Sonderdruck
`Reprint
`
`A rz n e i mitt e 1-F ors ch u n g /Drug Research
`ECV • Editio Cantor Verlag · Arzneim.-Forsch./ Drug Res. 42 (II), 12, 1439-1452 (1992)
`
`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, toxic effects were screened by histological
`examination of liver, kidney, adrenal, lung and spleen.
`The various iron complexes were classified into four
`groups according to their physicochemical properties (mo(cid:173)
`lecular mass, kinetic and thermodynamic stability). It was
`found that the toxic effects can be forecasted by the chem(cid:173)
`ical properties. The results clearly show that not all iron
`preparations tested can be recommended for intravenous
`application. After injection, the ideal iron preparation is
`deposited in the reticulo-endothelial system, and not in
`the parenchyma of the liver, nor mainly in the periportal
`area. Furthermore, its renal elimination rate should be
`below J % of the dose, and there should be practically no
`iron detectable in the tubuli. The molecular mass of an
`optimal product is between 30 000 and 100 000 Dal tons,
`and the preparation does not contain any slowly degrad(cid:173)
`able biopolymers, so that the incidence of allergic side
`effects is reduced to a minimum. Iron preparations con(cid:173)
`sisting only of weak iron complexes, which liberate iron
`ions stochastically, should not be used for intravenous ap(cid:173)
`plication.
`
`Zusammenfassung
`Struktur I Histotoxizitats-Beziehung von parenteralen Ei(cid:173)
`senpraparaten
`
`Handelsiibliche, zur parenteralen Amvendung empfohlene
`Eisenkomplexe mil verschiedenen chemischen Strukturen
`und unterschiedlichen Stabilitaten wurden analysiert und ·
`nach intravendser Gabe an A1ause histologisch auf ihre
`toxischen Wirkungen auf Leber, Niere, Nebenniere,
`Lunge und A1ilz untersucht. Die Eisenkomplexe wurden
`entsprechend ihren chemisch-physikalischen Eigenschaf
`ten (Jvlolmasse, kinetische und thermodynamische Sta(cid:173)
`bilitat) in vier Typen eingeteilt. Dabei stellte sich heraus,
`daj3 aufgrund der chemischen Eigenschaften die toxischen
`Auswirkungen gut vorhergesagt werden kdnnen. Die R e(cid:173)
`sultate zeigen, daj3 nicht a/le untersuchten Eisenpraparate
`zur intravendsen Applikation empfohlen werden kdnnen.
`Ein gutes Praparat wird nach Applikation vonviegend im
`retikuloendothelialen System, und weder im Parenchym
`der Leber noch bevorzugt in der periportalen Zone ge(cid:173)
`speichert. Im weiteren wire/ es renal unter 1 % ausgeschie(cid:173)
`den und lager/ sich nicht in den Tubuli der l'-liere ab. Die
`Molmasse eines optima/en Praparates liegt z wischen
`30 000 und JOO 000 und enthalt keine schlecht abbau(cid:173)
`baren Biopolymere, so daj3 die A1dglichkeit z u al/ergischen
`Reaktionen mdglichst k lein bleibt. Eisenpraparate, die
`nur schwache Eisenkomplexe enthalten und dadurch Ei(cid:173)
`senionen ungezielt freigeben kdnnen, sol/ten nicht intra(cid:173)
`vends verabreicht 1verden.
`
`Key words: Anaemex® • Ferrum Hausmann ® · Iron, structurelhistotoxicity relationship of parenteral preparations
`
`1. Introduction
`It is well known that in vi tro incubation of divalent iron
`ions (Fe 2 +) 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 (Spiro et al. 1969). Within this biochemical
`step highly toxic iron ions are converted into only
`slightly toxic, non-ionic, polynuclear iron(III)-hydroxide,
`
`Hausmann, Laboratories Inc. , R esearch Department,
`St. Gallen (Switzerland)
`
`which becomes water-soluble through ferritin complex
`formation (Islam et al. 1989). The formation ofnon-ionic
`iron(III)-hydroxide complex ferritin allowed to solve tox(cid:173)
`icity, tolerance and safety problems of iron stores in the
`evolution of animals and mammals (Theil et al. 1979).
`This can be demonstrated for instance by evaluating the
`LD 50-values of iron salts and mono- and oligonuclear
`iron complexes on one hand, which have a high toxicity,
`and of the polynuclear ferric hydroxide carbohydrate
`complexes on the other hand, which are of low toxicity
`(Mi.ilJer 1974, Berenbaum et al. 1960, Hoppe et al. 1955)
`(Table I).
