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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2807-2816
A Comparison of the Pharmacological Properties of Carbohydrate
Remodeled Recombinant and Placental-Derived  -Glucocerebrosidase:
Implications for Clinical Efficacy in Treatment of Gaucher Disease
By
BethAnn Friedman,
Kris Vaddi,
Constance Preston,
Elizabeth Mahon,
James R. Cataldo, and
John M. McPherson
From the Cell and Protein Therapeutics Department, Genzyme
Corporation, Framingham, MA.
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ABSTRACT |
The objective of these studies was to characterize the macrophage
mannose receptor binding and pharmacological properties of carbohydrate
remodeled human placental-derived and recombinant  -glucocerebrosidase (pGCR and rGCR, respectively). These are similar
but not identical molecules that were developed as enzyme replacement
therapies for Gaucher disease. Both undergo oligosaccharide remodeling
during purification to expose terminal mannose sugar residues.
Competitive binding data indicated carbohydrate remodeling improved
targeting to mannose receptors over native enzyme by two orders of
magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At
37°C, 95% of the total macrophage binding was mannose receptor
specific. In vivo, pGCR and rGCR were cleared from circulation by a
saturable pathway. The serum half-life (t1/2) was 3 minutes
when less than saturable amounts were injected intravenously (IV) into
mice. Twenty minutes postdose, -glucocerebrosidase activity
increased over endogenous levels in all tissues examined. Fifty percent
of the injected activity was recovered. Ninety-five percent of
recovered activity was in the liver. Parenchymal cells (PC), Kupffer
cells (KC), and liver endothelium cells (LEC) were responsible for
75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR
and for 76%, 11%, and 12%, respectively, of the hepatocellular
uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC
(t1/2 = 3 days).
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GAUCHER DISEASE is a rare genetic
disorder characterized by a functional deficiency of
-glucocerebrosidase activity. -Glucocerebrosidase is required for
hydrolysis of glucocerebroside to glucose and cerebroside. There are no
alternative degradative pathways. For reasons that are not well
understood, tissue macrophages are the predominant cell type that
accumulate glucocerebroside glycolipid under enzyme-deficient
conditions. Consequently, Gaucher disease is characterized by the
presence of lipid-laden macrophages (Gaucher cells) in the liver,
spleen, bone, and lungs. The clinical manifestations of Gaucher disease
reflect the multiorgan distribution of Gaucher cells. Individuals with
advanced Type I Gaucher disease usually present with
hepatosplenomegaly, life-threatening anemia, thrombocytopenia, and
severe bone disease (see Beutler,1 Morales,2 and Kingma3 for reviews).
Two enzyme replacement therapies have been approved by the Food and
Drug Administration for treatment of this genetic disease. The first,
Ceredase (Genzyme Corp, Cambridge, MA), contains carbohydrate remodeled
human placental-derived -glucocerebrosidase (pGCR). More recently, a
recombinant form of this protein, Cerezyme (Genzyme Corp),
was approved for human use. The recombinant enzyme (rGCR) differs
structurally from pGCR in that it contains histidine in place of
arginine at amino acid residue 495, a
Man3GlcNAc2Fuc in place of an oligomannose
structure at amino acid 19, and a Man3GlcNAc2Fuc in place of
Man3GlcNAc2 at amino acid 146.4 Despite these differences, both enzyme replacement therapies were shown
to be clinically effective in halting the progression of Gaucher
disease and, perhaps more importantly, in reversing many of the
debilitating clinical manifestations of this genetic
disorder.5-18
The effectiveness of enzyme replacement therapy for Gaucher disease is
believed to be dependent on the ability to deliver -glucocerebrosidase to macrophages, because these are the cells that
accumulate glycolipid in the enzyme-deficient state. It is for this
reason that during the production of pGCR and rGCR the terminal sialic
acid, galactose, and N-acetyl-glucosamine sugars are sequentially
removed from the glycoproteins to expose mannose sugars. It was
originally postulated that such a remodeling step would provide a means
to target -glucocerebrosidase to the mannose receptor-mediated
endocytotic system of macrophages.19
Here we provide strong evidence to support the premise that these
enzymes can be effectively delivered to macrophages in vivo by
presenting mannose receptor binding data, Kupffer cell uptake data, and
intracellular concentration-time data.
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MATERIALS AND METHODS |
Materials.
All chemicals were of the highest analytical grade commercially
available. Sources for individual reagents are noted when mentioned.
Placental and recombinant glucocerebrosidase (pGC and rGC) and the
respective carbohydrate remodeled proteins (pGCR and rGCR) were
obtained from Genzyme Corporation's protein purification and
manufacturing groups. These preparations, when not commercially available, were prepared as described elsewhere.4
-Glucocerebrosidase enzyme activity assay.
Glucocerebrosidase activity was measured using
p-nitrophenyl--D-glucopyranoside (Sigma Chemical Co, St Louis, MO)
as a substrate. Product (p-nitrophenyl; pNP) formation was
detected by absorbance at 405 nm. A reference standard curve, assayed
in parallel, was used to quantitate concentrations of
glucocerebrosidase in samples to be tested.
Protein quantitation.
125I-labeled GCR was quantitated using a micro-BCA reagent
kit (Pierce, Rockford, IL) with bovine serum albumin (BSA; Pierce, Rockford, IL) as a standard.
Protein iodination.
