British Journal of Haematology, 2003, 120, 853–859
`
`Pharmacokinetics and red cell utilization of 52Fe/59Fe-labelled
`iron polymaltose in anaemic patients using positron emission
`tomography
`
`Soheir Beshara,1 Jens So¨ rensen,2,3 Mark Lubberink,4 Vladimir Tolmachev,4 Bengt La˚ ngstro¨ m,3
`Gunnar Antoni,3 Bo G. Danielson5* and Hans Lundqvist4 1Department of Clinical Chemistry and
`2Department of Clinical Physiology, University Hospital, 3PET Centre and 4Department of Biomedical Radiation Sciences,
`Uppsala University, and 5Department of Internal Medicine, University Hospital, Uppsala, Sweden
`
`Received 13 August 2001; accepted for publication 6 February 2002
`
`iron–polysaccharide complexes are
`Summary. Parenteral
`increasingly applied. The pharmacokinetics of iron sucrose
`have been assessed by our group using positron emission
`tomography (PET). A single intravenous
`injection of
`100 mg iron as iron (III) hydroxide–polymaltose complex,
`labelled with a tracer in the form of 52Fe/59Fe, was similarly
`assessed in six patients using PET for about 8 h. Red cell
`utilization was followed for 4 weeks. Iron polymaltose was
`similarly distributed to the liver, spleen and bone marrow.
`However, a larger proportion of this complex was rapidly
`distributed to the bone marrow. The shorter equilibration
`phase for the liver, about 25 min, indicates the minimal role
`of the liver for direct distribution. Splenic uptake also
`reflected the reticuloendothelial handling of this complex.
`
`Red cell utilization ranged from 61% to 99%. Despite the
`relatively higher uptake by the bone marrow, there was no
`saturation of marrow transport systems at this dose level. In
`conclusion, high red cell utilization of
`iron polymaltose
`occurred in anaemic patients. The major portion of the
`injected dose was rapidly distributed to the bone marrow. In
`addition, the reticuloendothelial uptake of this complex may
`reflect the safety of polysaccharide complexes. Non-satura-
`tion of transport systems to the bone marrow indicated the
`presence of a large interstitial transport pool, which might
`possibly be transferrin.
`
`Keywords: positron emission tomography (PET), 52Fe, 59Fe,
`iron polymaltose, red cell utilization.
`
`Iron preparations for therapeutic administration include
`both iron salts and iron hydroxide–polysaccharide com-
`plexes. The latter group constitutes formulations for paren-
`teral administration, which have been increasingly applied
`over the last few years (Macdougall, 1994; Macdougall et al,
`1996).
`Two main concerns have, however, emerged as a
`consequence of the parenteral application of these com-
`plexes, namely, the iron distribution in the different organs,
`which may be related to late organ damage due to iron
`accumulation, and the second is the release of free iron that
`may result in oxidative stress (Figueiredo et al, 1993). The
`rate of iron release from a complex has been suggested to be
`closely related to the molecular weight of this particular
`complex. The pharmacokinetics of the individual complexes
`
`in terms of iron distribution as well as its availability for
`marrow utilization need to be further explored.
`In earlier
`studies,
`the pharmacokinetics and red
`cell utilization of 100 mg of
`iron hydroxide sucrose
`were
`studied using
`positron emission tomography
`(PET) (Beshara et al, 1999a,b). Iron (III) hydroxide poly-
`maltose is another polysaccharide iron preparation for
`intravenous administration with a molecular weight of
`about 150 kDa.
`The aim of the study was to assess the pharmacokinetics
`and red cell utilization of iron polymaltose by evaluation of
`the uptake and distribution characteristics of 52Fe/59Fe-
`labelled complex, using the PET technique as well as an
`extended follow-up of radioiron red cell utilization.
`
`Correspondence: Dr Soheir Beshara, Department of Clinical Chem-
`istry, University Hospital, SE-751 85 Uppsala, Sweden. E-mail:
`soheir.beshara@medsci.uu.se
`*Professor emeritus.
`
`Patients. Six patients (two men and four women), with
`a mean age ± SD of 45 ± 16 years (range 29–73), were
`included in the study. Baseline haemoglobin ranged from
`
`PATIENTS AND METHODS
`
`Ó 2003 Blackwell Publishing Ltd
`
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`
`854
`
`S. Beshara et al
`
`Table I. Baseline characteristics of the patients.