`The formation of the physiological iron depot ferritin
`represents a model of the synthesis of different iron prep-
`
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`

`Table 1: Toxicity of different iron compou nds.
`
`Compound
`
`Salts
`FeS04
`Fe(II)-gluconate
`Fe(Il)-fumarate
`FeCl3
`Mono- and oligonuclear
`complexes
`Fe(IIl)-EDTA
`Fe(IIl)-ammonium-citrat e
`Pol ynuclea r complexes
`Ferric hydroxide dex tran
`Ferric hydroxide dextrin
`Ferric hydroxid e sucrose
`
`LD 50 in white mice in mg Fe/kg
`body weight
`
`Oral
`
`Int ravenous
`
`230')
`429 3>
`630')
`500-840 3>
`
`11 2>
`I 3'>
`
`18.5 3)
`
`500ll
`IOOO J>
`
`> 2500 1>
`> 2500 1>
`> 2500 1>
`
`40-50 1>
`I 6.5 3>
`
`> 2500 1>
`> 2500 1>
`> 200 1>
`
`References: 1> MUiler 1974, 2> Berenbaum et al. 1960, 3> 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 (Millier 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 g/1), where a therapeutical effect must
`be achieved as quickly as possible, as for instance in
`the last trimester bf 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 va riou s 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)
`ical properties.
`
`2. Material and methods
`2.1. Animals
`ICR (Institute Charles Ri ver) mice of both sexes (our own breed
`with animals from the Animal Breeding Institute, University of
`Zurich, Switzerland) of 20- 24 g (about 4 weeks old) were used
`in all experiments without previous randomization. The animals
`were kept in stainless steel 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 animals 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 between ane(cid:173)
`mic and non anemic, or between male and female animals, as far
`as the typical characteristics of the histological findings are con(cid:173)
`cerned. The iron preparations were applied intravenously by in(cid:173)
`jection into the tail vein. Usually a solution, diluted with normal
`saline, containing 2 w/v % of iron , was used. The standard dose
`
`2
`
`was 200 mg Fe/kg body 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 inj ectionem).
`IO min , 4 h, 4 and 14 d after application the animals were sac(cid:173)
`rificed and dissected subsequently. Liver, kidney, adrenal, lung
`and spleen were isolated and placed on a round metal plate and
`fro zen at -12 to -15 ·c for 45 min. Two animals were used for
`each preparation at each check time. A minimum of 2 frozen
`sections of each type of tissue were prepared per an imal 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 thickness of the tissue sections was chosen as 4-5
`µm . The following sections were used: liver cross-sections from
`the upper third and from the middle pai;t of the liver from the
`lobuli sinister lateralis and dexter medialis, resp. ; longitudinal
`peripheral kidney cross-sections (cortex and medulla) and
`through the center (cortex - medulla - calix - medulla - cor(cid:173)
`tex); adrenal cross-sections 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-gl ycerol before use. Two pieces of tissue
`sections of each organ were fi xed on glass slides. After this they
`were colored 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 after 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 ind~cate the mean of at least 5 different microscopic pictures
`(sectors) per section. The severity grades were defined as follows:
`0 rU: no iron :
`No iron detectable with this method.
`I rU: very little: Only traces of iron , sometimes detectable only
`locally. Very fine-grained iron deposit or only
`individual iron particles.
`Several clearly detectable fine to medium(cid:173)
`sized iron deposits or only few medium-sized
`iron particles.
`Iron is distributed over the whole tissue; local
`agglomerations can appea r. Fine to medium(cid:173)
`sized iron particles.
`Clear
`iron deposits everywhere. Fine
`coarse-grained iron particles.
`Further increase in the frequency of iron par(cid:173)
`ticles and in the densit y of the agglomerations;
`often with coarse-clotted iron deposits.
`6 rU: ve ry much: Maxim um 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
`pictures (sectors) were used for each section.
`With 0.9 % NaCl as test solution , all tissue sections show a rel(cid:173)
`ati ve iron concentration of 0-1 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:
`
`to
`
`2.2. MateJi.als and method of analysis
`The iron preparations were taken from the market or from our
`own manufacturing lines. All preparations were reanalyzed with
`respect to their iron content. Further determinations of the car(cid:173)
`bohydrate content, the 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 Table 3). All preparations were
`used in parenteral iron therapy, except for Fe-AA and the low
`molecular mass iron dextrin complex Fe-Ma, which has been
`chosen for comparison with Fe-Am .
