Iron dextran formulations

Ferric oxyhydroxide-dextran compositions for treating iron deficiency having ellipsoidal particles with a preferred molecular weight range of about 250,000 to 300,000 daltons.

FIELD OF THE INVENTION 
The present invention relates to improved iron dextran formulations for the 
treatment of iron deficiency, and to methods for preparing such 
formulations. 
BACKGROUND OF THE INVENTION 
The intravenous or intramuscular injection of sterile solutions of an iron 
dextran complex is clinically indicated for the treatment of patients with 
documented iron deficiency in whom oral administration is unsatisfactory 
or impossible. 
Iron dextran is absorbed from the injection site after intramuscular 
injection, for example, into the capillaries and the lymphatic system. 
Circulating iron dextran is cleared from the plasma by cells of the 
reticuloendothelial system, which split the complex into its components of 
iron and dextran. IMFERON.RTM., for example, a product previously marketed 
by Fisons Pharmaceuticals, is released to the blood after uptake by the 
phagocytic activity of macrophages. See Henderson, et al., Blood 
34:357-375 (1969). The iron immediately is bound to available protein 
moieties to form hemosiderin or ferritin, the physiological forms of iron 
or, to a lesser extent, to transferrin. This iron, which is subject to 
physiological control, replenishes the iron component of hemoglobin and 
other depleted iron stores. 
The major benefit of the clinical use of iron dextran is that, due to its 
large molecular weight (i.e., greater than 70,000 daltons), the iron 
dextran complex is not excreted by the kidneys. Therefore almost the 
entire dose of iron dextran remains bioavailable as the iron dextran is 
metabolized in the liver. The major portion of an intramuscular injection 
of iron dextran is absorbed within 72 hours. Most of the remaining iron is 
absorbed over the ensuing 3 to 4 weeks. 
Iron dextran for parenteral administration currently is marketed by Steris 
Pharmaceuticals, Inc. under the brand name INFeD.RTM.. As formulated, this 
product is a dark brown and slightly viscous sterile liquid complex of 
ferric oxyhydroxide, beta-FeO(OH), and is a low molecular weight dextran 
derivative in approximately 0.9% weight per volume sodium chloride for 
intravenous or intramuscular use. It contains the equivalent of 50 mg of 
elemental iron (as an iron dextran complex) per ml. Sodium chloride may be 
added for tonicity. The pH of the solution is between 5.2 and 6.5. 
Under electron microscopy, IMFERON.RTM. has been shown to have an inner 
electron-dense FeO(OH) core with a diameter of approximately 3 nm and an 
outer moldable plastic dextran shell with a diameter of approximately 13 
nm. Almost all of the iron, about 98-99% is present as a stable 
ferric-dextran complex. The remaining iron represents a very weak ferrous 
complex. 
The dextran component of conventional iron dextran products is a 
polyglucose that either is metabolized or excreted. Negligible amounts of 
iron are lost via the urinary or alimentary pathways after administration 
of iron dextran. Staining from inadvertent deposition of iron dextran in 
subcutaneous and cutaneous tissues usually resolves or fades within 
several weeks or months. Various studies have reported that the half life 
of iron dextran in iron deficient subjects ranges from 5 to more than 20 
hours. Notably, these half-life values do not represent clearance of iron 
from the body because iron is not readily eliminated from the body. See, 
for example, the package inserts for IMFERON.RTM. and INFeD.RTM., or 
Hamstra, et al. JAMA 243:1726-1731 (1980). 
U.S. Pat. No. 2,820,740 and its reissue RE 24,642 to London et al. describe 
colloidal injectable iron preparations suitable for parenteral injection 
formed of a nonionic ferric hydroxide, partially depolymerized dextran 
complex. Current commercial iron dextran products, based on these two 
prior patents do not have sufficient purity (see FIGS. 1 and 2) and needed 
thermal stability (see FIGS. 3 and 4) to safeguard safety and sterility 
concerns. Also, these commercial products have a relatively short plasma 
residence time which could cause a potential risk of iron overload in 
specific organs. See, Carthew, R. E. , et al. Hepatology 13 (3) :534-538 
(1991); Pitts, T. O., et al. Nephron 22:316 (1978); Weintraub, L. R., et 
al. Brit. J. Hematology 59:321 (1985); and Fletcher, L. M., et al., 
Gastroenterology 97:1011 (1989). 
Similarly, U.S. Pat. No. 2,885,393 to Herb also discloses iron dextran 
complexes. The most suitable range in molecular weight of the partially 
depolymerized dextran for injection was found to be 30,000 to 80,000 
daltons or lower. A subsequent patent to Herb, U.S. Pat. No. 4,180,567, 
discloses other iron preparations and methods for making and administering 
such preparations; however, the method disclosed does not teach the 
heating of iron dextran complexes above 100.degree. C. 