`
`Age
`(years)
`
`No
`
`Sex
`
`Diagnosis
`
`Hb
`(g/dl)
`
`S-creatinine
`(lmol/l)
`
`S-Fe
`(lmol/l)
`
`S-ferritin
`(lg/l)
`
`TIBC
`(lmol/l)
`
`TS
`(%)
`
`dose
`(U/kg/w)
`
`duration
`(months)
`
`1
`2
`3
`4
`5
`6
`
`42
`35
`39
`54
`28
`73
`
`f
`f
`m
`m
`f
`f
`
`IDA
`ID
`IDA
`Diabetic nephropathy + ID
`Polycystic kidney + RA
`Interstitial nephritis + RA
`
`11Æ3
`12Æ4
`10Æ5
`13Æ2
`10Æ7
`12Æ5
`
`74
`80
`86
`266
`295
`207
`
`7
`9Æ7
`3Æ2
`4Æ6
`20
`19Æ4
`
`3Æ4
`4Æ4
`6Æ1
`178
`151
`224
`
`74
`88
`94
`55
`58
`48
`
`9Æ6
`11
`3Æ4
`8Æ4
`34
`40
`
`–
`–
`–
`20
`80
`30
`
`–
`–
`–
`48
`16
`46
`
`rHuEpo therapy
`
`S-Fe, serum iron; TIBC, total iron-binding capacity; TS, transferrin saturation; rHuEpo, recombinant human erythropoietin; U/kg/w,
`unit/kg/week; m, month; IDA, iron deficiency anaemia; RA, renal anaemia; ID, iron deficiency.
`
`10Æ5 to 13Æ2 g/dl. Three patients were treated with recom-
`binant human erythropoietin (rHuEpo) at a weekly dose of
`20–80 U/kg. None of the patients had received a blood
`transfusion during the 4 weeks prior to inclusion. Causes of
`anaemia other than iron deficiency or renal anaemia were
`excluded and iron supplementation therapy was not
`allowed during the study. Baseline and clinical character-
`istics of the patients are described in Table I. The study was
`approved by the Ethics Committee and the Isotopes Com-
`mittee of the Medical Faculty of Uppsala University, and
`informed consent was obtained from all the patients.
`Radiopharmaceutical method. The production of 52Fe was
`performed as described earlier (Beshara et al, 1999a).
`59FeCl3 was purchased from Amersham (London, UK) in
`the form of solution in 0Æ1 mol/l HCl. Before the synthesis of
`the iron polymaltose, 59FeCl3 (in 0Æ01 mol/l HCl) and
`52FeCl3 were mixed with a ratio of radioactivity of 1:40,
`respectively, and the total volume was adjusted to 1 ml.
`Radiolabelled iron polymaltose was prepared, according to
`the method developed by Vifor (St. Gallen, Switzerland).
`Sucrose (100 mg) was dissolved in the iron solution and the
`pH was adjusted to above 11Æ8 with 5 mol/l NaOH. Iron
`polymaltose (2 ml, corresponding to 100 mg of Fe III) was
`added to this solution. HCl was then added until the pH was
`between 4Æ5 and 7 and the solution was filtered through a
`sterilized 0Æ22 lm filter into a sterile injection bottle. The
`injection bolus was diluted to 20 ml using physiological
`saline and applied over 10 min using a constant volume
`infusion pump.
`The injected positron-derived radioactivity (52Fe/52mMn)
`given to the individual patients ranged from 17Æ4 to 22Æ1
`MBq. The amount of 59Fe was less than 0Æ5 MBq in all the
`patients. The effective dose, for the amounts of radioactivity
`given to the individual patients, was calculated using the
`model described by Robertson et al (1983) and was found to
`be about a total of 5 mSv for 52Fe and 3 mSv for 59Fe. These
`levels were approved for
`this
`study by the Isotopes
`Committee of the Medical Faculty.
`PET imaging, data acquisition and corrections. PET imaging
`and data analysis were done as described earlier (Beshara
`
`et al, 1999a,b). Data analysis was applied in order to
`obtain standardized uptake values. The PET camera
`measures radioactivity concentration (Bq/cc). When this
`is normalized for the injected radioactivity/g body weight,
`with the assumption that tissue density is 1 g/cc, a ratio
`value called the standardized uptake value (SUV)
`is
`obtained.
