomplexe wurden
`entsprechend ihren chemisch-physikalischen Eigenschaj(cid:173)
`ten (Molmasse, kinetische und thermodynamische Sta(cid:173)
`bilitiit) in vier Typen eingeteilt. Dabei steIlte sich h eraus,
`daj3 aufgrllnd der chemischen Eigenschaften die toxischen
`Auswirkungen gut mrhergesagt werden kan nen. Die Re(cid:173)
`sultate zeigen, daj3 nicht alle untersuchten Eisenpriiparate
`zllr intral'en6sen Applikation empfohlen werden k6nnen.
`Ein gutes Priiparat wird naeh Applikation vorwiegend im
`retikll/oendothelialen S)'stem, und weder im Parenchym
`der Leber noch bel'orztlgt in der periportalen Zone ge(cid:173)
`speichert. 1m weiteren wird es renal unter J % ausgeschie(cid:173)
`den lind lagert sich nicht in den Tubuli der Niere ab. Die
`Mo/masse eines optima/en Priiparates liegt zwischen
`30000 und JOa 000 und enthiilt keine schlecht abbau(cid:173)
`baren Biopolymere, so daj3 die Moglichkeit ZZl allergischen
`Reaktionen moglichst kleil/ bleibt. Eisenpriiparate, die
`nur schwache Eisenkomplexe enlha/ten lind dadurch Ei(cid:173)
`senionen IIngezielt ji-eigeben konnen, sollten nicht intra(cid:173)
`l'enos verabreicht werden.
`
`Key words: Anaemex®' Ferrum Hausmann® . Iran, structurelhistotoxicity relationship ofparenteral preparations
`
`1. Introduetion
`It is well known that in vitro incubation of divalent iron
`ions (Fe2 +) with the protein apoferritin leads to the for(cid:173)
`mation of ferritin in the presence of oxygen or other ox(cid:173)
`idizing agents (Spira et al. 1969). Within this bioehemieal
`step, highly toxie iran ions are eonverted wto only
`slightly toxie, non-ionie, polynuclear lron(III)-hydraxlde,
`
`Hausmalln, Laboratories [nc., Research Departmelll,
`SI. Gallen (Switzerland)
`
`Arzllcim.-Forsch./Drug Res. 42 (II), Nr 12 (1992)
`GciSSl"r
`leon prcparations
`
`which beeomes water-soluble through ferrit in eomplex
`formation (Islam et al. 1989). The formation ofnon-ionie
`iron(IlI)-hydraxide eomplex ferritin allowed to solv.e tox(cid:173)
`ieity, tolerance and safety problems of iron stores In the
`evolution of animais and mammals (Theil et al. 1979).
`This ean be demonstrated for instance by evaluating the
`LDso-values of iron salts and mono- and oligonu~lear
`iron eomplexes on one hand, whieh have a high toxlelty,
`and of the polynuclear fenic hydroxide carbohy~rate
`eomplexes on the other hand, which are of low tOXIClty
`(Muller 1974, Berenbaum et al. 1960, Hoppe et al. 1955)
`(Table l).
`. .
`The formation of the physiologieal iran depot ferntln
`repn;sents a model af the synthesis of different iron prep-
`
`1439
`
`Pharmacosmos, Exh. 1049, p. 1
`
`

`

`Table 1: Toxicity of different iron compounds.
`
`Compound
`
`Salts
`FeSO.
`Fe(Il)-gluconate
`Fe(ll)-fumarate
`FeCl,
`Mono- and oligonuclear
`complexes
`Fe(III)-EDTA
`Fe(lll)-ammonium-citrate
`Polynuclear complexes
`Ferric hydroxide dextran
`Ferric hydroxide dextrin
`Ferric hydroxide sucrose
`
`LD so in white mice in mg Fe/kg
`body weight
`
`Oral
`
`lntravenous
`
`2302)
`429"
`63021
`500-840')
`
`l!')
`133)
`
`18.S3)
`
`500 1)
`1000')
`
`> 25001)
`> 2500 1)
`> 2500"
`
`40-S0 1)
`16.S 31
`
`> 2S001)
`> 2500 1)
`> 200"
`
`References: I) Miiller 1974, 2) Berenbaum et al. 1960,1) Hoppe et al. 1955.
`
`arations with extremely low toxicity, good tolerance, a
`wide therapeutical range and a minimal danger of acci(cid:173)
`dental overdosing (Muller 1974).
`Parenteral iron therapy is said to be indicated in the fol(cid:173)
`lowing cases:
`known severe problems of intestinal iron absorption,
`absolute intestinal iron intolerance,
`severe or very severe iron deficiency conditions
`(Hb < 90-100 gll), where a therapeutical effect must
`be achieved as quickly as possible, as for instance in
`the last trimester of pregnancy or in pre-operative iron
`deficiency conditions (Hallberg et al. 1970),
`cases where regular intake of an oral preparation is not
`guaranteed,
`iron deficiency where there is no response to oral ther(cid:173)
`apy, e.g. in dialysis patients (Lawson et al. 1971),
`situations where iron stores are scarcely or not at all
`formed but would be important for further therapy,
`e.g. in combination with erythropoietin (Van Wyck
`1989).
