Nephrol Dial Transplant (2004) 19: 561–565
`DOI: 10.1093/ndt/gfg579
`
`Original Article
`
`Labile iron in parenteral iron formulations: a quantitative
`and comparative study
`
`David Van Wyck1, Jaime Anderson2 and Kevin Johnson2
`
`1University of Arizona College of Medicine, Tucson and 2J2 Laboratories, Tucson, AZ, USA
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`Abstract
`iron-mediated oxidative
`Background. Evidence of
`stress, neutrophil dysfunction and enhanced bacterial
`growth after intravenous (IV) iron administration has
`been ascribed to a labile or bioactive iron fraction
`present in all IV iron agents.
`Methods. To quantify and compare the size of the
`labile fraction in several classes of IV iron agents, we
`examined iron donation to transferrin (Tf) in vitro. We
`added dilutions of ferric gluconate, iron sucrose and
`each of two iron dextran preparations to serum in vitro,
`passed the resulting samples through alumina columns
`to remove iron agent and free organic iron, and
`measured Tf-bound iron in the resulting eluates.
`Comparing results to serum samples without added
`iron, we calculated delta Tf-bound iron for each agent
`at each concentration. Finally, we compared delta
`Tf-bound iron to the concentration of added agent and
`calculated the percent iron donation to Tf.
`Results. We found that Tf-bound iron increased
`with added iron concentration for each agent: delta
`Tf-bound iron was directly related to the concentration
`and type of iron agent (P<0.001). Mean percent iron
`donation to Tf ranged from 2.5 to 5.8% with the
`iron dextran-DexferrumÕ<
`following progression:
`iron dextran-INFeDÕ<iron sucrose<ferric gluconate.
`Pairwise differences between agents for percent iron
`donation were statistically significant (P<0.05) only
`between ferric gluconate and both iron dextran
`agents, and between iron sucrose and iron dextran-
`DexferrumÕ.
`iron in
`Conclusions. Approximately 2–6% of total
`commonly used IV iron compounds is available for
`in vitro iron donation to Tf. This fraction may con-
`tribute to evidence of bioactive iron in patients after IV
`iron administration.
`
`Correspondence and offprint requests to: David B. Van Wyck, MD,
`Kidney Health Institute, LLC, 6720 N. Nanini Drive, Tucson, AZ
`85704, USA. Email: dvanwyck@sprynet.com
`
`Keywords: adverse effects; ferric gluconate; iron; iron
`dextran; iron sucrose; transferrin
`
`Introduction
`
`Oxidant stress, atherogenesis, infection and inflamma-
`tion are hallmarks of the dialytic milieu [1]. Each
`process holds a plausible pathogenic role for biologi-
`cally active iron. Intravenous (IV) iron therapy, com-
`monly administered to dialysis patients as an adjunct to
`managing anaemia, provides a potentially rich source
`for intradialytic bioactive iron [2]. All IV iron agents
`tested, including iron dextran [3], iron polymaltose [4],
`iron sucrose [3–5] and ferric gluconate [3,4,6], show
`evidence of bioactive iron release in vitro and in vivo.
`IV iron agents have been found to induce oxidative
`stress [3,4,6], boost bacterial growth in vitro [2,5] and
`disturb neutrophil function [2].
`Bioavailability of IV iron agents (iron sucrose [7],
`ferric gluconate [8],
`iron polymaltose [9] and iron
`dextran [10]) stems primarily from intracellular release
`of
`low-molecular-weight
`iron after clearance from
`plasma and uptake by cells of RES. However, in vitro
`evidence of
`iron-mediated biological activity [3,4]
`suggests the presence of a labile iron fraction in IV iron
`agents capable of exerting biological impact prior to
`cellular uptake.
`Debate over the clinical implications of labile iron
`in IV iron agents is vigorous. Some recommenda-
`tions have been quite explicit, including caution to
`avoid use of 125 mg doses of ferric gluconate [6] or
`300 mg doses of iron sucrose in dialysis patients [2].
`Other commentators have been more general, suggest-
`ing that IV iron agents contain ‘free iron’ and that
`classes of IV iron agents differ in their capacity to
`release free iron [11]. Though the debate promotes
`alarm and confusion,
`it resists resolution in part
`because quantitative and comparative information
`is lacking. Specifically,
`the fraction of
`total
`iron
`represented by labile iron in IV iron agents has not
`
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`562
`
`been quantified and comparative data on labile iron
`generation has not been obtained.