`The net influx of the radioactivity from blood to tissue as
`well as the sizes of the reversible pools were analysed using
`a compartment model; namely blood, reversible and irre-
`versible tissue pools (Rutland, 1979; Patlak et al, 1983;
`Patlak & Blasberg, 1985). The relationship between the
`tracer concentration in the blood and tissue may be
`described as follows:
`C=Cb ¼ Vd þ Kt  T
`Cb ðsÞ ds=Cb ðtÞ:
`
`Z
`
`t
`
`in which T ¼
`
`0
`The time courses of C and Cb (tissue and blood concen-
`tration of the tracer respectively) are measured from the
`tomograms. Vd is the fractional distribution volume of the
`reversible compartments (no units),
`t
`is the real-time
`following tracer administration (min) and Kt is the transfer
`rate constant into the irreversible compartment (/min). The
`integrated part of the equation constitutes the transformed
`time, which is used to linearize the relationship between
`tissue and blood concentration. It has a dimension of time
`as it represents the time integration of the blood curve
`divided by the blood curve itself. As C/Cb is a ratio between
`tissue and blood concentration of the tracer, it therefore has
`no units. According to this model, the intercept of the linear
`part of the plot represents the distribution volume of the
`tracer in the reversible compartment and the slope repre-
`sents the influx constant, which describes the rate of
`transfer of
`iron from the blood into the irreversible
`compartment. This transfer may also be seen as a clearance
`process, in which Kt represents volume of blood cleared/unit
`time/volume of tissue.
`Radioactivity concentrations were corrected for 52mMn as
`described in an earlier study (Lubberink et al, 1999).
`
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`
`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe iron polymaltose
`Radioiron (59Fe) red cell utilization (%)
`This was performed as described earlier (Beshara et al,
`1999a,b), where the red cell utilization (%) was calculated
`as follows:
`Red cell utilization (%) on day n ¼
`WBA=ml on day n=Injected 59Fe radioactivity
` total blood volume ðmlÞ  100
`
`RESULTS
`
`855
`
`in which WBA is whole blood radioactivity.
`The total blood volume was based on the initial dilution
`of the administered isotope. A linear regression was applied,
`using the PET data from the heart, typically between 10 and
`30 min, and extrapolated to the time of injection. The PET
`data were corrected for 52mMn.
`Total blood volume (ml) ¼
`injected activity ðBqÞ=whole blood radioactivity
`concentration extrapolated to 0 min ðBq=mlÞ
`
`The blood-volume values as determined using the PET
`data from the heart were compared with predicted blood
`volume calculated from the weights and heights according
`to the equations described by Nadler et al (1962).
`
`PET measurements
`The time span of the kinetic studies in the patients ranged
`from 7Æ87 to 8Æ14 h. Changes in the radioactivity concen-
`tration in the different tissues over time are shown in
`Fig 1.
`Blood kinetics. Blood kinetics were similar to that of the
`iron–sucrose complex. However, the blood radioactivity,
`measured by PET in the left ventricle of the heart, reached
`values of 13–23% by the end of the study.
`Organ uptake. A distribution phase of about 25 min was
`noted in liver and spleen uptake of
`iron polymaltose.
`Standardized uptake values are calculated through normal-
`izing the measured radioactivity concentration (Bq/cc) for
`the injected radioactivity per gram body weight (Bq/g), with
`the assumption that tissue density is 1g/cc. A mean liver
`and spleen uptake of about 27 and 16, respectively, was
`shown. A slight decrease in liver radioactivity was seen by
`the end of the PET investigation in all the patients, where
`the radioactivity reached 55% of the peak value. In patients
`4 and 6, an increasing uptake throughout the whole first
`scanning session,
`i.e. 85 min, was followed by a slight
`
`Fig 1. Uptake values of labelled 52Fe-polymaltose in the liver (L), spleen (S) and bone marrow (BM) in one of the patients taken at 0Æ5, 3Æ5 and
`8 h after injection. The injected radioactivity (Bq) is assumed to be homogeneously distributed all over the body. With the assumption of a
`tissue density of 1 g/cc, standardized uptake values (SUV) therefore indicate tissue uptake per cc in relation to the injected radioactivity when
`normalized for the average body uptake per cc. In this patient, maximum radioactivity concentration in BM after 8 h is up to 103 times higher
`than average body concentration.
`
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`856
`
`S. Beshara et al
`
`Fig 2. 52Fe-polymaltose uptake curves in the
`liver, the spleen and the bone marrow in
`patient 3. Standardized uptake values are
`presented on the Y-axis. These are calculated
`through normalizing the measured
`radioactivity concentration (Bq/cc) for the
`injected radioactivity per gram (g) body
`weight, with the assumption that tissue
`density is 1 g/cc. The blood clearance curve is
`also presented.