`In clinical situations where parenteral iron preparations
`are indicated, a high safety margin is of paramount im(cid:173)
`portance. This implies that toxic as well as allergic side
`effects must be avoided.
`Nevertheless, the various iron preparations available on
`the market and used for parenteral application differ
`strongly in crucial parameters. Not all of them belong to
`the safest group of polynuclear iron complexes. As their
`chemical structure is different, a different toxic and his(cid:173)
`tological behavior is observed. This work will demon(cid:173)
`strate, analyze and explain the relationship between the
`chemical structure of iron complexes and their histolog(cid:173)
`icai properties.
`
`2. Material and methods
`2.1. Animals
`ICR (Institute Charles River) mice ofboth sexes (our own breed
`with animais from the Animal Breeding Institute, University of
`Zurich, Switzerland) af 20-24 g (aboul 4 weeks old) were used
`in all experiments without previous randomization. The animais
`were kept in stainless sted cages with bottom lattice for the pre(cid:173)
`vention of coprophagia. The light-dark interval was 12 h, tem(cid:173)
`perature 22 'C and humidity 55 %. The ani mais were fed with a
`standard diet from Nafag, Gossau (Switzerland) (Nr. 850) con(cid:173)
`taining 250 mg Fe/kg, and iron-free tap water, both ad libitum.
`In a test experiment no difference could be found bctweeo. ane(cid:173)
`mie and non anernic, or betwcen male and femalc animais, as far
`as the typical charactcristics of thc histological findings are con(cid:173)
`cerned. The iran preparations were applied intravenously by in(cid:173)
`jection into the tail vein. Usually a solution, dilutcd with normal
`saline, containing 2 w/v % af iron, was used. Thc standard dase
`
`1440
`
`was 200 mg Fe/kg bod y weight. (Preparations provoking liver
`necrosis caused apathies and breath troubles after 30 to 60 min,
`and led to death in some cases within 3-48 h post injectionem)_
`IO min, 4 h, 4 and 14 d after application the animais were sac(cid:173)
`rifked and dissected subsequently. Liver, kidney, adrenal, lung
`and spleen were isolated and placed on a round metal plate and
`frozen at -12 to -15 'C for 45 min. Two animais were used for
`each preparation at each check time. A minimum of 2 frozen
`sections of each type af tissue were prepared per animal with a
`microtome cryo-cut (American Optical Company, Buffalo, USA;
`General Representation: Leica AG, Glattbrugg, Switzerland),
`whereby it proved advantageous to let the lungs thaw at -10 to
`-12 ·C. The thiekness of the tissue seetions was ehosen as 4-5
`/lm. The folIowing sections were used: liver cross-sections from
`the upper third and from the middle part of the liver from the
`lobuli sinister lateralis and dexter medialis, resp.; longitudinal
`peripheral kidney eross-sections (cortex and medulla) and
`through the center (cortex - medulla - calix - medulla - cor(cid:173)
`tex); adrenal cross-seetioris through the middle part; lung cross(cid:173)
`sections through the middle part; longitudinal spleen sections
`through the middle part. The microscope slides were spread with
`a thin layer of albumen-glycerol before use. Two pieces of tissue
`sections of each organ were fixed on glass slides. After this they
`were eolored with Berliner blue and Kernecht-red I aluminium
`sulphate solution, dehydrated and embedded with Eukitt
`(mounting medium for microscopic preparations) and a cover
`glass. Three hours af ter embedding the dry preparations were
`ready to be examined under the microscope. The microscopic
`pictures were taken with a Zeiss Axioskop H DIC and a Minolta
`7000 camera with Ektachrome 50 EPY and 64 T films.
`A semiquantitative standard measure (relative unit = rU) was
`selected in order to estimate the distribution of colored particles
`in the tissues. The values represent grades of severity and were
`estimated as integers from the above mentioned tissue sections;
`they indicate the mean of at least 5 different mieroseopic pictures
`(seetors) per section. The severity grades were defined as follows:
`O rU: no iron:
`No iron detectable with this method.
`l rU: very little: Only traces ofiron, sometimes detectable only
`locally. Very fine-grained iron deposit or only
`individual iron particles.
`Several clearly deteetable fine to medium(cid:173)
`sized iron deposits or only few medium-sized
`iron partieles.
`Iron is distributed over the whole tissue; local
`agglomerations ean appear. Fine to medium(cid:173)
`sized iron particles.
`Clear
`iron deposits everywhere. Fine to
`coarse-grained iron partieles.
`Further increase in the frequency of iron par(cid:173)
`tieles and in the density ofthe agglomerations;
`often with coarse-c1otted iron deposits.
`6 rU: very much: Maximum iron deposit in the whole region of
`the tissue.
`Relative amounts of iron found in selected tissue areas and cells
`(e.g. reticulo-endothelial system (RES) and parenchyma) are ex(cid:173)
`pressed as proportions with integers for the description of the
`relative quantities. The same tissue sections and microscopic
`pietures (sectors) were used for each section.
`With 0.9 % N aCl as test solution, all tissue sections show a rel(cid:173)
`ative iran concentration of O-l rU.