`
`2.2.1 . Determination of the iron content
`Complex bound iron was mineralized with hydrochloric acid ( 10
`ml of HCI 37 w/w % for 5 ml iron complex solution), oxidized
`with l g potassium persulfate, diluted with l 00 ml distilled water
`
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`

`and IO ml glacial aceti c acid , adjusted wit h NaOH 30 w/ w % at
`pH 2.2- 2.5 and tit rated at 40-50 °C wit h 0.1 moll! EDTANa 2
`and pyrocatecholdi sulphonic acid disodium salt (trade name =
`Tiron) as indicator until th e color changed from red to green,
`and finally to yellow.
`
`2.2.2. Determination of the carbohydra te content
`2 x I m l distilled water, 2 x I ml standard solution ( 145 mg su(cid:173)
`crose dissol ved with distilled water to a vo lume of 100 m l solu(cid:173)
`tion) resp. 2 x I ml test solution (0.5-2.5 ml iron complex so(cid:173)
`lution , depending o n the expected carbohydrate co ntent, dis(cid:173)
`solved with distilled water to a volume of 100 ml solution) were
`added into 2 x 3 test tub es wi th gro und glass stoppers by means
`of a I ml Hamilton syringe. Thereafter 10 ml anthron e reagent
`were added and mixed thoroughly (200 mg anthron e we ighed
`into a 100 ml volumetri c fl ask, rinsed down with 20 ml distilled
`water; 60 ml concentrated sulfuri c acid were slowly added while
`continuous cooling was ensured. After co mplete dissolu tion it
`was fi lled up to I 00 ml with co ne. sulfuric acid). The test tubes
`we re placed into a boiling water bath; the stop wa tch was started
`and the test tubes closed with glass stoppers. The rack was placed
`in cold running water after exactl y 10 min. After cooling, the
`co ntent of the tes t tubes was mi xed thoroughly. The absorption
`spectrum was measured with a spectrophotometer in th e ran ge
`of 600-650 nm in IO mm glass cuvettes. The carboh ydrate co n(cid:173)
`tent was calcul ated from the m easured 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 co nt ent) of iron co mplex so(cid:173)
`lutio n was transferred into a 200 ml beaker and diluted with I 00
`ml of distilled water. 0.1 molll hydro chloric acid or 0. 1 moll!
`sodium hydroxide solution was slowl y added from a burette
`while magnetic stirring, potentiometric pH measurement and
`horizontal illumination through the beaker with a microscopic
`lamp were carri ed out in a darkened room , until a distin ct, per(cid:173)
`manent turbidit y appeared. At this point the pH was read . 'None'
`m ea ns that no turbidity or precipitation occurred (complexes
`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 determin ed according to th e
`method of Erni et al.
`(1984) at 25 °C. The k-values
`[k · 1000 · min- 1] 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 8 = 0.1 and 0.9 for mixtures were used resp ectivel y for
`th e correlat ion diagram with the molecul ar masses as shown in
`fig. 30 (cf. discussion).
`
`2.2.5. Determination of molecular masses
`The m ethod is based on the applicat ion of HPLC to permeation
`chromatography on poly(methylmethacrylate) gel. The following
`equipm ent was used: Waters HPLC-station, consisting of Waters
`590 programmable pump, WISP 710B autosampler, column
`oven co nn ected with Waters 4 IO differe ntial refractometer and
`a Waters system interface mod ul e. For operation an d data eval(cid:173)
`uati on, MAXIMA 820 software was used. The following columns
`were used: HEMA-Bio 100, IOµ , 8 x 30 mm , and H EMA-Bio
`1000, IOµ , 8 x 300 mm, by Stagroma AG (Wallisellen , Switz(cid:173)
`erland). These two columns were conn ected in series and tb er(cid:173)
`mostated at 45 °C. An aqeuous solution of 0.02 moll! Na 2HPO 4
`and 0.02 moll! NaH 2PO4 was used as solvent. The solven t flow
`was 0.5 ml/ min at a pressure of max . 2000 psi. The refractometer
`was set to a sensitivity of 32 an d a scale factor of 50.
`A Shodex stand ard ki t (Showa D enko K. K. , Tokyo, Jap an; Dis(cid:173)
`tributor Switzerland: Macherey and Nagel, Oensingen), contain(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 suppli er by means of
`an ultracentrifugal sedimentation equilibrium m et hod. The first
`and last point of th e calib ration curve was performed with a mix(cid:173)
`ture of dextran T 2000 (Pharmacia, Uppsala, Sweden) with a Mw
`of approx. 2.000.000, and glucose with a Mw of 180. A calibra(cid:173)
`tion curve was obta ined from the relat ionship log Mw versus re(cid:173)
`tention tim e. By means of this curve, th e molecular masses of
`th e iron complexes were calcul ated after integration of th e rele(cid:173)
`vant peaks.