`
`decrease towards the end of the study. In the bone marrow
`uptake curves, a fast radioactivity uptake was seen during
`the first 10 min followed by an influx of the radioactivity
`into the bone marrow at a lower but steady rate.
`Representative uptake curves in the liver, spleen and the
`bone marrow in patient 3 are shown in Fig 2.
`Graphical analysis. In the bone marrow, no equilibration
`phase was seen but a straight line was found from the very
`beginning in all the patients but patient 4. For the liver, the
`equilibration could be seen after about 20 min, while for
`the spleen, the equilibration varied in the different patients.
`
`The intercepts of the linear part of the liver in the individual
`patients ranged from 0Æ55 to 1Æ33, while that of the bone
`marrow gave a smaller intercept, which ranged from 0Æ02
`to 0Æ57. The slopes of the linear part of the regression
`analysis of the bone marrow reached up to 16 times that of
`the liver (range 1Æ9–15Æ7). Representative linear regression
`plots in patient 3 are shown in Fig 3.
`
`Radioiron (59Fe) red cell utilization (%)
`Predicted blood volumes were within ± 10% of the calcu-
`lated blood volumes in all patients (Nadler et al, 1962).
`
`Fig 3. Regression plots in the liver, spleen and bone marrow in patient 3. The radioactivity concentration on the Y-axis is a ratio between
`tissue and blood, and therefore dimensionless, whereas the transformed time on the X-axis represents the time integration of the blood–
`radioactivity curve divided by the blood–radioactivity curve itself. According to the applied compartment model, the intercept of the linear part
`represents the distribution volume and the slope represents the transfer rate constant from the blood to the irreversible compartment.
`
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`
`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe iron polymaltose
`
`857
`
`Fig 4. Follow-up data of the rate of
`59Fe-iron polymaltose red cell utilization
`in the individual patients. Day 0 is the
`day of PET investigation.
`
`The maximum radioiron red cell utilization ranged from
`61 to 99% and was reached after 16–24 d. Follow-up data
`of the rate of red cell utilization in the individual patients are
`presented in Fig 4.
`
`Changes in Hb levels, reticulocyte counts and iron status
`In all the patients but two (patients 4 and 6), Hb levels
`increased by 1Æ68 ± 0Æ8 g/dl (range 0Æ8–2Æ4 g/dl) within
`2–23 d following administration of the drug. Rapid increase
`in reticulocyte counts within 1–6 d was noted in patients 1,
`2 and 3. In patients 4 and 6, reticulocytosis was noted after
`20 and 14 d respectively.
`In patients 1, 2 and 3, there was a direct increase in
`serum (S)-ferritin levels of eightfold to twelvefold above the
`baseline within 2 d, followed by a decline to baseline levels
`by the end of the study. Minor changes in S-ferritin levels
`were noted in patients 4, 5 and 6.
`
`Adverse events
`No adverse events were experienced by any of the patients
`during or following the application of the drug. One patient
`acquired a haematoma at the site of injection with slowly
`resolving paresthesia. This was not deemed a drug-related
`side effect, however, it might be a drug delivery-related side
`effect.
`
`DISCUSSION
`
`The liver, spleen and bone marrow have been identified as
`the major pathways for endogenous iron metabolism
`(Finch et al, 1970). The relative distribution of
`the
`complex has shown a much higher uptake by the bone
`marrow in relation to the liver and spleen uptake,
`compared with the uptake pattern of the iron–sucrose
`complex.
`As the injected iron polymaltose was handled through
`these organs, a finding which was consistent with the
`
`iron–sucrose pattern (Beshara et al, 1999a,b), these organs
`seem to represent the major pathway for the pharmaco-
`kinetics of
`injected iron polysaccharides. Whether the
`injected iron was
`taken up by the macrophages or
`parenchymal cells is of relevance both to its potential
`toxicity and availability. The uptake by the macrophage-
`rich spleen of
`this iron complex, determined by PET
`technique, was consistent with our observation for the
`iron–sucrose complex. Such reticuloendothelial uptake of
`the injected iron polysaccharides was thought to indirectly
`illustrate the safety of these preparations with regard to
`the long-term effects on the parenchyma of the various
`tissues.
`The high red cell utilization in the different patients
`indicated the efficacy of this iron complex. The variation in
`the percentage red cell utilization was consistent with the
`observed variations in the early distribution of the complex,
`as noted in patients 4 and 6.