`Undue toxicity tests in white mice were carried out according to
`BP guidelines (British Pharmacopoeia 1988) relating to iron dex(cid:173)
`tran injection and iron sorbitol injection.
`
`2 rU: little:
`
`3 rU: moderate:
`
`4 rU: distinct:
`
`5 rU: much:
`
`2.2. Materials and method of analysis
`The iron preparations wcre taken from the market ar from our
`own manufacturing lines. All preparations were reanalyzed with
`respect to their iron con ten!. Further determinations of the car(cid:173)
`bohydrate content, lhe point of zero charge, the degradation ki(cid:173)
`netics, and the molecular mass by gelchromatography were car(cid:173)
`ried out (the results are given in lilble 3). All preparations were
`used in parenteral iron therapy, except for Fc-AA and the low
`molecular mass iron dextrin complex Fc-Ma, which has been
`chosen for comparison with Fe-Am.
`
`2.2.1. DetermiIlation of tile iron content
`Complcx bound iron was mineralized with hydrochloric acid (10
`ml af HCI 37 w/w % for 5 ml iron complex solution), oxidized
`with l g potassium persulfate, diluted with 100 ml distilled water
`
`ArnH'lm -Forsfh.lDrug Res. 42 (II), Nr. 12 (1992)
`lron preparatlons
`CielS\Cr
`
`I
`
`Pharmacosmos, Exh. 1049, p. 2
`
`

`

`and IO ml glacial acetic acid, adjusted with NaOH 30 w/w % at
`pH 2.2-2.5 and titrated at 40-50'C with 0.1 molll EDTANa2
`and pyrocatecholdisulphonic acid disodium salt (t rade name =
`Tiron) as indicator until the color changed from red to green,
`and fin ally to yellow.
`
`2.2.2. Determination of the carbohydrate content
`2 x l ml distilled water, 2 x I ml standard solution (145 mg su(cid:173)
`erose dissolved with distilled water to a volume of 100 ml solu(cid:173)
`tion) resp. 2 x I ml test solution (0.5-2.5 ml iron complex so(cid:173)
`lution, depending on the expected carbohydrate content, dis(cid:173)
`solved with distilled water to a volume of 100 ml solution) were
`added into 2 x 3 test tubes with ground glas s stoppers by means
`of a l ml Hamilton syringe. Thereafter IO ml anthrone reagent
`were added and mixed thoroughIy (200 mg anthrone weighed
`into a 100 ml voIumetric flask, rinsed down with 20 ml distilled
`water; 60 ml eoncentrated suIfuric acid were sIowly added while
`continuous cooling was ensured. Af ter complete dissolution it
`was filled up to 100 ml with conc. suIfuric acid). The test tubes
`were placed into a boiling water bath; the stop watch was started
`and the test tubes c10sed with glass stoppers. The rack was placed
`in coId running water af ter exactly 10 min. After cooling, the
`content of the test tubes was mixed thoroughly. The absorption
`spectrum was measured with a spectrophotometer in the range
`of 600-650 nm in IO mm glass cuvettes. The carbohydrate con(cid:173)
`tent was calculated from the measured absorption and the cali(cid:173)
`bration curve.
`
`2.2.3. Determination of the point of zero charge
`0.2-1.0 ml (depending on the iron content) of iron complex so(cid:173)
`lution was transferred into a 200 ml beaker and diluted with 100
`ml of distilled water. 0.1 moIII hydrochIoric acid or 0.1 molll
`sodium hydroxid e solution was sIowIy added from a burette
`while magnetic stirring, potentiometric pH measurement and
`horizontal illumination through the beaker with a microscopic
`lamp were carried out in a darkened room, until a distinet, per(cid:173)
`manent turbidity appeared. At this point the pH was read. 'None'
`means that no turbidity or precipitation occurred (compIexes
`which are stable enough not to precipitate still have a point of
`zero charge).
`
`2.2.4. Determination of the degradation kinetics
`The degradation kinetics were determined according to the
`(1984) at 25 'c. The k-values
`method of Erni et al.
`[k . 1000 . min· l ] given in Table 3 were calculated at 8 = 0.1,0.5
`and 0.9. The k-values at 8 = 0.5 for monodisperse systems, and
`those at e = 0.1 and 0.9 for mixtures were used respectively for
`the correlation diagram with the molecular masses as shown in
`fig. 30 (ef. discussion).
`
`2.2.5. Determination of molecular masses
`The method is based on the application of HPLC to permeation
`chromatography on poly(methylmethacrylate) gel. The folIowing
`equiprnent was used: Waters HPLC-station, consisting ofWaters
`590 programmable pump, WISP 710B autosampler, column
`oven connected with Waters 410 differential refractometer and
`a Waters system interface module. For operation and data eval(cid:173)
`uation, MAXIMA 820 software was used. The folIowing columns
`were used: HEMA-Bio 100, IO /-l, 8 x 30 mm, and HEMA-Bio
`1000, IO fl, 8 x 300 mm, by Stagroma AG (Wallisellen, Switz(cid:173)
`erland). These two columns were connected in series and ther(cid:173)
`mostated at 45 'c. An aqeuous solution of 0.02 molll Na2HPO.