Other methods for the production of iron dextran complexes have been 
described, for example, in U.S. Pat. No. 4,599,405 to Muller et al. 
regarding iron (III) hydroxy/dextran complexes that are produced using an 
alkali carbonate, ammonium carbonate or a carbonate of an organic base 
added to an acid solution containing a partially depolymerized dextran and 
an iron (III) salt. Thereafter, an alkali metal hydroxide or ammonium 
hydroxide is added. The suspension so formed is then converted into a 
solution by heating, and the solution worked up in a known manner. 
Alternatively, ferric chloride and dextran can be reacted in aqueous 
solution in the presence of citric acid as disclosed in U.S. Pat. No. 
3,697,502 or by treating reactive trivalent iron with a complex-forming 
agent consisting of sorbitol, gluconic acid and certain oligosaccharides, 
in particular proportions and amounts as taught in U.S. Pat. No. 
3,686,397. 
U.S. Pat. No. 4,749,695 and its divisional, U.S. Pat. No. 4,927,756, both 
to Schwengers, disclose a water-soluble iron dextran and a process for its 
manufacture. As disclosed, the dextran utilized has an average molar mass 
of from 2,000 to 4,000 daltons. Another alternative includes the 
complexation of ferric hydroxide with hexonic acid derivatives of dextran 
as in U.S. Pat. No. 4,788,281 to Tosoni. 
U.S. Pat. No. 3,908,004 to Kitching discloses the preparation of iron 
compositions to treat iron-deficiency anemia. Methods of formulating these 
compositions include the heating of an aqueous alkaline solution of a 
polysaccharide with a water soluble inorganic iron compound such as ferric 
oxychloride. The presence of the alkali is said to be necessary to bring 
about the formation of the complex. However, the alkaline conditions also 
cause some degradation of the polysaccharide and the low molecular-weight 
species so formed produce iron compounds which are responsible for 
undesirable effects. 
U.S. Pat. No. 4,659,697 to Tanaka discloses a process for producing an 
organoiron (II) compound-containing antianemic composition which through 
the cultivation of a yeast in a saccharide-containing nutrient medium, 
such as grape juice, in the presence of an iron compound to form a 
cultured broth comprising an organoiron(II) compound, alcohol and water 
and removing the alcohol from the cultured broth to an extent that the 
resulting cultured broth has an alcohol content of less than about 1% by 
volume, and an antianemic composition produced thereby. The antianemic 
composition was said to be very stable, with excellent absorbability into 
a living body and incorporation of iron into hemoglobin. 
Iron dextran complexes also have application as imaging agents. For 
example, dextran/magnetite is disclosed as a particulate solution 
specifically noted to be stabilized by polymeric dextran. (See Hasegawa et 
al., U.S. Pat. No. 4,101,435. Several others have used dextrans of various 
molecular weights as ingredients in the synthesis of magnetic colloids or 
particles. (See Hasegawa et al., U.S. Pat. No. 4,101,435; Molday, U.S. 
Pat. No. 4,454,773; and Schroder, U.S. Pat. No. 4,505,726. The resulting 
complexes of dextran and iron oxide have varying sizes and structures, but 
all have molecular weights of at least about 500,000 daltons. 
The incorporation of high molecular weight dextran into magnetic particles 
or colloids may, however, cause some patients to experience adverse 
reactions to the dextran, particularly when such complexes are 
administered as parenteral magnetic resonance contrast agents. These 
adverse reactions may also be due in part to problems of high molecular 
weight polymers such as dextran dissociating from the metal oxide colloid 
upon prolonged storage or under high temperatures, thereby leaving the 
metal oxide free to aggregate. 
Despite the variety of iron dextran formulations described in the prior 
act, current iron deficiency products are based on technology that has not 
satisfactorily resolved stability and purity concerns. What is needed in 
the therapeutic field of iron supplementation, is an improved 
next-generation iron dextran product with enhanced purity and thermal 
stability, as well as prolonged plasma residence time to minimize possible 
iron overload complications without compromising the efficacy of iron 
dextran therapy. 
SUMMARY OF THE INVENTION 
These and other objects are achieved by the iron dextran product prepared 
according to this invention. It has excellent attributes and thermal 
stability but also has prolonged plasma residence time to minimize 
possible iron overload problem without compromising the efficacy of iron 
dextran. 
It is an object of the present invention to provide methods for 
synthesizing iron dextran compositions useful in the treatment of iron 
deficiency. Associated compositions also are disclosed. Such compositions 
include aqueous colloidal suspensions or solutions of a ferric 
oxyhydroxide-dextran complex, having an average molecular weight of about 
100,000 to 600,000 daltons and a substantially uniform size distribution. 
Physiologically acceptable carriers for these compositions also are 
contemplated. The administration of such compositions to humans and other 
mammals for the treatment of iron deficiency or, in the case of non-human 
mammals, for medicinal as well as investigational purposes also are 
described. 