`Values of the calibration curve:
`Curve T ype= cubic; r2 = 0.99948; Standard Error= 0.033 13
`
`Ta ble 2: Pullulan standard kit characteristics.
`
`Grade
`
`P-800
`P-400
`P-200
`P-100
`P- 50
`P- 20
`P- 10
`P-
`5
`
`853.000
`380.000
`186.000
`100.000
`48.000
`23. 700
`12.200
`5.800
`
`1.14
`1. 12
`1.1 3
`1. 10
`1.09
`1.07
`1.06
`1.07
`
`Equatio n of th e Curve:
`log Mw = + 3.09E + 0 1 - 2. 07E + 100 x R + 5.72E-02 x R 2
`- 5.73E-04 X R 3
`(R = retention time)
`
`30 Characterization of the analyzed
`iron preparations
`3.1. Compilation of 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-Da5, Fe-Da20, Fe-Am
`These iron complexes are composed of a polynuclear iron
`hydroxide complexed with dextran (polyisomaltose) or
`with dextrin (polymaltose); (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 (cf. Table 3). This leads to
`the observed low toxicity (MUiler 1974). Thus the iron
`dextranates and dextrinates are suited especially for in(cid:173)
`tramuscular application , but they are also used for intra(cid:173)
`venous inj ection or infusion (Fe-Da-BP/USP, Fe-Da5 ,
`Fe-Am) or for TDI (total dose infusion) (Fe-Da-BP/USP,
`Fe-Da5, Fe-Am) (Hallberg et al. 19 70, Dresch 1976).
`
`3.2.2. Fe-Su-I, Fe-Su-II, Fe-SU-III, Fe-Ma
`These iron complexes are composed of a polynuclear iron
`hydroxide, 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
`(Millier 1974). But iron saccharates are still suited espe(cid:173)
`cially for intravenous application (cf. Discussion) .
`The molecular mass and the complex stability of Fe-Ma,
`an iron complex used for oral application, are lower in
`comparison to Fe-Am , resulting in the higher toxicity ob(cid:173)
`served (cf. Table 3).
`
`3.2.3. Fe -DiSoCi, Fe-SuGl, Fe-AA, Fe-ChS
`The first two of these iron complexes are composed of an
`oligonuclear iron hydroxide complexed with dextrin, sor(cid:173)
`bitol and citric acid (Fe-DiSoCi), and with sucrose and
`gluconi c acid (Fe-Su GI). Th e molecular masses and com(cid:173)
`plex stabilities are very low in comparison to iron dextran
`and iron saccharates. Citric acid and gluconic acid yield
`a substantially better complex with iron hydroxide than
`do sorbitol and sucrose, so that in these mixtures mainly
`low molecular mass iron(IIl)-hydroxide citric acid com(cid:173)
`plex and partially gluconic acid complex respectively are
`present (cf. Table 3). This results in a comparatively
`higher toxicity (Millier 1974).
`Fe-AA is composed mainly of mononuclear iron(II)- and
`iron(IIl)-ascorbate and -dehydroascorbate. The complex
`stability with alloxanic acid is neglectable in comparison
`to asco rbic acid. The system iron(Il)/(111) / ascorbic acid,
`which generates radicals, becomes toxic fo r liver and mu(cid:173)
`cosa cells (Zglinicki et al. 1990, Hiraishi et al. I 99 1 ).
`
`3
`
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`Petitioner Ex. 1043 - Page 3
`
`

`

`'fable 3: Colll pilation of the results .
`
`I ron preparation
`
`Iron
`content
`(mg/ml]
`
`Point of
`Ligand content pH of th e zero charge
`(mg/llll]
`solution
`(pH]
`
`Degradation
`kinetics
`(k x 10 min· 1l
`9 - 0. 1/0.5/0.9
`
`Molecular mass Undue toxicity in
`white mice, i.v.