`and 0.02 molll NaH,PO. was used as solvent. The solvent flow
`was O. 5 ml/min at a pressure ofmax. 2000 psi. The refractometer
`was set to a sensitivity of 32 and a scale factor of 50.
`A Shodex standard kit (Showa Denko K. K., Tokyo, Japan; Dis(cid:173)
`tributor Switzerland: Macherey and Nagel, Oensingen), con tai n(cid:173)
`ing pullulanes (polymaltotriose-polymer) with different Mw val(cid:173)
`ues was used for calibration (Table 2).
`The pullulanes had been calibrated by the supplier by means of
`an ultracentrifugal sedimentation equilibrium method. The first
`and last point ofthe calibration curve was performed with a mix(cid:173)
`ture of dextran T 2000 (Pharrnacia, Uppsala, Sweden) with a M~
`of approx. 2.000.000, and glucose with a Mw of 180. A calibra(cid:173)
`tion curve was obtained from the re1ationship log Mw versus re(cid:173)
`telltion time. By means of this curve, the molecular masses af
`the iron complexes were calculated af ter integration af the rele(cid:173)
`vant peaks.
`Values af the calibration curve:
`Curve Type ~ cubic; r2 ~ 0.99948; Standard Error = 0.03313
`
`Arlneim.-Forsch IDrug Res. 42 (Il), Nr 12 (1992)
`Geisser ~ Iron prcparatlOl1S
`
`Table 2: Pullulan standard kit characteristics.
`
`Grade
`
`P-800
`P·400
`P-200
`P·lOO
`P- 50
`P- 20
`P-
`IO
`P- 5
`
`853.000
`380.000
`186.000
`100.000
`48.000
`23.700
`12.200
`5.800
`
`1.14
`1.12
`1.13
`1.10
`1.09
`1.07
`1.06
`1.07
`
`Equation of the Curve:
`log Mw = + 3.09E + Ol - 2.07E + 00 x R + 5.72E-02 X R2
`- 5.73E-04 X R3
`(R = retention time)
`
`3. Characterizatian af the analyzed
`iran preparatians
`3.1. Compilation af the results
`The results are shown in Table 3.
`
`3.2. Description of the tested preparations
`(cf. Table 3 and Discussion)
`3.2.1. Fe-Da-BP/USP, Fe-DaS, Fe-Da20, Fe-Am
`These iron complexes are composed of a polynuclear iran
`hydroxide complexed with dextran (polyisomaltose) or
`with dextrin (po ly maltose); (amylum has a higher molec(cid:173)
`ular mass than maltrin of Fe-Ma). The molecular mass
`and the complex stability are higher in comparison to all
`other tested iron preparations (ef. Table 3). This leads to
`the observed 10w toxicity (Milller 1974). Thus the iran
`dextranates and dextrinates are suited especially for in(cid:173)
`tramuscular application, but they are also used for intra(cid:173)
`venous injection or infusion (Fe-Da-BP/USP, Fe-DaS,
`Fe-Am) or for TDI (total dose infusion) (Fe-Da-BP/USP,
`Fe-DaS, Fe-Am) (Hallberg et al. 1970, Dresch 1976).
`
`3.2.2. Fe-Su-I, Fe-Su-II, Fe-SU-III, Fe-Ma
`These iran complexes are composed of a polynuclear iron
`hydraxide, complexed with sucrose (Fe-Su-I, Fe-Su-II,
`Fe-Su-III), and with dextrin (Fe-Ma). The molecular
`mass and the complex stability are lower in comparison
`to iron dextran, resulting in the observed higher toxicity
`(MUller 1974). But iran saccharates are still suited espe(cid:173)
`ciaIly for intravenous application (ef. Discussion).
`The moleeular mass and the complex stability of Fe-Ma,
`an iron complex used for oral application, are lower in
`com pari son to Fe-Am, resulting in the higher toxicity ob(cid:173)
`served (ef. Table 3).
`
`3.2.3. Fe-DiSoCi, Fe-SuGI, Fe-AA, Fe-ChS
`The first two af these iron complexes are composed of an
`oligonuclear iran hydraxide complexed with dextrin, sor(cid:173)
`bitol and eitric acid (Fe-DiSoCi), and with sucrose and
`gluconic aeid (Fe-SuGI). The molecular masses and com(cid:173)
`plex stabilities are very low in comparison to iron dextran
`and iran saccharates. Citric acid and gluconic acid yield
`a substantially better complex with iran hydroxide than
`do sorbitol and suerose, so that in these mixtures mainly
`low moleeular mass iron(III)-hydroxide citric acid com(cid:173)
`plex and parti ally gluconic acid complex respectively are
`present (ef. Table 3). This results in a eomparatively
`higher toxicity (MUller 1974).
`Fe-AA is eomposed mainly of mononuclear iron(II)- and
`iron(I1I)-ascorbate and -dehydroaseorbate. The complex
`stability with alloxanic acid is negleetable in comparison
`to ascorbic aeid. The system iron(II)/(ITI) / aseorbic acid,
`whieh genera tes radicals, beeomes taxie for liver and mu(cid:173)
`cosa cells (Zglinieki et al. 1990, Hiraishi et al. 1991).