Mannosylated BSA (Man23 BSA; E.Y. Labs Inc, San Mateo, CA)
was iodinated by the chloramine T method20 and purified
from free 125I by size exclusion chromatography. Chloramine
T was obtained from Sigma Chemical Co. GCR was iodinated by the
Bolton-Hunter method.21 Approximately 8 µg of
Bolton-Hunter reagent (SHPP; N-succinimidyl-3-[-4-hydroxyphenyl]-propionate; Pierce) was labeled with 2 mCi 125I-Na (Amersham Lifescience Inc, Arlington
Heights, IL) using chloramine T. 125I-labeled-SHPP was
extracted into benzene, dried under nitrogen, and reacted with GCR at
4°C overnight. 125I-labeled GCR was separated from free
125I-labeled SHPP by size exclusion chromatography on G50
sephadex (Pharmacia Biotech AB, Uppsala, Sweden).
Rat alveolar macrophage harvesting.
Macrophages were harvested from Sprague Dawley rat lungs by lavage with
phosphate-buffered saline (PBS) according to the method of Brain and
Frank.22 Cells in pooled lavages were counted, concentrated
by centrifugation, resuspended at 1 × 107 cells/mL,
and used the same day of harvest.
Binding experiments.
Unless otherwise specified, binding experiments were performed by
incubating freshly isolated rat alveolar macrophages (5 × 105 cells/tube) with nmol/L concentrations of
125I-labeled GCR in the presence and absence of a
competitive inhibitor of mannose receptor binding (yeast mannan, final
concentration 1.25 mg/mL) for a specified amount of time at a specified
temperature. The total volume was 0.1 mL/tube. Bound was separated from
free by centrifuging 0.08 mL or 0.09 mL of suspended cells through 0.1 mL or 0.2 mL of a 4:1 mixture of silicone oil (Boss Products, Elizabeth
Town, KY) and mineral oil (Aldrich, Milwaukee, WI) in 0.4 mL Eppendorf
tubes for 1 minute × 14,000 rpm at 4°C. Each tube was then
cut through the oil layer just above the cell pellet and the counts per
minute (cpm) in the cell pellets were measured in a gamma counter. In
some cases, cpms in supernatants were also measured. Specific mannose
receptor binding was determined by calculating the difference between
binding in the absence of yeast mannan (total binding) and in the
presence of yeast mannan (nonspecific binding). Each time point and
condition was assayed in triplicate.
Competitive mannose receptor binding experiments.
Rat alveolar macrophages were incubated with nmol/L concentrations of
125I-mannosylated BSA in the presence of various
concentrations of the competitive ligands for 3 to 4 hours at 4°C.
Binding in the absence of any competitive ligand was used to calculate
maximum counts per minute cpm bound. The percent of the maximum cpm
bound that occurred in the presence of competitive ligand was then calculated.
Direct mannose receptor binding experiments.
Each preparation of rGCR and pGCR was iodinated on three separate
occasions with freshly prepared 125I-SHPP. The
concentration of each 125I-labeled preparation was
determined by both protein concentration measurement and
-glucocerebrosidase enzyme activity measurement. Binding experiments
were performed by incubating a series of 12 different concentrations of
125I-labeled rGCR or 125I-labeled pGCR (ranging
from 0.5 nmol/L to approximately 100 nmol/L) with rat alveolar
macrophages in the absence and presence of yeast mannan for 3 to 4 hours at 4°C. The data for specific mannose receptor binding then
underwent Scatchard analysis.23 The dissociation constant
(Kd) was equal to the negative reciprocal of the slope of
the best fit line, and the concentration of binding sites was equal to
the x-intercept of that line of the Scatchard plot. The number of
binding sites per cell was calculated by dividing the concentration of
binding sites (M/L) by the concentration of cells (cells/L) and
multiplying that by Avogadro's number (6.023 × 1023 molecules/mol). A statistical comparision of Kd values for
rGCR and pGCR was performed using one-way analysis of variance (ANOVA).
Mannose receptor-mediated association of 125I-labeled
rGCR and 125I-labeled pGCR at 37°C with rat alveolar
macrophages.
Binding experiments were performed by incubating rat alveolar
macrophages with 125I-labeled rGCR or
125I-labeled pGCR in the presence and absence of yeast
mannan at 37°C for times ranging from 0 to 10 minutes. The
concentrations of radiolabeled enzymes in these experiments were 1.5, 3, and 6 nmol/L. Reactions were stopped by transferring tubes to a
0°C ice bath. Bound ligand was separated from free by centrifuging cell suspensions through oil. The rate constants describing the association of 125I-labeled rGCR and
125I-labeled pGCR with mannose receptors on macrophages
(k1) were determined from specific mannose receptor binding
data obtained during the first few minutes of 37°C incubation with
macrophages. Because this portion of the binding curve was linear with
time, the association rate constants (k1) could be
determined from the slope of plots of specific 37°C binding to
mannose receptors versus time. Statistical analysis with ANOVA was used
to compare k1 values of 125I-labeled rGCR and
125I-labeled pGCR.
Animals.
In vivo studies were performed using 6- to 8-week-old female Balb/c
mice obtained from Charles River Laboratories, Wilmington, MA. The mice
weighed an average of 17 g.
Pharmacokinetics of pGCR and rGCR in Balb/c mice.
Tail vein bleeds ( 10 µL/bleed) were obtained at predetermined
times after bolus intravenous (IV) administration of pGCR and rGCR to
Balb/c mice. Sera from these bleeds were assayed for glucocerebrosidase using the glucocerebrosidase enzyme activity assay. Serum
concentration-time data was described by first order exponential
equations. The half-life (t1/2) in serum was calculated
from these equations.