In a preferred embodiment of the present invention, the molecular weight 
range of the iron dextran compositions are about 150,000 to 350,000 
daltons, and more particularly preferred are compositions with a molecular 
weight range of about 250,000 to 300,000 daltons. 
It is a further object of the present invention to provide iron dextran 
compositions having a beta-FeO(OH) core. A further object of the invention 
is to provide ellipsoidal iron-dextran particles with a length in the 
range of about 25 to 45 nanometers, more preferably about 31.5 to 36.5 
nanometers, and a width of about 3.5 to 5.5 nanometers, more preferably 
about 4 to 5 nanometers. 
It is a further object of the present invention to provide methods for 
synthesizing iron-dextran compositions as described above. The process of 
the present invention involves the initial production of iron-dextran 
particles by conventional methods. Applicants, however, have discovered 
that superior particles may be produced by the following process. 
Generally, as discussed in greater detail below, iron-dextran particles 
are purified by conventional techniques to remove various impurities, in 
particular, chloride iron, but also including any toxic by-products, 
uncomplexed dextran and, generally, any component of the initial 
iron-dextran reaction mixture which would not be appropriate or permitted 
to be administered to patients in an approvable composition.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present inventors have found that iron dextran formulations prepared 
according to the following specifications are surprisingly more 
temperature stable and/or exhibit a much greater degree of homogeneity 
than is evidenced by or would have been expected from iron dextran 
formulations of the prior art such as IMFERON.RTM. and INFED.RTM.. The 
improved methods and compositions disclosed for the preparation of these 
iron dextran formulations achieve uniform molecular weight distribution. 
Safety, reliability and quality of iron dextran injectable and infusible 
products can be significantly improved over previous products. Our product 
now in development is called DEXFERRUM.RTM.. DEXFERRUM.RTM. is a 
pharmaceutically-equivalent iron dextran characterized by a higher mean 
molecular weight (266,608.+-.1.4% daltons). 
In the following discussion and examples, certain calculations as set forth 
below are required to determine the amounts of active and inactive 
ingredients: 
The amount of iron dextran is based on its iron (Fe.sup.3+) content. The 
amount in mg/ml is calculated by dividing the desired iron concentration 
in mg/ml of elemental iron by the powder's % w/w iron content divided by 
100. This amount is then multiplied by the batch size in liters for the 
amount required in grams for that batch size. This value is then corrected 
for its moisture content. 
In general, a suitable iron III salt, such as ferric chloride, is 
neutralized with a suitable alkali to which a modified dextran is added 
either before, concomitantly or after neutralization to produce an iron 
dextran complex with a molecular weight in the range of about 100,000 to 
about 600,000 daltons. The resulting solution is purified of excess 
dextran, salts, toxic impurities, etc., such as are identified in Table 2 
by any suitable method to produce an iron dextran aqueous concentrate or 
powder with an elemental iron concentration of between about 5% to about 
50%. Purified iron dextran powder or concentrate is then used in the 
preparation of a final solution made of the foregoing iron dextran 
composition, with an elemental iron content of about 25 to about 100 
mg/ml. 
We have observed that in solution, dextran is not tightly bound to the iron 
core, and complexes formed of aggregates in which, e.g., two cores might 
be bound to the same dextran molecule, can be observed. The dextran serves 
to stabilize the core, but the purification process associated with the 
initial preparation of iron dextran particules in which, e.g., chloride 
iron is removed, also tends to remove some of the dextran. 
To a final solution made of the foregoing iron-dextran composition, an 
appropriate amount of oxidized dextran is added to provide a desired final 
ratio of the content of iron to dextran in the final iron dextran 
composition in a range from about 1:2 to 1:5, but preferably about 1:4 as 
described in greater detail below. The iron-dextran and oxidized dextran 
mixture is heated and reacted for an appropriate length of time with a 
suitable alkali. Generally, an appropriate length of time is not less than 
about one hour. The actual amount of time required to complete the 
reaction is dependent on the amounts and ratios of starting materials. 
Determination of the end point may be measured by the absence of dextran 
enhancement of the LAL endotoxins test. We have determined that oxidized 
dextran enhances the LAL gel clot method for assessing endotoxins, whereas 
reacted material, prepared according to our disclosure, demonstrates no 
such enhancement. Thus, in our manufacturing procedure, the reaction end 
point is determined by this technique to be complete when the amount of 
unreacted dextran does not exceed about 0.05 percent. After cooling and 
dilution to a final volume, the pH of the solution is adjusted to a 
physiologically acceptable pH range. This adjusted solution is then 
aseptically filled and/or terminally sterilized for administration, such 
as by injection. 