`
`1441
`
`Pharmacosmos, Exh. 1049, p. 3
`
`

`

`Table 3: Compilation of the results.
`
`lron preparation
`
`lron
`content
`[mg/ml]
`
`Ligand content pH ofthe
`[mg/ml]
`solution
`
`Point of
`zero charge
`[pH]
`
`Degradation
`kineties
`[k x IO min-']
`9 = 0.1/0.5/0.9
`
`Molecular mass
`ofeomplex
`[DaitonI
`
`U ndue toxieity in
`white miee, i. v.
`(mg Fe/kg body
`weight]
`
`Points in
`correlation
`diagram
`{Fig. 30)
`
`48.6
`
`49.3
`
`206
`
`64
`
`198.0
`
`205
`
`50.3
`
`20.2
`
`20.0
`
`6.0
`
`6.0
`
`6.0
`
`5.8
`
`10.8
`
`9.6
`
`9.7
`
`none4)
`
`20/34/67
`
`none4J
`
`23/34/66
`
`none4 )
`
`12127/50
`
`none4 )
`
`9/20!42
`
`107/89/117
`
`110/87/[29
`
`5.0
`
`4.5
`
`5.3
`
`103000
`
`523000
`
`445000
`
`462000
`
`43300
`
`31 100
`
`> 500
`
`> 1000
`
`> 1000
`> IO 000 (i.p.)
`> 1000
`
`> 200
`
`> 200
`
`I
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Fe-Da-BP/USP
`(Lot 015109)
`Fe-DaS
`(Lot 911308)
`Fe-Da20
`(Lot 985108)
`Fe-Am
`(Lot 962208)
`Fe-Su-I
`(Lot 750208)
`Fe-Su-Il
`(Lot 951108)
`Fe-Su-lll
`(Lot Y 288)
`Fe-Ma
`(Lot 919218)
`Fe-DiSoCi
`(Lot 09.109.431)
`FeSuGl
`(Lot 91145701)
`Fe-AA
`(Lot NN 348.102)
`Fe-ChS
`(Lot 91.1.55101)
`
`56
`dextrin
`318
`sue rose
`383
`suerose
`392
`suerose
`67
`dextrin
`160')
`
`197' )
`5 mg Ase.a."
`5 mg AILa.')
`-
`
`20.3
`
`50.4
`
`50.5
`
`12.5
`
`2.0
`
`3.8
`
`71181/107
`
`none4}
`
`51173/118
`
`2.2
`
`3.6
`
`2.6
`
`449/320/204
`
`136/130/130
`
`6151608/96
`
`none4 )
`
`144/9818
`
`6.0
`
`7.3
`
`8.4
`
`7.3
`
`7.4
`
`48200
`
`52300
`
`8700
`
`37500
`< 1000"
`< 1000
`
`47800
`1400000
`
`180')
`
`> 400
`
`> 50
`
`> 50
`
`> 50
`
`250"
`
`7
`
`8
`
`9
`
`IO
`
`II
`
`12
`13
`
`l) As sorbitol and dextrin (cf. Methods of analysis); con tai ns also citric aeid, which is not included. l) As sucrose (cf. Methods of analysis); contains also
`23 mg sodium gluconate/ml (value from the declaration) with a molecular mass of < 1000 Daltons. " Ascorbic acid and alloxanic acid (values from the
`declaration). 4) For the explanation of 'none' cf. methods of analysis. ') LD,o-value in white miee as indicated in the leaflet.
`Key to the abbreviations ofthe preparations: Fe-Da BP/USP: iron dextran BP/USP manufactured by Hausmann Laboratories; Fe-DaS: iron dextan 5 %
`human: Ferrum Hausmann'" i.m.: Dexferrum; Fe-Da20: iron dextran 20 %, Anaemex" (Hausmann); Fe-Am: iron dextrin (amylum) complex, Ferrum
`Hausmann® Lm.: Amyloferrum; Fe-Su-I: iron suerose complex, Ferrum Hausmann" Lv.: Venoferrum; Fe-Su-II: iran sucrose eomplex, Feppsol,
`manufactured by Hausmann Laboratories, distributed by Green Cross, Japan; Fe-Su-I1I: iron suerose complex; Fe-Ma: iron dextrin (maltrin) complex,
`Maltoferrum (aetive ingredient ofFerrum Hausmann® chewable tablets, syrup and drops); Fe-DiSoCi: iron dextrin/sorbitolleitrie aeid complex; Fe-SuGI:
`iron suerose/gluconic ae id complex; Fe-AA: iron ascorbic acid/alloxanic aeid; FecChS: iron chondroitinsulphate.
`
`Fe-ChS is a mixture of iron(III)-chondraitinsulphates
`with very different molecular masses (cf. Table 3). The
`high molecular mass fraction has a similar complex sta(cid:173)
`bility as iran dextran, resulting in the faet that this com(cid:173)
`plex is present in the serum for a long time after appli(cid:173)
`cation (half-life time in rats: approx. 4 h).