`A substantial impediment to quantifying the labile
`iron fraction arises from one of its intrinsic biochemical
`effects:
`in standard serum iron assays,
`labile iron
`is difficult
`to distinguish from transferrin-bound
`(Tf-bound) iron. Indeed, false elevation of serum iron
`determinations by IV iron agents was likely the first
`reported manifestation of labile iron [12,13]. Since
`standard serum iron assays erroneously detect 2–60%
`of added iron agent as serum iron [12], yet, according
`to preliminary reports [4], iron agents may also donate
`IV iron directly to Tf, quantifying the relative con-
`tribution of labile iron to Tf-bound iron in serum has
`heretofore not been possible.
`In the current studies, we sought to quantify and
`compare labile iron fractions in commonly used IV iron
`agents. We used direct in vitro donation of Tf-bound
`iron as a marker of labile iron. To measure iron don-
`ation to Tf but exclude interference in the iron assay,
`we first added, then removed, iron agent from serum.
`By comparing Tf-bound iron before and after adding
`iron agents to serum, we determined the relationship
`between the concentration of parenteral iron and the
`degree of in vitro iron donation to Tf. We thereby
`derived an estimate of a labile iron fraction in each iron
`agent formulation.
`
`Materials and methods
`
`To determine the magnitude of direct donation of iron from
`iron agents to Tf and to explore the potential role of this
`process in the saturation of Tf after IV iron administration,
`we added dilutions of each IV iron agent to fresh serum over
`a range of concentrations, passed the resulting samples
`through an alumina column to remove intact iron agent, and
`assayed the resulting eluate for Tf-bound iron. This assay has
`been shown to reliably exclude both iron agent and inorganic
`iron from interfering with the colorimetric assay of Tf-bound
`iron in serum [13].
`
`Parenteral iron formulations
`
`We examined ferric gluconate (sodium ferric gluconate
`in sucrose; FerrlecitÕ, 12.5 mg/ml in 5 ml ampules; Watson
`Pharmaceuticals, Inc., Corona, CA, USA), iron sucrose (iron
`sucrose injection, USP; VenoferÕ, 20 mg/ml in 5 ml vials;
`American Regent,
`Inc., Shirley, NY, USA) and both
`available formulations of iron dextran (INFeDÕ; Watson
`Pharmaceuticals; and DexferrumÕ; American Regent; both
`50 mg/ml in 2 ml vials). For each experiment, we examined all
`agents at all experimental concentrations on the same day.
`For each concentration of iron agent studied, we prepared
`equimolar stock solutions of each of four agents on the day
`of use, employing successive dilutions (1:10) in 0.9% NaCl.
`All agents were used before lot expiration dates.
`
`Experimental iron concentrations
`
`We examined concentrations of iron formulations from 859 to
`6875 mg/dl (153–1228 mmol/l), a range expected to include the
`
`D. Van Wyck et al.
`
`maximum plasma concentration of agent after IV push
`injection (Cmax) of 125 mg of ferric gluconate [1900 mg/dl
`(339 mmo/l)], 100 mg of iron sucrose [3000 mg/dl (536 mmol/l)]
`or 100 mg of iron dextran [3080–3396 mg/dl (550–606 mmol/l)],
`according to data from the respective product package inserts.