We believe that the reaction of the iron dextran complex with an oxidized 
dextran under alkaline conditions converts the terminal unit of oxidized 
dextran from .delta.-Gluconolactone to sodium gluconate. The resulting 
solution contains dextran that is both bound and unbound to the iron 
complex where the molecular weight distributions of the bound and unbound 
dextrans are in equilibrium. Without wishing to be bound by any particular 
mechanism of action, we believe that the oxidized dextran at this stage of 
processing of iron dextran compositions minimizes or substantially 
eliminates aggregate complexes in which two iron cores might be bound to 
the same dextran molecule. Moreover, oxidized dextran has a terminal 
carboxyl group and has superior chelating abilities. 
The amount of oxidized dextran required to produce the desired product 
meeting its desired nonvolatile residue is calculated by subtracting the 
calculated # mg/ml iron dextran (dry weight) from the theoretical total 
weight based on the nonvolatile residue of the desired product. That is, 
for a nonvolatile residue of 28-43 % w/v, the theoretical total weight 
would range from 280 to 430 mg/ml. The value obtained is then corrected 
for the oxidized dextran's loss on drying by dividing this value by 
(1-(loss on drying/100)). This amount is then multiplied by the batch 
volume in liters for the amount of grams for that batch size. 
The amount of alkali (such as sodium hydroxide) is dependent on the amount 
of oxidized dextran since it reacts with the alkali to form a carboxylic 
acid. The reaction is 1:1. To determine the appropriate amount of alkali 
(such as NaOH) in grams, the molecular weight of the alkali is multiplied 
by the number of grams of oxidized dextran required for the desired 
product which is then divided by the average molecular weight of the 
oxidized dextran. 
A maximum limit for the hydrochloric acid used to adjust pH is calculated 
using the desired product's upper limit for chloride content. The amount 
of chloride supplied by the starting materials (iron dextran and oxidized 
dextran) is calculated, then the maximum amount of hydrochloric acid added 
is determined by subtracting the total amount of chloride supplied from 
the starting materials from the desired product's upper limit for chloride 
content, then multiplying the value obtained by the batch size in liters, 
divide this value by the atomic weight of chloride (35.5) and then divide 
by the normality of the hydrochloric acid solution to be used for the 
final value. 
The low molecular weight carbohydrates of the invention must be oxidized in 
order to avoid problems in lack of uniformity and with the presence of 
endotoxins. Such carbohydrates preferably have a molecular weight in the 
range of about 2,000 to 15,000 daltons, most preferably around 6,000 to 
7,000 daltons. The preferred concentrations of the carbohydrates of the 
invention which effectively impart stabilization to the carrier phase of 
the metal oxide composition are in the range of about 0.001M to about 2M, 
most preferably about 0.05M to about 0.5M, but optimal concentrations can 
be determined by those skilled in the art according to conventional 
techniques. 
Some preferred low molecular weight stabilizing agents include, but are not 
limited to, mannitol, sorbitol, glycerol, inositol, dextran 1 (Pharmacia 
Inc., Piscataway, N.J.) and ascorbate. Other useful agents include 
dextrins, celluloses, hydroxyethylstarches, heparins, starches, dextran 
sulfates, carboxylmethylated dextran and carboxymethyl cellulose. In the 
case of dextran 1, which has a molecular weight of about 1,000 daltons, 
the same compound can both stabilize the colloid or particulate suspension 
against unwanted physical changes and block possible adverse reactions. 
The simultaneous injection of dextran 1 and a complex of dextran and the 
magnetic iron oxide decreases adverse reactions to high molecular weight 
dextran alone. 
Preferred methods of manufacture of iron dextran solutions involve the 
neutralization of ferric chloride solution with an alkaline solution of 
dextran. The mixture is heated, then cooled to room temperature and 
clarified by centrifugation. The resulting solution is then concentrated 
to the desired iron content by dialysis against running water. The iron 
dextran is composed of a beta-FeO(OH) core formed by the neutralization of 
an acidic ferric chloride/dextran solution with alkaline sodium 
bicarbonate. The by-products of this reaction are sodium chloride and 
carbon dioxide. During neutralization, the modified dextran is absorbed 
(complexes) to the iron core's surface where the dextran's hydroxyl groups 
provide the "OH" needed for stabilization of the core's beta-FeO(OH) 
structure. 
EXAMPLES 
Experimental studies describing the use of low molecular weight 
carbohydrates as stabilizing agents for metal oxide compositions prepared 
according to the present invention are presented below. These examples are 
to be considered as illustrative of the present invention rather than 
limitative of its scope in any way. 
The preferred dextran formulation for the production of iron dextran 
formulations according to the present invention are prepared by 
fermentation of sucrose using Leuconostoc mesenteroides bacteria (NRRL 
B-512 (F)). The crude dextran is precipitated, hydrolyzed, and 
fractionated by conventional means. The dextran fraction is oxidized with 
an oxidizing agent under alkaline conditions, then purified. 