`
`4. Results
`4.1. General remarks about histology
`Histological tests were carrie d through in order to deter(cid:173)
`mine the distribution of intravenously applied iron in
`liver, kidney, adrenal, lung and spleen. At the same time
`the tissue sections involved were careful!y checked for
`damages such as necrosis. It is to be noted that with cer(cid:173)
`tain iran preparations the selected standard dose of 200
`mg Fe/kg b.w. was aiready dose to the LDso-value (cf.
`Table 3), so that cell damages were likely to appear.
`
`4.2. Results in detail
`Th.e deposited quantities of iron (in relative units, cf.
`Methods) in liver, kidney, adrenal, lung and spleen, in
`correlation with the time after application (10 min, 4 h,
`4 d and 14 d) are shown in Fig. 1-5.
`
`4.2.1. Comments an liver sections
`Fe-DaS, Fe-Da20, Fe-Am (cf. rable 4)
`Th e distribution and relative con cent rations of iran de(cid:173)
`posits correspond largely to the picture of iran dextran
`BP/USP (Fig. 6-9).
`
`Fe-Su-II, Fe-Su-IlI
`The distribution and relative concentrations of iran de(cid:173)
`posits correspond largely to the picture of Fe-Su-I (Fig.
`10-12).
`
`Fe-Ma
`After 4 d (Fig. 13) and 14 d, necroses were found over
`the whole tis sue. After 14 d a phase of regeneration was
`observed.
`
`Fe-AA
`Dose: The folIowing doses had to be selected for toxico(cid:173)
`logi cal reasons: 200 mg Fe/kg b.w. (acute toxic region) for
`IO min and 4 h. IOD mg Fe/kg b.w. for 4 d and 14 d.
`Necrotization beg an IO min post injectionem. After 4 h
`there wc re severe necroses in the periportal region with
`the most part of deposited iran in the parenchyma (Fig.
`16).
`
`Fe-ChS
`Necrotization began 4 h post injectionem. After 4 d there
`were aiready some necroses in the periportal region (Fig.
`17).
`In those mice which survived only 2 days because of the
`high toxicity ofthe iron injected, 4 rU of iron were found
`in the liver: homogeneously distributed, partly coarse(cid:173)
`grained, generally more in the RES than in parenchyma.
`The proportion between periportal and central are a was
`about I : l. Smal! to medium-sized necrases were visible
`all over the tissue.
`
`4.2.2. Comments an kidney sections
`Fe-DiSoCi (cf. Table 5)
`A dark brown colQl-ation of the Ufine appearing 10 min
`after the i.y. application is the most noticeable phenom(cid:173)
`enon and is caused by the excretion of low molecular iran
`complexes, which are detectable in the histological prep(cid:173)
`aration of the calix (Fig. 21). All tested organs are free
`from iran after 4 days aiready.
`
`1442
`
`ArLnl:im -Forsch !\)nJg Res. 42 (Il), Nr. 12 ([992)
`Geisser
`lron preparations
`
`Pharmacosmos, Exh. 1049, p. 4
`
`

`

`Cl>
`~.;J
`~ ;:;
`~ 3
`I ,
`:;'0
`o ~
`;:j ~
`'0"-
`
`~~ ::~
`o·;::::
`" ~
`~ v ... ,~
`-::
`z
`
`'-O
`oD
`~
`
`.j:>.
`.j:>.
`w
`
`_
`D
`
`RES (endothelium, Kupffer's cells)
`pcrenchymo
`
`cortex
`_
`D medulla, zona reticularis
`
`5
`
`4
`
`2
`'2
`::>
`
`Q)
`
`.~
`.!'!
`~
`
`4
`
`'" ~ c
`" .,
`~ 3
`o
`~
`
`o
`
`Y':"UI UWVY UUWR • • • •
`
`Cl.
`(f)
`:::>
`"-Cl.
`al
`I
`o
`o
`I
`"
`"-
`
`"'
`o
`o
`I
`"
`"-
`
`D
`-
`o
`e
`...
`N
`o
`E
`(f)
`"
`"
`"
`o
`Ci
`o
`:l?
`(f)
`(f)
`(f)
`r
`r
`I
`I
`r
`r
`I
`"
`"
`"
`"
`"
`"
`"
`u..
`u..
`u..
`u..
`u..
`u..
`u..
`distribution af ter 1 Omin/ 4h/ 4d/ 14d
`>(after 10min/105min/4h/4d)
`Fig. 1: Distribution of iron deposits in histological 5ections af the liver. The distribution is
`given in relative units after 10min, 4 h, 4 d and 14 d C corresponds to 10min, 105 min, 4 h
`and 4 d). For further details ef. 4.2. These details apply a150 to Fig. 2-5. For a clinically safe
`iron preparation the iron deposit should mainly be in the RES (cf. Discussion).
`
`13
`"
`Vl
`r
`"
`u..
`
`~
`r
`"
`u..
`
`(f)
`.c
`u
`r
`"
`u..
`
`Cl.
`m
`I
`o
`o
`I
`~
`
`'"
`o
`o
`I
`"
`.....
`
`"
`Ul
`I
`~
`
`D
`o
`-
`o
`N
`o
`Ul
`o
`"
`"
`~
`Ci
`:l?
`VI
`VI
`Cl
`I
`I
`I
`I
`I
`I
`"
`"
`"
`"
`"
`.....
`.....