Organ distribution of pGCR and rGCR in Balb/c mice.
Animals administered a bolus tail vein injection of pGCR, rGCR, or
mannosylated BSA (controls) were killed 20 minutes postinjection. The
liver, spleen, heart, lung, brain, and kidneys were excised, weighed,
and tissue homogenates were prepared and assayed for glucocerebrosidase
activity. The organ distribution of pGCR and rGCR after IV
administration was assessed by comparing glucocerebrosidase activity in
organs from pGCR and rGCR injected animals with activity in organs from
mannosylated BSA-injected controls. Biodistribution data were expressed
as pGCR and rGCR activity recovered per gram wet weight tissue and per
organ. In addition, the ratios of total glucocerebrosidase activity in
organs from pGCR and rGCR-injected animals relative to endogenous
levels in the respective organs (from control animals) were calculated.
The percent of injected dose and the percent of recovered dose in each
organ were also calculated. Statistical analysis was performed using
ANOVA. Statistical significance was set at P < .05.
Hepatocellular distribution of pGCR and rGCR after IV administration
to mice.
Mice were injected with pGCR, rGCR, or mannosylated BSA to identify the
cell type(s) responsible for hepatic uptake. At 20 minutes after
administration, livers of anesthetized mice were perfused in situ with
PBS and then collagenase D (0.5 U/mL; Boehringer Mannheim,
Indianapolis, IN). The livers were then excised and gently teased apart
with forceps to free cells. These cell suspensions were filtered
through Spectra/Mesh 60-µm filter sheets (Spectrum Laboratory
Products, Houston, TX) into 15-mL centrifuge tubes and centrifuged at
500 rpm for 5 minutes at room temperature. Three populations enriched
in either parenchymal (PC), Kupffer (KC), or liver endothelial
cells/stellate cells (LEC/SC) were separated on the basis of
differences in size, cell density, and phagocytic properties. A
PC-enriched preparation was obtained from the pellet from the initial
centrifugation step by two additional sequential sedimentation steps.
The supernatant from the initial centrifugation step was layered over 3 mL of 25% Percoll (Sigma Chemical Co) and centrifuged at 1,000 rpm for
15 minutes at 20°C to remove debris. The supernatants were
discarded, and the cell pellets from this step were resuspended in 1 mL
90% Waymouth media (Sigma Chemical Co), 10% bovine serum, pH 7.2, and
were incubated with latex-coated iron particles (Advanced Magnetics,
Cambridge, MA) for 5 minutes at room temperature. The iron particles
and any cells that had bound and/or phagocytosed the iron particles (mostly KC) were separated from solution by a 5-minute exposure to a
magnet. The cells that remained in suspension (mostly LEC/SC) were
removed and saved. Those bound to the magnet were resuspended in 1 mL
90% Waymouth media, 10% bovine serum, pH 7.2. Each population was
analyzed for cell number, cell composition using morphology, esterase
staining and antibody staining of cytospins (see below), and
glucocerebrosidase activity. The amount of glucocerebrosidase activity
in 1 × 106 cells of each cell type was then
calculated from algebraic equations. Endogenous glucocerebrosidase
activity, determined in liver cells isolated from control mice (n = 8),
was subtracted from these values. The data were expressed as mU
glucocerebrosidase per 1 × 106 cells, mU
glucocerebrosidase per total liver, % injected dose, and % recovered
dose in each cell population examined. Literature values for the number
of each cell type per gram liver (1.15 × 108
PC, 1.5 × 107 KC, and 4.8 × 107 LEC/SC per gram liver,
respectively24) were used to estimate the amounts of pGCR
and rGCR recovered per liver and to estimate the percent injected dose
recovered in each cell type evaluated. ANOVA was used to compare cell
type distribution of pGCR and rGCR.
Cell identification.
At least 200 cells/cytospin slide/preparation were classified as a PC,
LEC/SC, or KC by the following criteria: mouse PC are very large, often
binucleated cells with large round, centrally located nuclei, which
stain lightly to moderately with hematoxylin. The cytoplasm often
appears "foamy" and the plasma membrane is well-defined by
hematoxylin staining. These cells stain positive for esterase activity
using 1-naphthol-acetate (Sigma Chemical Co) as a substrate according
to the method of Geissler et al.25 Mouse LEC/SC are the
smallest cells, with round, well-defined, often centrally located
nuclei that stain moderately to intensely with hematoxylin. The
cytoplasm appears "smooth" and the amount of cytoplasm ranges
from barely detectable to less than 50% of the total cell area. The
plasma membranes are distinct and smooth. These cells do not stain
positively for esterase activity using 1-naphthol-acetate as a
substrate. Mouse KC are larger than LEC/SC, but not as large as PC. The
nuclei are often crescent shaped, noncentrally located, often
"foamy" in appearance, and stain lightly to moderately with
hematoxylin. The cytoplasm often appears "foamy" and makes up at
least 50% of the total cell area. The plasma membranes are ruffled and
ill-defined. KC stain positively for esterase activity using
1-naphthol-acetate as a substrate and stain positively with a variety
of antimouse macrophage antibodies including MCA 519 (Serotec,
Kidlington, Oxford, UK) and F4/80 (a gift of Dr Siamon Gordon,
University of Oxford, Oxford, UK).
Intracellular stability of pGCR and rGCR in parenchymal and Kupffer
cells of Balb/c mice.