`
`Determination of transferrin-bound iron
`
`Using a previous method [13], we prepared final experimental
`samples by adding 0.1 ml of stock solution to 1.5 ml of fresh
`pooled serum [average TIBC 370 mg/dl (66.1 mmol/l)] and
`incubating for 5 min. We then passed 1.5 ml of the resulting
`sample over a 2.0 g alumina column to absorb inorganic
`and drug-bound iron, collected the eluate, reconstituted the
`eluate to a total volume of 1.5 ml, and determined the final
`iron concentration on a Hitachi 717 chemistry analyser
`(Boehringer Mannheim Corporation,
`Indianapolis,
`IN,
`USA) using Hitachi-specified ferrozine reagents (Boehringer
`Mannheim) which include detergent, buffers of citric acid
`and thiourea, ascorbate and ferrozine. Briefly, this is a non-
`deproteinizing method in which detergent serves to clarify
`lipaemic samples, buffers lower pH to <2.0 to free iron as
`Fe3þ
`from Tf, ascorbate reduces Fe3þ
`to Fe2þ
`and ferrozine
`reacts with Fe2þ
`to form a coloured complex measured
`spectrophotometrically at 560 nm. We processed blank serum
`samples (0.1 ml of 0.9% NaCl plus 1.5 ml of serum, no added
`iron agent) in a similar manner. To determine the rise, if any,
`in serum iron (delta iron, mg/dl), we subtracted the serum
`blank value from those obtained after iron agent addition
`and column extraction. To determine percent iron donation
`to Tf, we divided delta iron by the concentration of iron
`agent and multiplied by 100.
`
`Statistical analysis
`
`to
`We used two-way analysis of variance (ANOVA)
`determine the effect of the agent and concentration on
`delta iron (SigmaStat Version 2.03; SPSS Science, Chicago,
`IL, USA) and one-way repeated measures ANOVA on ranks
`(Friedman) with pairwise multiple comparison procedures
`(Tukey test) to determine effect of the agent on percent iron
`donation.
`
`Results
`
`To determine the reliability of the method to exclude
`iron agent or inorganic iron from the measurement of
`Tf-bound iron, we prepared plasma-free (Tf-free)
`solutions of each iron agent at a high concentration
`(6875 mg/dl)
`in 0.9% sodium chloride, passed the
`solutions through alumina columns, and assayed the
`eluate. Results (Table 1) showed that this method of
`sample preparation and assay detects <1% of added
`iron agent, regardless of the agent assayed.
`To further evaluate the reliability of the method,
`we next determined the within-test variability of
`Tf-bound iron results in serum with iron agent added.
`We assayed 12 samples of serum after adding ferric
`gluconate to a final concentration of 1719 mg/dl,
`a concentration approximating the maximum expected
`after administering 125 mg of ferric gluconate IV over
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`Labile iron in IV iron agents
`
`563
`
`Table 1. Evaluation of reliability of the method to exclude contaminating iron agent
`
`Iron agent
`
`Detected iron (mg/dl, mmol/l)
`
`Iron agent detected (%)
`
`Ferric gluconate
`Iron sucrose
`Iron dextran-INFeDÕ
`Iron dextran-DexferrumÕ
`
`42 (7.3)
`30 (5.4)
`36 (6.4)
`27 (4.8)
`
`0.61
`0.44
`0.52
`0.39
`
`Serum-free (Tf-free) solutions of each iron agent at 6875 mg/dl (1228 mmol/l) concentration in 0.9% sodium chloride were prepared, the
`solutions were passed through alumina columns, and the eluate was assayed. In the absence of serum, the assay detected <1% of the
`added iron.
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`Fig. 1. Relationship between the change in Tf-bound iron (Delta Tf-Bound Iron) and concentration of added iron for each of four iron
`formulations. The two iron dextrans examined include iron dextran-I (INFeDÕ) and iron dextran-D (DexferrumÕ). Each data point
`represents the mean of six replicate experiments.
`
`10 min. The 12 replicate samples processed with the
`test method yielded a within-test coefficient of variation
`of 9.0%.
`We then used the column separation method to
`determine the relationship between the concentration
`of added iron agent and change, if any, in serum Tf iron
`concentration. Results, expressed as delta Tf-bound
`iron, are shown in Figure 1. At low levels of added iron
`agent, delta iron results were low regardless of the class
`of agent. Delta iron increased with added iron con-
`centration for all agents. The degree of increase in delta
`iron differed according to class and identity of agent.
`The effect of concentration and class of agent were
`each significant (P¼ <0.001). There was a statistically
`significant interaction between concentration and agent
`(P¼ 0.002).
`We then calculated the delta Tf-bound iron as a
`percent of the total concentration of iron agent added.