`The increase in S-ferritin levels illustrated the replenish-
`ment of the depleted iron stores, which is a well-identified
`and desired effect of iron therapy.
`An interesting observation in this study was that the
`early distribution to the liver was quantitatively much
`less for this complex compared with that of the iron–
`sucrose complex. This,
`in turn, reflected on marrow
`uptake, which reached higher
`levels at earlier
`time
`points. The different preparations, although having a
`similar general distribution pattern, clearly differed in
`their early distribution components. The early pharma-
`cokinetics of the iron sucrose, molecular weight 43 kDa,
`involves the liver and spleen as a buffer to a relatively
`high degree before reaching the marrow, an observation
`which was not found for the iron polymaltose, molecular
`weight 150 kDa.
`The graphical analysis provided two values, namely the
`intercept and the slope, which reflected the distribution
`volume of 52Fe equilibrating with 52Fe in the blood, and the
`
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`
`S. Beshara et al
`858
`influx-rate constant of 52Fe to a compartment which binds
`52Fe irreversibly within the time frame of the study. An
`observation of interest in this study, which was performed
`in vivo, was that the blood to bone marrow transfer-
`rate constant was steady, irrespective of the large variation
`in the blood–iron concentration over the range observed.
`This indicates that despite the high doses of iron admin-
`istered,
`saturation of
`the transport process was not
`reached.
`Under physiological conditions, transport iron is com-
`posed almost exclusively of transferrin-bound iron (Huebers
`& Finch, 1984). One interesting observation, as shown by
`the compartment analysis, which is consistent with our
`earlier studies, is the non-saturation of the transport system
`to the bone marrow at the applied dose level. During the
`study period of 8 h, about 80% of the injected dose was
`cleared from the circulation, most of which was distributed
`in the bone marrow. This exceeded the capacity of plasma
`transferrin by several
`fold. This raises the question of
`possible transport pathways into the bone marrow. One
`possible mechanism is that the iron complex is rapidly
`transported from the plasma into the interstitium, where
`the iron is
`then delivered to an interstitial pool of
`transferrin. This can, in turn, be the source of cellular iron
`uptake. The transport of plasma transferrin over the blood–
`marrow membrane, which can be thought a rate-limiting
`step, is thus of less significance. Direct transferrin uptake of
`iron from polysaccharide complexes has been shown in vitro
`using the iron–sucrose complex (Danielson et al, 1996). A
`similar model has been suggested to explain tumour uptake
`of 67Ga through the transferrin system (Weiner, 1996). The
`buffer capacity of the iron complex, as well as the presence
`of such an interstitial pool of
`transferrin in the bone
`marrow, would explain the tolerance of the injected doses of
`iron complex without having the hazards of free radical
`formation when exceeding the plasma transferrin transport
`capacity. In fact, a parallel elimination process of the iron–
`polymaltose from the blood could be postulated. On one
`hand, uptake of the iron complex by the reticuloendothelial
`system in the macrophage-rich organs could take place,
`with redistribution to the bone marrow, although at a
`relatively low proportion. On the other hand, a direct
`uptake of
`iron by transferrin within the bone marrow
`interstitium can be suggested. Studying the distribution
`characteristics of the various iron complexes is therefore of
`great importance. We cannot exclude other mechanisms of
`cellular iron uptake directly from the complex (Conrad et al,
`1999; Conrad & Umbreit, 2000).
`iron
`In conclusion,
`the early pharmacokinetics of
`polymaltose using the PET technique showed a distribution
`through the liver, spleen and bone marrow, with the
`major portion of the injected dose preferentially distributed
`to the bone marrow. High red cell utilization (up to 99%)
`was found. The least utilization was found in the patient
`with functional iron deficiency, indicating the possible role
`of subtle inflammation.
`In this particular patient,
`late
`utilization was consistent with the early distribution
`pattern. There was no saturation of the transport system
`to the bone marrow at this dose level. Different transport
`
`systems might be involved in iron transport through the
`interstitium.
`
`ACKNOWLEDGMENTS
`
`The authors acknowledge the contribution to the study of
`Margareta Forsman for her assistance with patient data
`collection and Iva Kulhanek for secretarial assistance. We
`also acknowledge the Swedberg Laboratory for technical
`assistance with 52Fe production and the PET Centre staff for
`assistance with the patients.