`o f complex
`[ lllg Fe/kg body
`[Dalton]
`weight]
`
`Points in
`correlation
`diagram
`(Fig. 30)
`
`48.6
`
`206
`
`49.3
`
`64
`
`198.0
`
`205
`
`50.3
`
`20.2
`
`20.0
`
`20. 3
`
`none'>
`
`20/34/67
`
`none4>
`
`23/ 34/66
`
`none4>
`
`12/27/50
`
`none4>
`
`9/20/42
`
`5.0
`
`4.5
`
`107/89/1 17
`
`I 10/ 87/129
`
`6.0
`
`6.0
`
`6.0
`
`5.8
`
`10.8
`
`9.6
`
`9.7
`
`103 ODO
`
`523 000
`
`445 000
`
`462 000
`
`43 300
`
`31 100
`
`> 500
`
`> I 000
`
`> I 000
`> 10 000 (i.p.)
`> I 000
`
`> 200
`
`I
`
`> 200
`
`180')
`
`I
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Fe-Da-BP/ USP
`(Lot O I 5109)
`Fe-Das
`(Lot 9 I I 308)
`Fe-Da20
`(Lot 985108)
`Fe-Am
`(Lot 962208)
`Fe-Su- I
`(Lot 7 50208)
`Fe-Su-II
`(Lot 951108)
`Fe-Su-III
`(Lot Y 288)
`Fe-Ma
`(Lot 919218)
`Fe-DiSoCi
`(Lot 09.109.43 I)
`FeSuG I
`(Lot 9 114570 1)_
`Fe-AA
`(Lot NN 348.102)
`Fe-ChS
`(Lot 91.1.5510 I)
`
`56
`dextrin
`318
`sucrose
`383
`sucrose
`392
`sucrose
`67
`dextrin
`)6Ql)
`
`197')
`
`5 mg Ase.a. 3>
`5 mg All.a .3>
`-
`
`50. 4
`
`50.5
`
`12.5
`
`2.0
`
`3.8
`
`5.3
`
`77/8 1/ 107
`
`none4>
`
`5 1/7 3/ 11 8
`
`2.2
`
`3.6
`
`2.6
`
`449/ 320/ 204
`
`i 36/ 130/ 130
`
`615/608/96
`
`none4l
`
`144/ 98/8
`
`6.0
`
`7.3
`
`8.4
`
`7.3
`
`7.4
`
`48 200
`
`52 300
`
`8 700
`
`37 500
`< I OOO'l
`< I 000
`
`47 800
`I 400 000
`
`> 400
`
`> 50
`
`> 50
`
`> 50
`
`250')
`
`7
`
`8
`
`9
`
`10
`
`II
`
`12
`13
`
`1> As sorbitol and dextrin (cf. Methods of ana lysis); contains also citric acid, wh ich is not included. 2> As sucrose (cf. Methods of analysis); contains also
`23 mg sodium gluconate/ml (value from th e declaration) with a lllolecular mass of < 1000 Daltons. 3> Ascorbic acid and alloxanic acid (values from the
`declaration). •> For the explanation of 'none' cf. methods of analysis. 1l LD , 0-value in white mice as indicated in the leaflet.
`Key to the abbreviations of the preparations: Fe-Da BP/USP: iron dex tran BP/ USP manufactured by Hausma nn Laboratories; Fe-DaS: iron dextan 5 %
`hulllan: Ferrum HausI11ann"' i.lll .: Dexfcrrulll ; Fe-Da20: iron dextran 20 %, Anaemex® (Hausmann); Fe-Am: iron dextrin (amylum) complex, Ferrum
`Hau smann® i.lll.: Amylofcrrum; Fe-Su-I: iron sucrose colllpl ex, Ferrulll Hau sman n"' i.v.: Venoferrum; Fe-Su-II: iron sucrose complex, Feppsol,
`manufactured by Hau smann Laboratories, distributed by Green Cross, Japan ; Fe-Su-Ill: iron sucrose colllplex ; Fe-Ma: iron dextrin (maltrin) com plex,
`Maltofcrrum (active ingredient ofFcrrum Hausmann® chewable tablets, syrup and drops); Fe-DiSoCi: iron dextrin/sorbitol/ citric acid colllplex; Fe-SuGI:
`iron sucrose/gluconic acid complex; Fe-AA: iron ascorbic acid/ allox an ic acid; Fe-ChS: iron chondroitinsu lphate.
`
`Fe-ChS is a mixture of iron(III)-chondroitinsulphates
`with very different molecular masses (cf. Table 3). The
`high molecular mass fraction has a similar complex sta(cid:173)
`bility as iron dextran, resulting in the fact that this com(cid:173)
`plex is present in the serum fo r a long time after appli(cid:173)
`cation (half-life time in rats: approx . 4 h).
`
`I
`
`4. Results
`4.1. General remarks about histology
`Histological tests were carried 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 carefully checked for
`damages such as necrosis. It is to be noted that with cer(cid:173)
`tain iron preparations the selected standard dose of 200
`mg Fe/kg b.w. was already close to the LD 50-value (cf.