`(1719–6875 mg/dl)
`At concentrations of
`iron agent
`expected to be achieved after USFDA-recommended
`doses are administered IV push in adults, the median
`increase in Tf-bound iron represented 2.5–5.8% of
`
`added iron depending on the agent added (Figure 2). In
`general, the effect of agent on percent iron donation
`was highly significant (P¼ <0.001), with means pro-
`gressing as follows: ferric gluconate > iron sucrose>
`iron dextran-INFeDÕ>iron dextran-DexferrumÕ. Dif-
`ferences between agents for percent iron donation were
`statistically significant (P<0.05) only between ferric
`gluconate and both iron dextran agents, and between
`iron sucrose and iron dextran-DexferrumÕ.
`
`Discussion
`
`to
`the first
`to our knowledge,
`These results are,
`demonstrate that IV iron formulations donate iron
`directly to Tf in vitro, to show that the degree of iron
`donation is concentration-dependent, to estimate the
`size of the labile iron fraction, and to compare the
`size of labile iron fractions among commonly used
`IV iron agents. We found that the biologically available
`or labile iron fraction estimated by our methods
`represents 2.5–5.8% of total iron in IV iron agents
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`D. Van Wyck et al.
`
`significantly interfere with results of the assay we
`used to determine Tf-bound iron. In our assay, the
`magnitude of the increase in serum Tf-bound iron we
`observed, at drug iron concentrations likely to have
`been achieved after doses used in the in vivo studies, is
`10-fold higher than the expected level of drug iron
`interference. This finding prompts two conclusions
`about the delta Tf-bound iron results we observed.
`First, our results do not arise from contamination by
`iron agents passing through the column. Secondly, the
`absolute values of delta Tf-bound iron we observed
`are theoretically sufficient to predict Tf over-satura-
`tion after IV iron administration in patients if iron
`doses are high and given rapidly and pretreatment
`unbound iron-binding capacity (UIBC) is low.
`The role of the UIBC of plasma in preventing
`consequences of labile iron release should be considered
`crucial. Tf binds iron at either or both of two binding
`sites. At physiological pH, the binding of iron to Tf is
`sufficiently stable (stability constant 1024) to assure that
`it would take nearly 10 000 years for one atom of iron
`to dissociate spontaneously. The avidity of Tf for
`ferric (Fe3þ
`) iron renders unbound (apo) Tf a potent
`endogenous iron chelator. In vitro addition of apo-
`transferrin effectively blocks the oxidant damage [4],
`bacterial growth enhancement [5] and anti-phagocytic
`effects [14] of labile iron associated with IV iron
`agents. In vivo administration of apotransferrin binds
`free iron and removes redox-active non-Tf-bound iron
`in patients after chemotherapy for haematological
`malignancies [15].
`If IV iron agents indeed include a labile iron fraction,
`as our data and others demonstrate, and if adverse
`clinical outcomes are shown to result, then care should
`be taken in patients to assure sufficient UIBC to
`accommodate the delta iron expected after rapid IV
`administration of iron agents.
`That labile iron is not free iron is an important
`distinction. Dialysable free iron has not been detected
`in any formulation of IV iron agent thus far examined,
`including iron dextran, iron sucrose or ferric gluconate.
`Labile iron lacks biochemical characterization and is
`therefore functionally described as that portion of the
`total IV iron agent that exerts a disproportionate early
`biological activity. Furthermore, recent results suggest
`that the bioactivity of labile iron in the extracellular
`space extends to the intracellular compartment. The
`response of human hepatoma cells to the addition of
`IV iron agents to culture media suggests early release of
`bioactive iron from iron preparations [16]. As antici-
`pated by our current results, intracellular iron effects
`follow the progression ferric gluconate  iron sucrose
`> iron dextran.
`Despite the abundant and longstanding albeit
`indirect evidence of labile iron in all
`iron agents
`examined here, and despite widespread therapeutic
`use of these agents for more than 50 years, no adverse
`patient outcomes attributable to labile IV iron have
`been demonstrated in patients when the agents are
`given in recommended doses and within recommended
`laboratory iron indices. Prospective multicentre cohort
`
`564
`
`Fig. 2. Percent iron donation to Tf by parenteral iron agent (mean
`± 95% CI). Differences in iron donation between agents reached
`significance (P<0.05) as follows:
`ferric gluconate greater than
`either iron dextran-I (INFeDÕ) or iron dextran-D (DexferrumÕ);
`iron sucrose greater than iron dextran-D only.