To determine the intracellular stability of exogenously added enzymes
in parenchymal and Kupffer cells, mice were injected with rGCR or pGCR
and killed at various times postdose administration. Two experimental
animals were used per test article per time point for times greater
than 20 minutes postdose administration. Cells were separated,
identified, and quantitated for glucocerebrosidase as outlined above
for initial cellular distribution studies. Concentration-time data for
PC and KC were respectively best fit by one and two component first
order exponential equations using curve-stripping techniques. Half-life
(t1/2) estimates were determined from these mathematical expressions.
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RESULTS |
Effect of carbohydrate remodeling on the ability of
-glucocerebrosidase to compete for
125I-mannosylated BSA binding sites on macrophages.
Successive removal of terminal sialylic acid, galactose, and
N-acetyl-glucosamine sugars from the complex carbohydrate chains of
human placental-derived glucocerebrosidase (pGC) and recombinant glucocerebrosidase (rGC) significantly improved their abilities to
compete for 125I-mannosylated BSA binding sites on rat
alveolar macrophages (Fig 1). In this
experiment, the concentrations of pGC, rGC, pGCR, and rGCR that
inhibited 125I-mannosylated BSA binding by 50%
(IC50) were 30, 80, 0.2, and 0.3 µg/mL, respectively.
Thus, the difference in mannose receptor binding of pGC and rGC, which
contain mostly complex carbohydrate chains, and the carbohydrate
remodeled forms of these enzymes (rGCR and pGCR) was almost two orders
of magnitude. In addition, competitive binding studies indicated the
IC50 of pGCR and rGCR for 125I-mannosylated BSA
binding sites were comparable to that of nonradiolabeled mannosylated
BSA (data not shown).
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| Fig 1.
Competitive binding of native and carbohydrate remodeled
placental-derived and recombinant glucocerebrosidase to rat alveolar
macrophages. Rat alveolar macrophages were incubated with
125I-mannosylated BSA (0.25 µg/mL) at 4°C in the
presence of increasing concentrations of rGC ( ), pGC (x),
rGCR ( ), and pGCR ( ). Binding of the radiolabeled ligand to the
cells was determined as described in Materials and Methods.
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Direct binding of 125I-labeled pGCR and
125I-labeled rGCR to mannose receptors on rat alveolar
macrophages.
Rat alveolar macrophages were incubated at 4°C with
125I-labeled pGCR and 125I-labeled rGCR,
in the presence and absence of yeast mannan, to assess the
affinity of these ligands for mannose receptors. Under these
conditions, approximately 50% of the total cellular binding of
both ligands was mannose receptor-specific. A representative experiment
showing binding of pGCR to rat alveolar macrophages is shown in
Fig 2. Scatchard plots of the specific
mannose receptor binding data were linear, indicating mannose receptor
binding of 125I-labeled GCR was uniform and
consistent with a single class of binding sites. A total of three
independent direct binding studies were performed with both rGCR
and pGCR. Data for specific 125I-labeled GCR binding to
mannose receptors were analyzed using 125I-labeled GCR
concentrations determined from protein measurements and
125I-labeled GCR concentrations determined from enzymatic
activity measurements (Table 1). Both
methods of analysis yielded equivalent values, within assay
precision, which indicated the enzyme activity of GCR was not affected
by iodination. Using ANOVA, the Kd values for
rGCR and pGCR were compared and shown to be statistically equivalent.
The mean (Kd for GCR binding to mannose receptors was 13 nmol/L. The mean number of mannose receptor binding sites per
cell using these preparations was 3.3 × 104.
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| Fig 2.
Direct binding of pGCR to mannose receptors on rat
alveolar macrophages. (A) Increasing concentrations of
125I-labeled pGCR were incubated with rat alveolar
macrophages at 4°C in the presence () or absence () of yeast
mannan as described in Materials and Methods. Based on these data,
specific binding () of pGCR to mannose receptors was calculated. (B)
Scatchard analysis of the specific binding data provided a straight
line indicative of a single class of binding sites. In this experiment,
Scatchard analysis indicated the Kd for the binding of pGCR
to the mannose receptor was 14 nmol/L and the number of binding sites
per cell was 2.5 × 104.
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Table 1.
Summary of Dissociation Constants (kd) and Numbers
of Mannose Receptor Binding Sites/Cell for rGCR and pGCR
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Mannose receptor-mediated uptake of 125I-labeled rGCR at
37°C by rat alveolar macrophages.
At 37°C, under nonequilibrium binding conditions, binding and
uptake of 125I-labeled rGCR by macrophages was directly
proportional to the concentrations of rGCR tested at all time points
measured (Fig 3A). After a 10-minute
incubation of macrophages with 125I-labeled rGCR at
concentrations of 1.5, 3, and 6 nmol/L, approximately 7% to 8% of the
radiolabel was cell associated. Under these incubation conditions,
greater than 95% of the overall binding of 125I-labeled
rGCR to rat alveolar macrophages was specific for mannose receptors
(Fig 3B). The mannose receptor concentration (300 pmol/L) was estimated
by dividing the product of the mean number of mannose receptor binding
sites per cell (3.3 × 104) and the number
of cells used per liter (5 × 109) by Avogadro's
number (6.023 × 1023 receptors/mol). The
concentration of 125I-labeled rGCR that was specifically
associated with mannose receptors of macrophages after a 10-minute
incubation at 37°C was 600 pmol/L (Fig 3B). Considerable mannose
receptor recycling must have occurred during the elapsed time to have
mediated this amount of mannose receptor-specific uptake of
125I-labeled rGCR.