`Table 3), so that ceU damages were likely to appear.
`
`4.2. Results in detail
`The deposited quantities of iron (in relative units, cf.
`Methods) in liver, kidney, adrena l, lung and spleen, in
`correlation with the time after appli cation (IO min, 4 h,
`4 d and 14 d) are shown in Fig. 1-5.
`
`4.2.1. Comments on liver sections (cf. Table 4)
`Fe-Da5, Fe-Da20, Fe-Am
`The distribution and relative concentrations of iron de(cid:173)
`posits correspond largely to the picture of iron dextran
`BP/USP (Fig. 6-9).
`
`Fe-Su-II, Fe-Su-III
`Th e distribution and relative concentrations of iron de(cid:173)
`posits correspond largely to the picture of Fe-Su-I (Fig.
`10-1 2).
`
`Fe-Ma
`After 4 d (Fig. 13) and 14 d, necroses were found over
`the whole tissue. After 14 d a phase of regeneration was
`observed.
`
`Fe-AA
`Dose: The following doses had to be selected for toxico(cid:173)
`logical reasons: 200 mg Fe/ kg b.w. (acute toxic region) for
`10 min and 4 h. 100 mg Fe/ kg b.w. for 4 d and 14 d.
`Necrotization began 10 min post injectionem. After 4 h
`there were severe necroses in the periportal region with
`the most part of deposited iron in the parenchyrna (Fig.
`16).
`
`Fe-ChS
`Necrotization bega n 4 h post injectionem. After 4 d there
`were already some necroses in the periportal region (Fig.
`17).
`In those mice which survived only 2 days because of the
`high toxicity of the 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 area was
`about I : I. Small to medium-sized necroses were visible
`all over the tissue.
`
`4.2.2. Comments on kidney sections (cf. Table 5)
`Fe-DiSoCi
`A dark brown coloration of the urine appearing IO min
`after the i.v. application is the most noticeable phenom(cid:173)
`enon and is caused by the excretion oflow molecular iron
`complexes, which are detectable in the histological prep(cid:173)
`aration of the ca lix (Fig. 21). All tested organs are free
`from iron after 4 days already.
`
`4
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1043 - Page 4
`
`

`

`-
`D
`
`RES (endothelium, Kupffe r's cells)
`porenchymo
`
`cortex
`-
`c=J medulla, zono reticuloris
`
`5
`
`4
`
`2 ·c
`
`:,
`
`Q)
`
`~ 3
`0
`~
`
`2
`
`0
`
`0..
`(/1
`::,
`
`'-0.. w
`
`I
`0
`0
`I
`"
`"-
`
`U")
`
`0
`0
`I
`"
`"-
`
`-
`
`=
`
`:,
`(/1
`I
`"-
`
`c3
`:,
`(/1
`I
`"
`"-
`
`::i:
`I
`"
`"-
`
`(/1
`JC
`
`u
`I
`"
`"-
`
`.
`
`0
`0
`0
`N
`E
`(/1
`0
`:,
`:,
`0
`i5
`::!,
`0
`(/1
`(/1
`<(
`I
`I
`I
`I
`I
`I
`"
`"
`"
`"
`"
`"
`"
`"-
`"-
`"-
`"-
`"-
`"-
`dist ribution afte r 1 Omi n/ 4h/ 4d/1 4d
`•( ofter 1Omin/ 105min/ 4h/ 4d)
`Fig. 1: Distribution or iro n depos its in histological section s of the li ver. The di stributi o n is
`given in relative units afte r IO min , 4 h, 4 d and 14 d (" corresponds to IO min, I 05 min , 4 h
`and 4 d). For further details cf. 4.2. These details app ly also to Fig. 2-5. For a cli nicall y safe
`iron preparation the iron deposit shou ld mainl y be in the RES (cf. Discussion).
`
`~
`C
`:,
`
`Q)
`
`.2:
`0
`~
`
`5
`
`4
`
`3
`
`2
`
`0
`
`0..
`(/1
`::,
`'-0..
`w
`I
`0
`0
`I
`"
`"-
`
`"'
`0
`0
`I
`"
`"-
`
`0
`0
`0
`N
`E
`(/1
`:,
`:,
`0
`:,
`0
`i5
`::!,
`0
`(/1
`<(
`(/1
`(/1
`I
`I
`I
`I
`I
`I
`I
`"
`"
`"
`"
`"
`"
`"
`"-
`"-
`"-
`"-
`"-
`"-
`"-
`distribution after 1 Omin/ 4h/ 4d/14d
`•(ofter 1Omin/ 105min/ 4h/ 4d)
`Fig. 3: Di stribut ion or iron deposits in histo logical sectio ns of the adrena l gland.