Studies on the structure of the iron dextran complex report that it is 
composed of a beta-FeO(OH) core complexed with low molecular weight 
dextrans ranging from 3,500 to 7,500 daltons. The oxidized dextran used in 
this invention is the dextran which is depolymerized to an average 
molecular weight ranging from 3,500 to 7,500 daltons. The dextran's 
terminal unit, D-glucose, is then oxidized to gluconolactone. During the 
manufacturing process described in this invention the oxidized dextran's 
terminal unit, gluconolactone, is converted to D-glucuronic acid via 
alkaline hydrolysis. 
The oxidized dextran used to produce iron dextran products according to the 
present invention has the following physical properties as set forth in 
Table 1: 
TABLE 1 
______________________________________ 
Parameter Tolerance 
______________________________________ 
Description White, amorphous powder 
odor Odorless 
Loss on Drying (w/w %) 
Not more than 5.0% 
Sodium chloride content 
Not more than 2.0% 
(w/w %) 
Nitrogenous Impurities 
Not more than 0.015% 
Bromide content Less than 5 ppm 
Alcohol and Related 
Less than 0.05% w/w 
Impurities 
Relative Viscosity of a 10 
Less than 4.0 centistokes 
% sol 
Average Molecular Weight 
Between 3,000 and 7,000 
Phosphate (w/w %) Not more than 0.28% 
Reducing Sugars (w/w %) 
Not more than 7.0% 
Pyrogen Test Passes test 
______________________________________ 
The characteristics and physical properties of the preferred iron dextran 
powder used to produce iron dextran formulations of the present invention 
are as follows in Table 2. This composition is commercially available from 
Laboratorien Hausmann AG in Switzerland, and U.S. Pat. No. 4,599,405, 
discussed above, is relevant to the preparation of such compositions. U.S. 
Pat. No. 3,697,502 also is relevant. 
TABLE 2 
______________________________________ 
Parameter Tolerance 
______________________________________ 
Description Brown, amorphous powder 
Identification Complies 
Loss on Drying (w/w %) 
Not more than 10.0% 
Sodium chloride content 
Not more than 6.0% 
(w/w %) 
Dextran content Between 29.0 and 36.0% 
Iron Content Between 28.0 and 35.0% 
Bromide content Less than 5 ppm 
Alcohol and Related 
Less than 0.05% w/w 
Impurities 
pH of a 5% Solution 
5.2 to 6.5 
Molecular Weight 
Determination by GPC 
M.sub.w Between 255,000-520,000 
M.sub.n Between 200,000-365,000 
M.sub.w /M.sub.n Not more than 1.7 
Arsenic Not more than 2 ppm 
Lead Not more than 100 ppm 
Copper Not more than 100 ppm 
Zinc Not more than 100 ppm 
Bacterial Endotoxins 
Passes test 
______________________________________ 
EXAMPLE 1 
Preparation of Iron dextran Compositions 
In a 200 liter steam-jacket reaction vessel, 114 liter of hot (70.degree. 
C. -90.degree. C.) water was added. Next, 30.0 kg of iron dextran, 
satisfying the parameters described above, along with 28.3 kg oxidized 
dextran, also satisfying the parameters discussed above. The mixture was 
diluted up to 175 liters. Next, 185 g of NaOH was added and mixed with the 
iron dextran mixture. The vessel was sealed and then heated to a range of 
110.degree. C.-115.degree. C. using a steam jacket for three hours. The 
vessel was then cooled to approximately 25.degree. C. and vented during 
the cooling process. The pH was tested and adjusted to the range of 
5.7-6.0. 
The reaction solution was prefiltered through a 1.0 micron membrane into a 
holding vessel. Next, the filtered solution was passed through a 0.2 
micron filter into sterilized receiving vessels, and depyrogenated vials 
were filled and stoppered with aliquots of the sterilized solution. 
EXAMPLE 2 
Evaluation of Process Results to Determine Molecular Weight Using HP-GPC 
The molecular weight of the iron dextran complex of Example 1 was 
determined by gel permeation chromatography in a HP-GPC system equipped 
with a differential refractometer as the detector and an integrator with a 
GPC program for molecular weight calculations. The HP-GPC column was 
packed with porous particles of polyacrylic acid containing pore sizes up 
to 1000 angstroms. The pores act as sieves where smaller molecules 
permeate through in the packing's pores while the larger molecules are 
excluded from the packing and are eluted by the more mobile phase. Thus, 
macromolecules elute from the columns, from largest to smallest. 
FIGS. 1-4 show comparisons between the iron dextran formulations of the 
present invention and two commercial preparations. These figures present 
data generated by a refractive index detector. This detector measures the 
concentration of the iron dextran, dextran and other molecules and the 
integrator's GPC program interprets the data and calculates the relative: 
weight average molecular weight (Mw), number average molecular weight (Mn) 
and polydispersity index (Mw/Mn) of the sample. The reported values are 
based on polyethyleneglycol (PEG) and polyethylenoxide (PEO) standards 
used for calibration of the instrument, and are considered relative 
molecular weights which should be within 5% of the actual values. 