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| Fig 3.
Uptake of 125I-labeled rGCR by rat alveolar
macrophages at 37°C. (A) The concentration of
125I-labeled pGCR specifically bound to mannose receptors
plotted as a function of time after incubation with 1.5 (), 3 (),
and 6 () nmol/L 125I-labeled pGCR. (B) The binding of 6 nmol/L 125I-labeled pGCR to rat alveolar macrophages in the
presence () and absence () of yeast mannan, and the specific
mannose receptor-mediated binding and/or uptake (), plotted as a
function of time.
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Comparision of the rate constants describing the association between
rat alveolar macrophage mannose receptors and 125I-labeled
pGCR and 125I-labeled rGCR at 37°C.
Binding experiments performed at 37°C showed GCR binding to mannose
receptors was only directly proportional to time for the initial 2 minutes of incubation (see Fig 3). Therefore, rate constants describing
the association of 125I-labeled rGCR and
125I-labeled pGCR with mannose receptors on macrophages
(k1) were calculated from the slope of specific mannose
receptor binding versus time plots at these early timepoints.
Representative plots from such an experiment using
125I-labeled rGCR are shown in
Fig 4. Similar plots were obtained with
pGCR (data not shown). Based on the results of these experiments, the
mean k1 for 125I-labeled rGCR was 22.8 ± 9.0 µmol/L 1min1 (n = 5) and the
mean k1 for 125I-labeled pGCR was 25.8 ± 4.8 µmol/L1min1 (n = 3). These
values were statistically equivalent (P = .18).
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| Fig 4.
Rate of binding of 125I-labeled rGCR to rat
alveolar macrophages at 37°C. Binding of 125I-labeled
rGCR to rat alveolar macrophages was measured at early time points as
described in Materials and Methods. Binding of the radiolabeled enzyme
was performed in the presence () and absence () of yeast mannan.
Specific binding () of the enzyme to mannose receptors was
calculated from these data.
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Pharmacokinetics of pGCR and rGCR in Balb/c mice.
To assess the effect of dose on the concentration of GCR in serum over
time, pGCR was administered by bolus tail vein injections to Balb/c
mice at doses of 10, 20, 40, or 50 U/kg. Because these were initial
experiments, bleeds were only taken up to 3 minutes postdose
administration. The serum concentration-time data suggested that
saturation kinetics occurred after bolus administration of pGCR at 50 U/kg, but not at doses of 40 U/kg or less
(Fig 5). In addition, the data suggested
that pGCR, when injected at 40 U/kg or less, had a serum half-life
(t1/2) of between 1 and 3 minutes. When injected at 50 U/kg, the estimated t1/2 was 15 minutes. In subsequent
experiments, mice were injected with pGCR or rGCR at a dose of 40 U/kg
and bleeds were obtained out to 8 minutes postdose for more accurate
determinations of serum t1/2 values. Under these
conditions, the mean t1/2 in serum of
-glucocerebrosidase enzymatic activity after bolus administration
was 2.6 ± 0.6 minutes for pGCR (n = 21) and 3.0 ± 0.4 minutes
for rGCR (n = 12). The serum t1/2 values for rGCR
and pGCR were statistically equivalent.
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| Fig 5.
The effect of dose on the pharmacokinetics of pGCR in
mice. pGCR, at doses of 50 (), 40 (), 20 (*), or 10 ()
U/kg, was injected into the tail vein of mice. Tail vein bleeds were
collected at 0.25-minute intervals and analyzed for glucocerebrosidase
activity. Best fit exponential curves are plotted.
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Biodistribution of pGCR and rGCR in Balb/c mice.
The biodistribution of pGCR and rGCR was assessed 20 minutes postdose
after bolus IV administration of 40 U/kg. Under these conditions, less
than 1% of the initial activity remained in circulation at the time of
sacrifice (data not shown). For both test articles, increases in
-glucocerebrosidase activity over endogenous levels were sixfold in
liver, 2.5-fold to 3.5-fold in spleen and brain, and 1.5-fold to 2-fold
in kidneys and lungs (Fig 6A). Recoveries, calculated by subtracting endogenous levels (control mice) from total
levels of -glucocerebrosidase activity (GCR injected mice) in each
tissue examined, averaged 50% of the injected dose. Of the material
recovered, 94% to 97% was in the liver, 2% to 3% was in the brain,
and 1% to 2% was in the spleen (Fig 6B). There were no statistically
significant differences between the tissue distribution of pGCR and
rGCR.
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| Fig 6.
Biodistribution of pGCR (open bars) and rGCR (solid bars)
after IV administration to Balb/c mice at doses of 40 U/kg. Control
mice were injected with 1 mg/kg mannosylated BSA. Tissues were
harvested 20 minutes postdose and analyzed for glucocerebrosidase
activity. (A) Ratio of glucocerebrosidase activity in tissues from
pGCR- and rGCR-injected animals relative to activity in tissues from
mannosylated BSA-injected controls (denoted by a horizontal line). (B)
Glucocerebrosidase activity recovered from individual organs as a
percent of injected dose. Values presented are the averages and
standard deviations of determinations made on 12 animals per treatment
group.
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Hepatocellular distribution of pGCR and rGCR after IV administration
to mice.
The initial time point for evaluating the cell type(s) responsible for
hepatic uptake of rGCR and pGCR in mice was 20 minutes postdose.