`~
`u-
`"-
`"-
`distribution af ter 10min/4h/4d/14d
`.(after 10min/l05min/4h/4d)
`Fig, 3: Distribution af iran deposits in histological sections of the adrenal gi and.
`
`13
`"
`VI
`I
`~
`
`(f)
`.c
`u
`I
`~
`
`~
`r
`"
`.....
`
`gJemeruli
`_
`IIIIIIII in\ers\i\ium, loop cells
`D
`\ubul;
`
`_
`D
`
`tissue (septa)
`interstitium, intravasculor
`
`2
`"2
`:J
`
`Q)
`>
`~
`~
`
`o
`
`n..
`V1
`:::>
`"-Cl.
`al
`r
`o
`o
`r
`"
`u..
`
`"'
`o
`o
`r
`"
`u..
`
`D
`-
`o
`o
`N
`o
`Ul
`"
`"
`"
`o
`~
`Ci
`:l?
`o
`VI
`Ul
`V1
`I
`I
`r
`I
`r
`r
`r
`"
`"
`"
`"
`"
`"
`.....
`.....
`~
`u..
`"-
`u..
`u..
`distribution af ter 1 Omin/ 4h/ 4d/14d
`-(af ter 10min/105min/4h/4d)
`Fig. 2: Distribution of iron deposits in histologienl seetions ofthe kidney. For a c1inically safe
`and well utilizcd iron preparation the iron deposit should not be in the tubuli (ef. D;~ru5sion).
`
`13
`"
`Ul
`I
`~
`
`~
`r
`"
`U-
`
`Ul
`.<:
`u
`r
`"
`U-
`
`5
`
`4
`
`J
`
`2
`'2
`"
`" >
`:;J
`o
`Qj ...
`
`o
`
`"-VI
`:;,
`"-"-
`m
`I
`o
`o
`I
`"
`u-
`
`'"
`o
`Cl
`I
`"
`.....
`
`c::;
`::
`o
`o
`N
`"
`"
`"
`~
`"
`"
`~
`:l?
`o
`Ul
`Ul
`Ul
`I
`I
`I
`I
`I
`I
`I
`"
`"
`"
`"
`.....
`.....
`II
`~
`u-
`u-
`~
`u-
`distribution ofter 10min/4h/4d/14d
`.( af ter 1 Omin/1 OSmin/ 4h/ 4d)
`Fig. 4: Distribution of iron dcposits in hislOlogical scetions af the lung.
`
`C3
`.il
`I
`.!:
`
`~
`I
`.!:
`
`VI
`.c
`U
`I
`.!:
`
`.iiIIIIIII!I!I'
`
`I:\,
`
`Q~
`
`Pharmacosmos, Exh. 1049, p. 5
`
`

`

`red pulpa, marginal zone
`_
`D white pulpa
`
`5
`
`2
`'c
`::>
`Q)
`i; 3
`Qi ...
`
`4
`
`2
`
`O
`
`a..
`III
`::>
`"-a..
`m
`'"
`I
`o
`o
`Cl
`Cl
`I
`I
`~ ~
`
`o
`N
`Q
`Cl
`I
`~
`
`::>
`III
`I
`
`.. LL
`
`-
`U
`o
`E
`o
`!!l
`"
`::>
`..:
`'"
`::lE
`III
`Cl
`I
`I
`I
`I
`I
`~
`~ ~ ~
`~
`distribution ofter 1 Omin/ 4h/ 4d/ 14d
`.(after 10min/l05min/4h/4d)
`Fig. 5: Distribution of iron deposits in histological seetions ofthe spleen.
`
`(3
`::J
`III
`I
`~
`
`~
`I
`~
`
`Ul
`.r.
`(J
`
`I æ
`
`Table 4: Liver - Whole iron concentrations in liver tissue in relative units (rU), and proportions of iron deposits between RES and parenchyma (par.)
`and between periportal (pport.) and central (cent.) area in function of time af ter application.
`
`Code and time
`
`rU
`
`RES: par.
`
`pport.: cent.
`
`Necroses
`
`Deposits
`
`fine gr.
`med.-coa.-gr. (Fig. 6)
`med.-coa.-gr.
`
`-f
`
`ine-med.-gr.
`fine-med.-gr. (Fig. 7)
`med.-gr.
`
`-m
`
`ed.-gr.
`med.-coa.-gr. (Fig. 8)
`med.-coa.-gr.
`
`fine-med.-gr.
`med.-gr.
`med.-gr. (Fig. 9)
`med.-gr.
`
`med.siz.
`med.-coa.-gr. (Fig. IO)
`med.-coa.-gr.
`med.-coa.-gr.
`
`fine gr.
`med.-coa.-gr. (Fig. II)
`med.-coa.-gr
`med.-coa.-gr.
`
`med.siz.
`med.-siz. (Fig. 12)
`med.-coa.-gr.
`
`- f
`
`ine-med.-gr.
`fine-med.-gr.
`med.-coa.-gr. (Fig. 13)
`med.-coa.-gr.