Ellipsoidal particles of the present invention are shown in FIG. 5. This 
shows DEXFERRUM.RTM. at a magnification of about 140,000.times.. In 
comparison, FIG. 6 shows particles sold under the name INFeD.RTM.. The 
unique conformation and consistency of the DEXFERRUM.RTM. particles, as 
compared with another iron dextran supplement product, is evident from the 
foregoing figures and comparative electron photomicrographs. This 
information is consistent with the literature analyses of prior art 
iron-dextran complexes as reflected in the paper by Cog, et al, from J. 
Pharm. Pharmac 24:513-517 (1972). 
The DEXFERRUM.RTM. particles typically range in length from about 31.5 to 
about 36.5 nanometers and are approximately 4.5 nanometers in width. The 
IMFERRON.RTM. particles by photomicrograph have a core also in an 
ellipsoid shape but ranging in size from about 13.5 to 18 nanometers in 
length with a width ranging from about 9 to about 13.5 nanometers. These 
electron photomicrographs are not shown. FIG. 6, which shows the 
INFeD.RTM.product, reveals iron cores also in the form of thin ellipsoids 
with a length of about 13.5 to 18 nanometers with an average width of 
about 4.5 nanometers. As FIG. 5 indicates, the DEXFERRUM.RTM. particle is 
substantially uniform in terms of particle size and shape. FIG. 6 shows a 
relative heterogeneity of the comparable INFeD.RTM. product. 
EXAMPLE 3 
Human Plasma Residence Time 
The following Table 3 demonstrates that the plasma residence time of the 
new iron dextran prepared according to the present invention is 
significantly longer than that of other commercial iron dextran 
formulations. 
TABLE 3 
______________________________________ 
Plasma Residence Time of Iron Dextrans* 
Products Half life (hours) 
______________________________________ 
IMFERON 5.9 
INFED 34.2 
DEXFERRUM 58.9 
______________________________________ 
*The plasma halflife figures assume a standard intravenous dose of 100 mg 
of elemental iron. IMFERON.RTM. determination used a radioisotope label o 
iron .sup.59 Fe, while INFeD.RTM. and DEXFERRUM.RTM. had direct 
measurement of iron dextran in plasma. 
EXAMPLE 4 
Comparison of Indicators of Iron Dextran Efficacy 
Measurements of transferrin, plasma ferritin and hemoglobin levels are the 
major indicators of iron dextran efficacy. The following Tables 4 and 5 
demonstrate that the iron dextran according to the present invention are 
biologically comparable to an existing commercial preparation. Levels of 
hemoglobin, serum ferritin, serum iron and total iron binding capacity 
(the serum iron divided by the total iron binding capacity times 100%) 
were determined by standard CLIA monitored commercial clinical laboratory 
assays. 
TABLE 4 
______________________________________ 
Comparison of Transferrin Levels 
Transferrin AUC 0-96 hours (ug*hr/dL) 
Iron Dextran Invention 
Commercial #2 
______________________________________ 
11,510 11,316 
______________________________________ 
TABLE 5 
______________________________________ 
Comparison of Hemoglobin and Ferritin Levels 
Hemoglobin Ferritin 
Hemoglobin New Iron Ferritin New Iron 
Days Comm. #2 Dextran Comm. #2 Dextran 
______________________________________ 
0 10.7 10.3 122.8 104.1 
7 10.9 11.1 255.5 619.8 
14 11.3 11.2 205.8 233.8 
21 11.0 11.4 186.8 213.3 
28 11.0 11.4 194.5 193.2 
______________________________________ 
EXAMPLE 5 
Comparison of Biological Equivalence Between INFeD.RTM. and DEXFERRUM.RTM. 
To examine the pharmacokinetics of iron dextran in hemodialysis patients, 
we serially determined iron dextran concentrations in the serum of 20 
patients after 100 mg IV (intravenous) iron dextran was administered. By 
this study, we determined whether treatment with DEXFERRUM.RTM. versus 
INFeD.RTM. was biologically equivalent for the pharmacokinetic parameters, 
since DEXFERRUM.RTM. is an iron dextran preparation, according to the 
process of the present invention. DEXFERRUM.RTM. has a higher average 
molecular weight than INFed.RTM., i.e., about 300,000 daltons to 180,000 
daltons. The clinical design was a 2-period crossover study with patients 
randomized to receive either DEXFERRUM.RTM. followed by INFed.RTM. or 
INFeD.RTM. followed by DEXFERRUM.RTM.. Blood samples were obtained at 
specified times after the end of drug infusion. 