Analysis of the cell types responsible for rGCR uptake indicated that,
per 106 cells, KC took up twice as much rGCR as PC and 20 times as much rGCR as LEC/SC (see Table 2).
The methods used did not separate or differentiate between LEC and SC,
and therefore these cell types are grouped together. KC have
approximately four tenths the surface area of PC and 1.25 times the
surface area of LEC/SC.24 When differences in surface area
were taken into account, the relative uptake of rGCR by KC, PC, and
LEC/SC was 1, 0.19, and 0.056, respectively, which suggested
preferential uptake of rGCR by KC over the other liver cell types.
However, in the entire liver, there are approximately three times more
LEC/SC and eight times more PC than KC.24 When cell numbers
were taken into account, it appeared that PC were responsible for three
times more uptake of rGCR in the liver than KC and LEC/SC were
responsible for very little. It was estimated that 15% of the injected
rGCR activity was recovered in KC, 50% in PC, and 2% in LEC/SC. The
total recovery was estimated at 66%. Of the recovered rGCR activity,
23% was recovered in KC, 75% was recovered in PC, and only 2% was
recovered in LEC/SC.
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Table 2.
Recovery of Exogenous Glucocerebrosidase Activity in
Different Liver Cell Types 20 Minutes After a Bolus IV Injection of
rGCR to Balb/c Mice at a Dose of 40 U/kg Body Weight (approximately 680 mU/mouse)
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Analysis of the cell types responsible for pGCR uptake by the liver is
summarized in Table 3. Because there was no
difference in the hepatocellular distribution of pGCR formulated in as
Ceredase (1% human serum albumin [HSA], 50 mmol/L
citrate, pH 5.9) and pGCR formulated as Cerezyme (3% mannitol, 0.01%
polysorbate-80, 50 mmol/L citrtatye, pH 5.9), that data was pooled. Per
106 cells, KC took up slightly more pGCR than PC and about
three times more than LEC. When differences in surface area were taken into account, there appeared to be preferential uptake of pGCR by KC
over PC and LEC. Overall, it was estimated that 8% of the injected
pGCR activity was recovered in KC, 57% in PC, and 9% in LEC/SC. Total
recovery was estimated at 74%, with 76% of that going to PC and the
rest distributing equally to KC and LEC.
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Table 3.
Recovery of Exogenous Glucocerebrosidase Activity in
Different Liver Cell Types 20 Minutes After a Bolus IV Injection of
pGCR to Balb/c Mice at a Dose of 40 U/kg Body Weight (approximately 680 mU/mouse)
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The differences in the hepatocellular distribution between rGCR and
pGCR appeared to be due to the relative amounts of these two enzymes
that distributed to KC and LEC/SC. Almost twice as much rGCR as
compared with pGCR was recovered from KC (P = .00055) and
considerably less rGCR compared with pGCR was recovered in the LEC/SC
population (P = .025). There was no statistical difference between recoveries of rGCR and pGCR in PC (P = .14), or in
overall recovery of rGCR and pGCR in the liver (P = .27).
Differences between the targeting of rGCR and pGCR was most apparent
when the data were normalized to the percent of total recovered
activity in the different cell populations. The percent of total
recovered rGCR and pGCR activities were 75% and 76%, respectively, in
PC, 22% and 11%, respectively, in KC, and 3% and 12%, respectively, in LEC/SC. These similarities and differences in targeting were noted
at all time points examined (see below).
Intracellular stability of pGCR and rGCR in Kupffer cells of Balb/c
mice.
Equivalent doses (40 U/kg) of pGCR and rGCR were administered to mice
and, at various times postdose, mice were killed and the concentration
of GCR in different populations of liver cells evaluated. At most time
points evaluated, essentially the same amounts of rGCR and pGCR were
recovered from PC and twice as much rGCR compared with pGCR was
recovered from KC (Fig 7). This observation suggested that the intracellular stabilities of these two test articles
were similar in these cell populations. Estimates of half-life
(t1/2) values were made by best-fitting the PC and KC data,
respectively, to one and two component first order exponential equations. The intracellular t1/2 values for rGCR and pGCR
in PC were estimated to be approximately 2 days (44 hours and 50 hours)
for the two test articles, respectively. In KC, approximately the same
percentages (45%) of each enzyme that was initially taken up was
rapidly lost, with t1/2 values of 3 to 4 hours, and
approximately the same percentages (55%) were fairly stable, with
t1/2 values of approximately 3 days (79 to 84 hours). The
amounts of rGCR and pGCR in LEC/SC populations had considerable
variability and were essentially the same as endogenous levels within a
few hours postinjection. Because of large standard deviations and low
levels of exogenous activity (relative to endogenous activity),
intracellular stability of rGCR and pGCR in this cell population was
not assessed.
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| Fig 7.
Uptake and stability of rGCR in (A) KC and (B) PC after
IV administration in Balb/c mice. Mice were injected IV with pGCR ()
or rGCR () at doses of 40 U/kg. At designated times postinjection,
two animals from each group were killed, collagenase perfused, and
hepatocellular populations separated as described in Materials and
Methods. Values presented are the average and range of the duplicate
determinations. Solid lines are theoretical plots.
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DISCUSSION |
The effectiveness of enzyme replacement therapy for Gaucher disease
hinges on the ability to target -glucocerebrosidase to Gaucher
cells, the lipid-laden macrophages that reside in the liver, spleen,
bone marrow, and lungs of affected individuals. Liver endothelial cells
as well as macrophages express mannose receptors.26 Here we
present data that provide strong evidence that -glucocerebrosidase
molecules that have exposed mannose sugars on carbohydrate chains bind
to mannose receptors in vitro and effectively target to macrophages in vivo.