`
`-
`
`=
`
`-
`
`c3
`:,
`(/1
`I
`"
`"-
`
`::i:
`I
`"
`"-
`
`(/1
`
`JC u
`I
`" "-
`
`~
`C
`:,
`
`Q)
`
`> :g
`~
`
`5
`
`4
`
`3
`
`2
`
`0
`
`0..
`(/1
`::,
`'-0..
`w
`I
`0
`0
`I
`"
`"-
`
`U")
`
`0
`0
`I
`"
`"-
`
`0
`N
`0
`0
`I
`"-
`
`:,
`(/1
`I
`"-
`
`-
`
`c3
`:,
`(/1
`I
`
`" "-
`
`::i:
`I
`" "-
`
`(/1
`
`JC u
`t "-
`
`0
`=
`0
`(/1
`~
`0
`:,
`:,
`i5
`::!,
`(/1
`(/1
`I
`I
`I
`I
`I
`"
`"
`"
`"
`"
`"
`"
`"-
`"-
`"-
`"-
`"-
`distribution after 1 Om in/ 4h/ 4d/14d
`•(ofter 1Omin/ 105min/ 4h/ 4d)
`Fig. 4: Distribution or iron deposits· in histo logical sections of the lung.
`
`glomeruli
`-
`lllllilllllll inte rstitium. loop cells
`D
`tubuli
`
`tissue (septa)
`-
`c=J interstitium, introvosculor
`
`2 ·c
`
`:,
`
`Q)
`
`l
`0
`~
`
`5
`
`4
`
`3
`
`2
`
`0
`
`0..
`(/1
`::,
`
`'-0.. w
`
`I
`0
`0
`I
`"
`"-
`
`U")
`
`0
`0
`I
`"
`"-
`
`=
`
`-
`
`c3
`:,
`(/1
`t
`
`"-
`
`::i:
`I
`"
`"-
`
`(/1
`JC
`
`u
`I
`"
`"-
`
`0
`0
`0
`N
`E
`(/1
`:,
`0
`:,
`:,
`0
`i5
`::!,
`0
`(/1
`<(
`(/1
`(/1
`I
`I
`I
`I
`I
`I
`I
`"
`"
`"
`"
`"
`"
`"
`"-
`"-
`"-
`"-
`"-
`"-
`"-
`distrib ution after 1 Omin/ 4 h/ 4d/14d
`•(ofter 1Omin/ 105min/ 4h/ 4d)
`Fig. 2: Distribution of iron deposits in histologica l sect ions of the kidney. For a clin ica lly safe
`and well utilized iron preparation the iron deposit should not be in th e tubu li (cf. Discussion).
`
`v,
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1043 - Page 5
`
`

`

`red pulpo, marginal zone
`-
`CJ white pulpo
`
`00
`n.
`V,
`::::,
`'-n.
`m
`I
`0
`0
`.,
`I
`LL
`
`0
`
`"'
`0
`0
`.,
`I
`LL
`
`0
`N
`0
`0
`I
`LL
`
`.,
`
`E
`<(
`.,
`I
`LL
`
`:,
`V,
`.,
`I
`LL
`
`:,
`V,
`.,
`I
`LL
`
`.e ·2
`
`:::,
`
`Q)
`
`> :g
`~
`
`5
`
`4
`
`3
`
`2
`
`0
`
`-
`
`:,
`V,
`I
`LL
`
`0
`::;
`.,
`I
`LL
`
`u
`0
`V,
`0
`.,
`I
`LL
`
`c3
`:,
`V,
`I .,
`
`LL
`
`::;
`.,
`I
`LL
`
`V,
`.i=
`0
`.,
`I
`LL
`
`"
`d istribution after 1 Om in/ 4h/ 4d/14d
`•(ofte r 1Omin/105min/ 4h/ 4d)
`Fig. 5: Dislribution of iron deposits in histological sections of the splee n.
`
`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 periporta l (pport.) and central (cent.) area in function of time after application.
`
`Code and time
`
`rU
`
`RES: par.
`
`pport.: cent.