A comparison of the results for area-under-the-curve suggested a 
statistically significant difference between the two treatments, with no 
statistically significant difference in the observed maximum blood 
concentration. Analysis of secondary parameters, suggested a statistically 
significant difference in the half-lives, but no difference in the volumes 
observed for the two treatments. 
Iron deficiency in dialysis-associated anemia is heralded by a falling 
hematocrit, or increasing Epoetin alfa requirements to maintain target 
hematocrit, coupled with a declining serum transferrin saturation and 
serum ferritin. See, e.g., Van Wyck DB, Iron Balance in Dialysis Patients, 
Healthmark, N.Y. (1989); Eschbach, J. W. et al., Ann. Intern. Med. 11:992 
(1989); McEvory, G. K. ed. AHES: Drug Information '92, American Society of 
Hospital Pharmacists, pages 766-768 (1992); and Gimenez, L. F. et al., 
Hematology/Oncology Clinics 8:913 (1995). 
Unfortunately, oral iron supplements do not reliably restore iron balance, 
probably because intestinal absorption of low doses is limited, high doses 
promote GI toxicity and noncompliance, and any benefit to body iron 
balance is outstripped by iron deficits due to dialysis-associated or 
pathologic blood loss. When oral supplementation fails to prevent iron 
deficiency in dialysis-associated anemia, therapy with intravenous iron 
dextran is indicated. See, Eschbach, J. W. et al., cited above; and Van 
Wyck, D. B., et al., Kid. Int. 35:712 (1989). 
The effective bioavailability of iron dextran given intravenously depends 
on clearance of the iron dextran colloid from the plasma space. Previous 
information in patients with normal renal function has shown that 
radiolabelled iron dextran after IV administration is removed from the 
plasma by the reticuloendothelial system. See, Eschbach, J. W. et al., and 
Henderson, et al., cited above. Though iron deficiency in patients with 
dialysis-associated anemia is a frequent indication for iron dextran 
therapy, information on pharmacokinetics of iron dextran in patients with 
renal failure is lacking. Nor are data available describing 
pharmacokinetics of an unlabelled product. 
The physiologic response to anemia in individuals with normal renal 
function is characterized by increased production of erythropoietin by the 
kidney. In chronic renal failure, erythropoietin production fails, and 
progressive anemia routinely ensues. Prior to the introduction of 
recombinant human erythropoietin (in North America, Epoetin alfa; produced 
by Amgen and OrthoBiotech), virtually all chronic hemodialysis patients 
suffered dialysis-associated anemia, and 25% required frequent 
transfusions to maintain the hematocrit in a life-sustaining range. 
The use of Epoetin alfa successfully reverses transfusion dependency and 
raises hemoglobin and hematocrit into a range compatible with health. 
Nevertheless, the therapeutic efficacy of Epoetin alfa is frequently 
thwarted in practice by the development of iron deficiency. Iron 
deficiency in dialysis-associated anemia is heralded by a falling 
hematocrit, or an increasing Epoetin alfa requirement to maintain target 
hematocrit, coupled with a declining serum transferrin saturation and 
serum ferritin. 
Several other factors also contribute to the ongoing negative iron balance 
experienced by hemodialysis patients. First and foremost, the dialysis 
procedure itself is associated with blood loss, from the needle stick and 
from retention of red cells within the dialyzer microtubules. Though the 
volume lost with each dialysis is small, the cumulative loss of iron is 
estimated to amount to greater than 1 gram annually. Since the diet of the 
dialysis patient is restricted by prescription in the foods richest in 
iron (red meat), little iron is available to dialysis patients from 
nutritional sources. 
Oral iron is commonly prescribed. However, despite the observation that 
intestinal iron absorption in chronic renal failure is intact, meals, 
antacids, a multiplicity of medications, and a high incidence of gastritis 
and constipation conspire against the effectiveness of oral iron 
supplements. Iron deficiency marked initially by a fall in ferritin level, 
followed by a drop in the transferrin saturation, and eventually, as iron 
deficiency erythropoiesis slows red cell production, by iron deficiency 
anemia or an increasing demand for Epoetin alfa. When oral supplementation 
fails to prevent iron deficiency in dialysis-associated anemia, therapy 
with intravenous iron dextran is indicated. 
Evidence in patients with iron deficiency anemia and normal renal function 
suggests that recovery of iron for hemoglobin synthesis or iron stores 
early after intravenous iron dextran infusion is incomplete. Our previous 
retrospective analysis in patients with dialysis-associated anemia 
confirmed that quantitative iron utilization for hemoglobin or 
ferritin-related stores is highly variable and incomplete within the first 
90 days after iron dextran infusion. 