The competitive binding experiments presented here clearly show that
treatment of pGC and rGC with neuraminidase, galactosidase, and
-hexosaminidase, which resulted in mannose-terminated
oligosaccharides,4 improved binding of these enzymes to the
mannose receptors on rat alveolar macrophages by approximately two
orders of magnitude. Scatchard analyses of equilibrium binding data
indicated that the receptor dissociation constants for rGCR and pGCR
were approximately 13 nmol/L and that there were approximately 3.3 × 104 mannose receptors per cell. These values are
comparable to those reported for the binding of
Man24-AI-BSA, the classic mannose receptor ligand, to
mannose receptors on rabbit alveolar macrophages at 2°C
(Kd = 12.8 nmol/L and receptor number = 3.8 × 104 sites/cell27).
The pGCR and rGCR binding data presented here are significantly
different from those reported by Sato and Beutler,28 who determined that the dissociation constant for 125I-labeled
pGCR binding to mannose receptors on mouse peritoneal macrophages was
100 nmol/L and that there were approximately 5 × 105
mannose-dependent receptors for pGCR per cell. The basis for these
differences in experimental results is not certain, but several
possible explanations exist. Sato and Beutler28 isolated pGCR from residue of commercial product using concanavalin A-Sepharose affinity chromatography followed by methyl- -pyranoside elution. Contamination of the pGCR with low levels of either the affinity matrix
or the eluting ligand may have interfered with their binding experiments. Alternatively, protein aggregation or oxidation, both of
which pGCR and rGCR are susceptible, may have occurred during their
manipulations of the protein before their binding studies. This is
particularly relevant because we and others19 have noted
that oxidative iodination procedures can inactivate the enzyme. In our
experience, only the use of Bolton-Hunter reagent for iodination
maintains the enzyme in its native form. Sato and Beutler used an
oxidative method to iodinate pGCR for their binding studies. In the
studies presented here, the specific enzyme activity of radiolabeled
and nonlabeled preparations were the same, indicating that the method
of iodination used did not result in structural effects that could
adversely affect binding properties. Furthermore, competitive binding
studies using nonlabeled material also indicated the affinity of GCR
for mannose receptors was similar to the classic mannose receptor
ligand, Man24-AI-BSA.
Binding experiments performed at 37°C provided additional,
independent verification that pGCR and rGCR effectively bound to mannose receptors on macrophages and were endocytosed via the mannose-receptor pathway. At 37°C, greater than 95% of the total binding of 125I-labeled rGCR and 125I-labeled
pGCR to macrophages was specifically mediated by mannose receptors
(Figs 3 and 4). Both rGCR and pGCR exhibited statistically equivalent
association rate constants. The data indicated that within 10 minutes
at the 37°C incubation temperature, 7% to 8% of the added rGCR
(at concentrations of 1.5 to 6 nmol/L), was specifically associated
with macrophages via the mannose receptor endocytotic system. Although
the amount of ligand endocytosed in these 37°C experiments was not
quantitated, to account for the amount of specific mannose
receptor-mediated cell association observed, considerable endocytosis
of mannose receptor-ligand complexes and receptor recycling would have
had to occur. In addition, when cells were incubated at 4°C with 6 nmol/L 125I-pGCR, the amount bound to mannose receptors was
one fourth that bound within 10 minutes at 37°C (see Figs 2A and
3). Taken together, these data indicated that binding and uptake of
rGCR and pGCR into macrophages in vitro was very efficient and
suggested that delivery of these enzymes to macrophages in vivo was feasible.
Evidence that GCR might target to mannose receptors in vivo was first
provided by Furbish et al.19 These investigators injected male Osborne-Mendel rats with pGCR alone and in combination with mannose-terminal fetuin or ahexo-orosomucoid, two competitors for
mannose receptor binding. The t1/2 of pGCR in serum
increased from 2.3 minutes to 36 minutes and 17 minutes in the presence of the respective competitors. Furbish et al19 also showed
a shift in the hepatocellular distribution from hepatocytes (PC) to
nonparenchymal cells (KC and LEC) when pGC was converted to pGCR by
sequential treatment with neuraminidase, galactosidase, and
N-acetylglucosaminidase. The data in this report confirm and extend
those initial observations.
In Balb/c mice, pharmacokinetic experiments provided evidence that rGCR
and pGCR were cleared from circulation by a saturable (receptor-mediated) pathway. At doses of 50 U/kg, pGCR had an estimated
t1/2 of 15 minutes, whereas at 40 U/kg, the
t1/2 of rGCR and pGCR was estimated between 2.5 and 3 minutes. Xu et al29 also observed saturation kinetics of
pGCR in Balb/c mice.