`
`REFERENCES
`
`Beshara, S., Lundqvist, H., Sundin, J., Lubberink, M., Tolmachev,
`V., Valind, S., Antoni, G., La˚ngstro¨m, B. & Danielson, B.G.
`(1999a) Kinetic analysis of 52Fe-labelled iron (III) hydroxide–
`sucrose complex following bolus administration using positron
`emission tomography. British Journal of Haematology, 104, 288–
`295.
`Beshara, S., Lundqvist, H., Sundin, J., Lubberink, M., Tolmachev,
`V., Valind, S., Antoni, G., La˚ngstro¨m, B. & Danielson, B.G.
`(1999b) Pharmacokinetics and red cell utilization of iron (III)
`hydroxide–sucrose complex in anaemic patients – a study using
`positron emission tomography. British Journal of Haematology,
`104, 296–302.
`Conrad, M.E. & Umbreit, J.N. (2000) Iron absorption and transport
`– an update. American Journal of Hematology, 64, 287–298.
`Conrad, M.E., Umbreit, J.N. & Moore, E.G. (1999) Iron absorption and
`transport. American Journal of Medical Sciences, 318, 213–229.
`Danielson, B.G., Salmonson, T., Derendorf, H. & Geisser, P. (1996)
`Pharmacokinetics of iron (III)-hydroxide sucrose complex after a
`single intravenous dose in healthy volunteers. Drug Research, 46,
`615–621.
`Figueiredo, M.S., Baffa, O., Barbieri Neto, J. & Zago, M.A. (1993)
`Liver injury and generation of hydroxyl free radicals in experi-
`mental secondary hemochromatosis. Research in Experimental
`Medicine, 193, 27–37.
`Finch, C.A., Deubelbeiss, K., Cook, J.D., Eschbach, J.W., Harker,
`L.A., Funk, D.D., Marsaglia, G., Hillman, R.S., Slichter, S.,
`Adamson, J.W., Ganzoni, A. & Giblett, E.R. (1970) Ferrokinetics
`in man. Medicine, 49, 17–53.
`Huebers, H.A. & Finch, C.A. (1984) Transferrin: physiologic beha-
`viour and clinical implication. Blood, 64, 763–767.
`Lubberink, M., Tolmachev, V., Beshara, S. & Lundqvist, H. (1999)
`Quantification aspects of patient studies with 52Fe in positron
`emission tomography. Applied Radiation and
`Isotopes, 51,
`707–715.
`Macdougall, I.C. (1994) Monitoring of iron status and iron sup-
`plementation in patients treated with erythropoietin. Current
`Opinion in Nephrology and Hypertension, 3, 620–625.
`Macdougall, I.C., Tucker, B., Thompson, J., Tomson, C.R., Baker,
`L.R. & Raine, A.E. (1996) A randomized controlled study of iron
`supplementation in patients treated with erythropoietin. Kidney
`International, 50, 1694–1699.
`Nadler, S.B., Hidalgo, J.U. & Bloch, T. (1962) Prediction of blood
`volume in normal human adults. Surgery, 51, 224–232.
`Patlak, C.S. & Blasberg, R.G. (1985) Graphical evaluation of blood-
`to-brain transfer constants from multiple-time uptake data.
`Generalizations. Journal of Cerebral Blood Flow and Metabolism, 5,
`584–590.
`(1983)
`J.D.
`Patlak, C.S., Blasberg, R.G. & Fenestermacher,
`Graphical evaluation of blood-to-brain transfer constants from
`
`Ó 2003 Blackwell Publishing Ltd, British Journal of Haematology 120: 853–859
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2041, P. 6
`
`

`
`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe iron polymaltose
`
`859
`
`multiple-time uptake data. Journal of Cerebral Blood Flow and
`Metabolism, 3, 1–7.
`Robertson,
`J.S., Price, R.R., Budinger, T.F., Fairbanks, V.F. &
`Pollycove, M. (1983) Radiation absorbed doses from Iron-52,
`Iron-55, and Iron-59 used to study ferrokinetics. Journal of
`Nuclear Medicine, 24, 339–348.
`
`Rutland, M.D. (1979) A single injection technique for subtraction of
`blood background in 131I hippuran renograms. British Journal of
`Radiology, 52, 134–137.
`(1996) The mechanism of 67Ga localization in
`Weiner, R.E.
`malignant disease. Nuclear Medicine and Biology, 23, 745–751.
`
`Ó 2003 Blackwell Publishing Ltd, British Journal of Haematology 120: 853–859
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
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