To forestall declining hematocrit or increasing Epoetin alfa doses, iron 
dextran is administered early in iron deficiency, whenever the ferritin 
falls below 100 ug/L or the transferrin saturation falls below 20%. Our 
data confirm that, when iron dextran is given in this early stage of iron 
deficiency, when storage iron depletion is present but worsening anemia or 
Epoetin alfa resistance has not yet occurred, therapeutic efficacy is 
marked by a rise in serum ferritin, signifying repletion of iron stores, 
without a concomitant increase in hemoglobin. 
In the current study, we examined iron utilization after infusion of five 
100 mg infusions of iron dextran, INFeD.RTM., in iron deficient patients 
receiving Epoetin alfa for dialysis-associated anemia. We compared results 
with those seen in patients after an equimolar dose of iron dextran, 
DEXFERRUM.RTM.. The 500 mg is a standard therapeutic dose for iron 
deficiency in iron anemic dialysis patients. 
This was an active treatment control study using a randomized, unblinded 
design. The purpose of the study was to determine whether treatment with 
DEXFERRUM.RTM., when compared with INFeD.RTM., is biologically equivalent 
for hemoglobin synthesis and ferritin-related stores in patients 
undergoing hemodialysis for end-stage renal disease who meet the 
requirements for parenteral iron supplementation. The primary study 
outcome was the percent mobilization of iron from iron dextran. Results 
after iron dextran INFeD.RTM. (Schein Pharmaceuticals, Phoenix, Az.) were 
compared to those after equimolar administration of DEXFERRUM.RTM. 
(Luitpold Pharmaceuticals, Shirley, N.Y.). 
Secondary study outcomes included serum ferritin, total body iron, 
hemoglobin, serum iron, total iron binding capacity (TIBC), and serum 
transferrin saturation. We also examined adverse events after 
administration of each test dose and each therapeutic dose of iron 
dextran, and compared results after DEXFERRUM.RTM. to those after 
INFeD.RTM.. Five (5) single 100 mg IV doses (total dose: 500 mg) of each 
drug were administered to the patients in each group during five 
sequential dialysis sessions (see FIG. 1 in section titled "Study 
Design"). 
EXAMPLE 6 
Iron Mobilization Early After Iron Dextran Infusion in Hemodialysis 
Patients 
To determine the reliability of serum iron indices and the degree of iron 
utilization early after iron dextran infusion, we measured iron status 
before and at weekly intervals after a total course of 500 mg IV iron 
dextran INFeD.RTM. in 11 iron-deficient patients receiving chronic 
hemodialysis and Epoetin alfa for dialysis associated anemia. Oral iron 
therapy was withheld and evidence of bleeding, infection, inflammation, 
recent surgery or transfusions was absent. Mobilization was calculated by 
expressing the increase in body iron as a percent of total iron 
administered (Van Wyck, et al. cited above): 
Iron stores=400.times.[log(ferritin)-log(3)] 
Red cell iron=150.times.(Hbg) 
% Mobilization={[(A.sub.0 -A.sub.1)! (B.sub.0 -B.sub.1)]/500}=100% 
where A.sub.0 and B.sub.0 are values for stores and red cell iron, 
respectively, at time zero, and A.sub.1 and B.sub.1 are values at 
intervals afterwards. Results.+-.SD) are as follows in Table 6: 
TABLE 6 
______________________________________ 
Day Hgb % Saturation 
Ferritin % Mobilization 
______________________________________ 
0 10.8 .+-. 0.9 
17.2 .+-. 7.4 
104.7 .+-. 84 
-- 
7 11.1 .+-. 1.1 
22.1 .+-. 9.5 
215.6 .+-. 107 
38.6 .+-. 26 
14 11.6 .+-. 1.0 
19.9 .+-. 7.6 
198.6 .+-. 108 
50.8 .+-. 29 
21 11.2 .+-. 1.0 
20.1 .+-. 7.1 
176.7 .+-. 102 
32.7 .+-. 28 
29 11.3 .+-. 0.9 
18.9 .+-. 6.9 
182.9 .+-. 117 
37.8 .+-. 25 
______________________________________ 
The increase in hemoglobin and ferritin was statistically significant 
(&lt;0.02). Thus, in the presence of Epoetin alfa therapy, 1) ferritin and 
hemoglobin rise quickly after IV iron dextran, and 2) an early rise in 
transferrin saturation is transient, due to early incorporation of iron 
into hemoglobin and iron stores, 3) which is, in the first four weeks, 
highly variable and predictably incomplete. Accordingly, decisions to 
repeat iron dextran therapy based on low transferrin saturation should be 
weighed against the observation that, within the first month after IV 
administration, most of the original iron dose remains physiologically 
unavailable. 
Based on the foregoing discussion and experimental data, one skilled in the 
art would readily be able to modify the production processes in order to 
optimize reaction and administration conditions for particular 
compositions of iron dextran. Thus, the following claims should be 
considered as defining our invention, rather than the foregoing specific 
examples. All articles and patent references are hereby incorporated by 
reference in their entireties.