`
`Necroses
`
`Deposits
`
`Fe-Da-BP/US P
`10 min
`4h
`4d
`14 d
`Fe-Da5
`IO min
`4 h
`4d
`14 d
`Fe-Da20
`10 min
`4h
`4d
`14 d
`Fe-Am
`IO min
`4 h
`4d
`14 d
`Fe-Su-I
`10 min
`4h
`4d
`14 d
`Fe-Su-II
`IO.n1in
`4h
`4d
`14 d
`Fe-Su-III
`10 min
`4 h
`4d
`14 d
`Fe-Ma
`IO min
`4h
`4d
`14 d
`Fe-DiSoCi
`IO min
`13/4 h
`4h
`4d
`Fe-SuGI
`10 min
`4 h
`4d
`14 d
`
`6
`
`0
`I
`4
`3
`
`0
`2
`4
`3
`
`0
`3
`4
`2
`
`I
`2
`3
`3
`
`4
`4
`4
`4
`
`I
`4
`4
`4
`
`3
`5
`4
`4
`
`I
`3
`5
`4
`
`0
`3
`0
`0
`
`4
`4
`5
`3
`
`0
`1.0
`20:1
`20:1
`
`0
`6: I
`6: I
`6: I
`
`0
`7: I
`6:1
`4:1
`
`5: I
`5: I
`4:1
`7: I
`
`4:1
`9: 1
`9:1
`9:1
`
`0
`9:1
`9:1
`9:1
`
`20: I
`3: I
`9: I
`20: I
`
`4: I
`5: I
`1:2
`4:1
`
`0
`1:2
`0
`0
`
`1:2
`1:2
`1:2
`2: I
`
`0
`1: 1
`1:1
`1: 1
`
`0
`1:1
`2: I
`1:1
`
`0
`1:1
`2: 1
`1:1
`
`1:1
`l;l
`I:I
`1:1
`
`2:1
`2:1
`2:1
`2: I
`
`1:1
`I;}
`1:1
`1:1
`
`2:1
`2: 1
`2: 1
`1:1
`
`1:1
`1:1
`2:1
`1:1
`
`0
`10:1
`0
`0
`
`5 I
`5 I
`4 I
`4 I
`
`none
`none
`few small
`few small
`
`-
`non e
`none
`non e
`
`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
`
`fine gr.
`med.-coa.-gr. (Fig. 6)
`med.-coa.-gr.
`
`-
`fine-med.-gr.
`fine-med.-gr. (Fig. 7)
`med. -gr.
`
`-
`med.-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. 10)
`med.-coa.-gr.
`med.-coa.-gr.
`
`fine gr.
`med.-coa.-gr. (Fig. 11)
`med .-coa.-gr
`med .-coa.-gr.
`
`med.siz.
`med.-siz. (Fig. 12)
`med.-coa .-gr.
`-
`
`fine-med.-gr.
`fine-m ed.-gr.
`med.-coa.-gr. (Fig. I 3)
`med.-coa.-gr.
`
`-
`fine gr. (Fig. 14)
`-
`-
`
`few small
`many big
`many small and med.
`few small
`
`small and med.-gr.
`med.- and coa.-gr. (Fig. 15)
`fine-med.-gr. and coa. -gr.
`med.-coa.-gr.
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1043 - Page 6
`
`

`

`Table 4 continued.
`
`Code a nd time
`
`rU
`
`RES: pa r.
`
`pport.: cent.
`
`Necroses
`
`Deposits
`
`Fe-AA
`10 min
`4 h
`4d
`14 d
`Fe-ChS
`10 min
`4h
`4d
`14 d
`
`4
`3
`I
`l
`
`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
`l : l
`1:1
`
`many sm all
`many med . a nd big
`few very small
`none
`
`none
`few small
`many m ed.
`few small
`
`fin e gr.
`fine gr. and med .-coa .-gr. (Fi g. 16)
`fine-med.-gr.
`fine gr.
`
`fin e-gr.
`fine -med.-gr.
`fin e, med. and coa. -gr. (Fig. l 7)
`few fine, more m ed.
`
`Table 5: Kidney - Whole iron concentrati ons in kidney tissue in relative units (rU), a nd proportions of iron deposits be tween glomeruli , tubuli, and
`interstitium in fun ction of time after appli cation .
`
`Code and time
`
`rU
`
`Glomeruli
`
`lntersti tium
`
`Tubuli
`
`Comment s on the iron depos it s found
`
`Fe-Da-BP/USP
`10 min
`4h
`4d
`14 d
`
`Fe-Da 5
`10 min
`4h
`4d
`14 d
`
`Fe-Da20
`10 min
`4h
`4d
`14 d
`
`Fe-Am
`