`
`fine gr. (Fig. 14)
`
`-
`
`--
`
`none
`none
`few small
`few small
`
`-n
`
`one
`none
`none
`
`none
`none
`none
`none
`
`none
`none
`none
`none
`
`few small
`few small
`few small
`.no more
`
`none
`none
`few small
`no more
`
`none
`few small
`few small
`no more
`
`none
`none
`many med.-big
`many med.-big
`
`none
`none
`none
`none
`
`few small
`many hig
`many smal! and med.
`fcw small
`
`srnall and med.-gr.
`med.- and coa.-gr. (Fig. 15)
`fine-med.-gr. and coa.-gr.
`med.-coa.-gr.
`
`Arzneim -Forsch.lDrug Res. 42 (II), Nr. 12 (1992)
`Iran preparations
`Geisser -
`
`Fe-Da-BP/USP
`IOmin
`4h
`4d
`14 d
`Fe-Da5
`10 min
`4h
`4d
`14 d
`Fe-Da20
`10min
`4h
`4d
`14 d
`Fe-Am
`10 min
`4h
`4d
`14 d
`Fe-Su-l
`10min
`4h
`4d
`14 d
`Fe-Su-I1
`IO min
`4h
`4d
`14 d
`Fe-Su-III
`IO min
`4h
`4d
`14 d
`Fe-Ma
`IO min
`4h
`4d
`14 d
`Fe-DiSoCi
`IO min
`13/4 h
`4h
`4d
`Fe-SuGl
`IO min
`4h
`4d
`14 d
`
`1444
`
`O
`I
`4
`3
`
`O
`2
`4
`3
`
`O
`3
`4
`2
`
`I
`2
`3
`3
`
`4
`4
`4
`4
`
`I
`4
`4
`4
`
`3
`5
`4
`4
`
`I
`3
`5
`4
`
`O
`3
`O
`O
`
`4
`4
`5
`3
`
`O
`1.0
`20:1
`20:1
`
`O
`6:1
`6:1
`6:1
`
`O
`7:1
`6:1
`4:1
`
`5: l
`5: l
`4:1
`7:1
`
`4:1
`9:1
`9:1
`9:1
`
`O
`9:1
`9:1
`9:1
`
`20:1
`3:1
`9:1
`20:1
`
`4:1
`5: I
`1:2
`4: I
`
`O
`1:2
`O
`O
`
`1:2
`1:2
`1:2
`2:1
`
`O
`1:1
`1:1
`1:1
`
`O
`1:1
`2:1
`1:1
`
`O
`1:1
`2:1
`1:1
`
`1:1
`1:1
`1:1
`1:1
`
`2:1
`2: l
`2:1
`2:1
`
`1:1
`1:1
`1:1
`1:1
`
`2:1
`2:1
`2:1
`1:1
`
`1:1
`1:1
`2:1
`1:1
`
`O
`10:1
`O
`O
`
`-
`
`5: I
`5: I
`4: I
`4: l
`
`Pharmacosmos, Exh. 1049, p. 6
`
`

`

`Table 4 continued.
`
`Code and time
`
`rU
`
`RES: par.
`
`pport.: cent.
`
`Necroses
`
`Deposits
`
`Fe-AA
`IO min
`4h
`4d
`14 d
`Fe-ChS
`IO min
`4h
`4d
`14 d
`
`4
`3
`I
`I
`
`3
`4
`5
`2
`
`1:5
`1:4
`1:2
`1:2
`
`1:6
`3:1
`2:1
`4:1
`
`10:1
`10:1
`10:1
`20:1
`
`10:1
`1:1
`1:1
`1:1
`
`many small
`many med. and big
`few very small
`none
`
`fine gr.
`fine gr. and med.-coa.-gr. (Fig. 16)
`fine-med.-gr.
`fine gr.
`
`none
`few small
`many med.
`few small
`
`fine-gr.
`fine-med.-gr.
`fine. med. and eoa.-gr. (Fig. 17)
`few fine. more med.
`
`Table 5: Kidney - Whole iron concentrations in kidney tissue in relative units (rU), and proportions of iron deposits between glomeruli. tubuli. and
`interstitium in function of time after application.
`
`Code and time
`
`rU
`
`Glomeruli
`
`Interstitium
`
`Tubuli
`
`Comments on the iron deposits found
`
`Fe-Da-BP/USP
`IO min
`4h
`4d
`14 d
`
`Fe-DaS
`10min
`4h
`4d
`14 d
`
`Fe-Da20
`10min
`4h
`4d
`14 d
`
`Fe-Am
`IOmin
`4h
`4d
`14d
`
`Fe-Su-!
`10min
`4h
`4d
`14d
`
`Fe-Su-II
`10min
`4h
`4d
`14 d
`
`Fe-Su-III
`IO min
`4h
`4d
`14 d
`
`Fe-Ma
`IO min
`4h
`4d
`14 d
`
`Fe-DiSoCi
`IO min
`11/4 h
`4h
`14 d
`
`Fe-SuGI
`IO min
`4 h
`4 d
`14 d
`
`Fe-A A
`IO min
`4 h
`4 d
`14 d
`
`Fe·ChS
`IO min
`4 h
`4 d
`14 d
`
`O
`O
`I
`O
`
`I
`2
`I
`I
`
`O
`I
`I
`I
`
`I
`2
`3
`2
`
`3
`3