Our studies showed that at 20 minutes post-rGCR or pGCR injection into
Balb/c mice (6 to 8 serum half-lives for a 40 U/kg dose), the liver,
spleen, kidney, heart, lung, and brain exhibited enhanced
-glucocerebrosidase activity compared with controls. Overall
recoveries averaged 50% for pGCR and rGCR. There were no discernable
differences in the organ distribution patterns of these two test
articles. The greatest enhancement in -glucocerebrosidase activity
was noted in the liver. In this organ, -glucocerebrosidase activity
levels were elevated sixfold relative to controls. This value is larger
than previously reported values after administration of pGCR to
mice.29,30 The lower values observed in previous studies
was possibly due to the use of a fluorescent assay
(4MU--D-glucopyranoside) to quantify enzyme activity in organs
retrieved from the experimental animals. Heme, present in nonperfused
organs of these animals, could quench the fluorescent product of such
an enzyme-based assay. In contrast to previous investigators, a
colorimetric assay (pNP--D-glucopyranoside; see Materials and
Methods) was used in the studies presented here to quantify
glucocerebrosidase activity. Based on spiking experiments, the accuracy
of the colorimetric assay was not significantly affected by heme
contamination in the tissue homogenates.
Evidence that pGCR and rGCR could be effectively delivered to tissue
resident macrophages in liver (KC) was derived from the cell type
distribution studies. Our results clearly showed that significant
amounts of rGCR and pGCR were taken up by KC 20 minutes postadministration. Given the differences in cell surface areas, it
also appeared that KC exhibited enhanced uptake of rGCR and pGCR
compared with surrounding cells.
Interestingly, uptake of pGCR and rGCR by KC and LEC/SC differed. The
only structural differences between pGCR and rGCR that have been
identified are the single amino acid difference at sequence position
495, the presence of oligomannose structures in lieu of complex
carbohydrate structures at Asn 19, and a lower overall level of
fucosylation in pGCR compared with rGCR.4 Presumably one or
more of these differences are responsible for the differences observed
in uptake of these enzymes by KC and LEC.
In the studies reported here, the PC population contributed to 75% of
the hepatocellular uptake of pGCR, and the KC and LEC populations
contributed 11% and 12%, respectively. These results agreed with a
previous report by Furbish et al.19 Willemsen et
al,30 Murray and Jin,31 and Bijsterbosch et
al32 reported relatively little uptake by PC and
considerable uptake by LEC. The reasons for the disparate data are not
clear and are difficult to discern, given all of the differences in the
study designs. These differences include differences in animal species,
strain, and sex, differences in pGCR dose and formulation, and
differences in in-life procedures, methods of tissue and cell
preparation, time points evaluated and methods of analyses. We and
Furbish et al19 used similar methods of cell separation,
similar methods for pGCR detection, but different test animals (female
Balb/c mice and male Osborne-Mendel rats, respectively) and different formulations of the test article (commercial preparation that contained
substantial HSA and research grade material, respectively). Despite the
differences, our results and those of Furbish et al19 were
similar. We and Bijsterbosch et al32 used similar methods of cell separation (except for temperature), similar methods for pGCR
detection, and the same formulation, yet our results were different.
Bijsterbosch et al,32 Willemsen et al,30 and
Murray and Jin31 used different methods for pGCR detection,
and different test animals (male Wistar rats, female Balb/c mice and
male Sprague Dawley rat, respectively) yet their results were similar
to each other and different from ours. Thus, there is not an obvious
explanation for why our results and those of Furbish et
al19 differ from results in other published reports.
Once taken up by KC, rGCR and pGCR exhibited similar intracellular
stability. The terminal intracellular t1/2 in PC and KC were estimated at 2 and 3 days, respectively. In KC, a biphasic intracellular stability profile was noted for both rGCR and pGCR, where
the initial phase had a t1/2 of 3 to 4 hours. The
intracellular stabilities reported here for the terminal phase are much
longer than those reported in a previous study by Xu et
al29 in the whole liver. Those investigators report an
initial stability t1/2 of about 1 hour and a secondary,
slower t1/2 of approximately 12 to 20 hours. Although there
were many similarities in study design (dose, test animal, etc), the
studies did differ in the assays used to detect pGCR activity. As
discussed above, heme quenches fluorescence of 4 MU (the product in the
fluorescent assay used by Xu et al29), but not pNP (the
product in the colorimetric assay used in our studies). Quenching would
result in an underestimation of residual pGCR activity in various
organs and the perception of a lower intracellular enzyme stability
than actually exists.
Interestingly, gamma scintography data obtained in humans after bolus
injections of tracer amounts of 123I-labeled
pGCR or rGCR also had a biphasic stability profile.33 Approximately half of the tracer was cleared rapidly from the viscera
(mean t1/2 = 1 to 2 hours), and approximately half was cleared slowly (mean t1/2 = 34 to 42 hours). In humans, as
reported here for mice, no difference in stability was noted between
the two tracer enzymes. The terminal t1/2 for pGCR and rGCR
in mice and in humans was comparable to the half-life of endogenous
glucocerebrosidase in cultured cells (t1/2 = 50 hours for
macrophages, t1/2 = 39 hours for
fibroblasts34).
Collectively, these in vitro and in vivo data provide evidence that
carbohydrate-remodeled glucocerebrosidase derived from human placental
or recombinant sources is effectively taken up by macrophages, the cell
type that accumulates lipid in patients with Gaucher disease.
Furthermore, the intracellular stability data indicate that the
enzymatic activity persists for adequate periods of time to clear
macrophages of their glucocerebroside burden.
| |
ACKNOWLEDGMENT |
The authors thank Edward S. Cole for his helpful editorial comments.
| |
FOOTNOTES |
Submitted July 27, 1998; accepted December 22, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to BethAnn Friedman, PhD, Cell and Protein
Therapeutics Department, Genzyme Corporation, PO Box 9322, Framingham,
MA 01701-9322; e-mail: bfriedman{at}genzyme.com.
| |
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ABSTRACT
INTRODUCTION