`
`and varies according to the sequence iron dextran-
`DexferrumÕ<iron dextran-INFeDÕ<iron sucrose<
`ferric gluconate.
`Our findings are consistent with the hypothesis that
`iron bioavailability among commonly used IV iron
`agents is not determined solely by intracellular meta-
`bolism and may be manifested prior to RES uptake of
`the IV iron compound. The manifestation of labile iron
`we examined in the current assay was an increase in
`Tf-bound iron. Previous studies suggesting a labile iron
`fraction revealed induction of oxidative stress [3,4],
`generation of bleomycin-detectable or redox-active
`iron [5], promotion of bacterial growth [5] and
`impairment of neutrophil phagocytic function [2]. A
`recent unreviewed report, using sophisticated fluores-
`cent methods to measure in vitro Tf-iron binding,
`showed that uptake of iron by apotransferrin from
`IV iron agents is rapid in the presence of ascorbate [4].
`Quantitative determinations were not made and quali-
`tative comparisons were not offered, but the degree of
`iron donation appeared to follow the sequence we
`observed, i.e. ferric gluconate > iron sucrose  iron
`polymaltose. Iron dextran was not examined.
`The acute effect of IV iron on Tf saturation has
`previously been examined in patients. Administration
`of ferric gluconate at a rate of 62.5–125 mg over
`30 min or 125 mg over 240 min or 100 mg of iron
`saccharate (Ferrivenin; Laevosan, Austria; this was
`not iron sucrose) over 1 min are associated with a Tf
`saturation >100% [2]. That is, measured serum iron
`levels after IV iron injection exceeded serum iron-
`binding capacity. These results, however, have been
`difficult to interpret because of potential interference
`of drug-bound iron with the assay for serum Tf-bound
`iron [12]. Thus, the possibility that drug iron inter-
`ference accounted for all or part of the rise in serum
`iron immediately after IV iron infusion could not
`be excluded. As others have demonstrated [13], even
`high concentrations of
`iron formulations do not
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`Labile iron in IV iron agents
`
`studies in haemodialysis patients found no relationship
`between the incidence of bacteraemia and either the
`serum ferritin or the total dose of IV iron [17]. In a
`retrospective study, although an increased risk of mor-
`tality was reported in haemodialysis patients receiving
`more than 10 vials (equivalent to 1.0 g) of iron dextran
`in a 6 month period [18], this conclusion was not
`sustained when similar data were assessed using more
`sophisticated statistical
`techniques
`(H.I. Feldman,
`submitted for publication). Higher doses of IV iron
`dextran were described among non-survivors compared
`with survivors of the Normal Hematocrit Heart Trial,
`but the significance of this effect is controversial [2].
`More recently,
`iron sucrose was administered to
`patients in the Scandinavian Hemoglobin Normaliza-
`tion trial [19]. Despite the need for high IV iron doses
`among haemodialysis patients randomized to the
`normal compared with the low haemoglobin treatment
`group, there was no difference in mortality or morb-
`idity between treatment groups and no difference in
`iron sucrose dose between survivors and non-survivors
`[19]. Since iron sucrose doses averaged80 mg per week,
`these latest findings are particularly reassuring.
`Taken together with the results of the foregoing
`clinical trials, our findings confirming and quantifying a
`labile iron fraction in IV iron agents, support the
`overall safety of IV iron therapy. Our results predict,
`however,
`that Tf
`super-saturation is theoretically
`possible after IV iron infusion. If so, occasionally,
`patients with low serum UIBC may experience super-
`saturation, manifesting a labile iron reaction (hypoten-
`sion, cramping, diarrhoea or chest pain) at
`the
`recommended upper limits of IV iron infusion rates:
`200 mg of iron sucrose over 5 min or 125 mg of ferric
`gluconate over 10 min. In such patients, caution, lower
`doses and slower infusion rates should accompany
`subsequent IV iron administration.
`
`Acknowledgements. This work was supported by an unrestricted
`grant from American Regent, Shirley, NY.
`
`Conflict of interest statement. D. Van Wyck is a consultant to
`American Regent, Inc., Amgen Inc., Gambro Healthcare and Shire
`Pharmaceuticals. He serves on the speakers boards for American
`Regent and Amgen.
`
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`Received for publication: 25.7.03
`Accepted in revised form: 1.10.03
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