of the Pharmacokinetics and Pharmacodynamics of Proteins by Polyethylene
received February 25, 2000, Revised April 17th, 2000; Accepted April 18th,
of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter,
Amarillo, Texas, USA
With the rapid advances in the
field of biotechnology during the last decade, many peptides and
proteins have been produced and evaluated for therapy of various
diseases, including cancer. However, rapid clearance and the possibility
of immunogenicity after the in vivo administration of these
biotechnology-driven products have impeded their marketing. To
circumvent these problems, synthetic and natural polymers such as
polyethylene glycol (PEG) and dextrans, respectively, have been
covalently attached to proteins, and some of these protein-polymer
conjugates have shown promising therapeutic results. The conjugation of
proteins with polymers usually causes a reduction in the recognition of
the protein by the immune system, resulting in a decrease in protein
clearance and immunogenicity. Most of the protein-polymer conjugates
retain the pharmacologic activity of the protein, although to a lesser
extent than the native protein. Additionally, in most of the examples in
the literature, a significant increase in the plasma half life of the
protein more than compensates for any reduction in the pharmacologic
effects of the polymer-protein conjugates. Therefore, polymer
conjugation in most cases would result in a net increase in the
pharmacologic activity of the protein.
The intent of this article is to
review the pharmacokinetics and pharmacodynamics of proteins conjugated
to PEG which is one of the most widely used synthetic polymers for
Polyethylene glycol (PEG) is a
polymer with the structure (–CH2CH2O–)n
that is synthesized normally by ring opening polymerization of ethylene
oxide. The polymer is usually linear
at molecular weights (MWs) £
10 kD. However, the higher MW PEGs may have some degree of branching.
Polyethylene glycols of different MWs have already been used in
pharmaceutical products for different reasons (e.g., increase in
solubility of drugs). Therefore, from the regulatory standpoint, they
are very attractive for further development as drug or protein carriers.
For coupling proteins to PEG,
usually monomethoxy PEG [CH3 (–O–CH2–CH2)n–OH]
is first activated by means of cyanuric chloride,
1,1'-carbonyldiimidazole, phenylchloroformate, or succidinimidyl active
ester (1) before the addition of the protein. In most cases, the
activating agent acts as a linker between PEG and the protein, and
several PEG molecules may be attached to one molecule of protein as
depicted in Figure 1. Therefore, pharmacokinetics and pharmacodynamics
of the PEG-protein conjugates are dependent on the MW of the PEG used
for conjugation, the number of PEG molecules per each molecule of
protein, and the nature of the bond between the protein and the linker.
Interested readers are referred to a comprehensive review of the
PEG-protein coupling methods by Deluged et al. (1).
1. Schematic presentation of a protein-PEG conjugate. The number of
PEG molecules per each protein molecule varies for different
Vivo Disposition of PEG Backbone
It is believed that the kinetics
of proteins attached to polymers are substantially affected by the
kinetics of the polymer itself. Therefore, before reviewing specific
PEG-protein conjugates, an analysis of the plasma kinetics and tissue
distribution of PEGs is necessary.
The plasma kinetics of PEGs are
reported (2, 3) to be dependent on both the MW of the polymer and the
site of injection. Yamaoka et al. (2) investigated the disposition of
radiolabeled PEGs with MWs of 6 kD (PEG-6), 20 kD (PEG-20), 50 kD
(PEG-50), and 170 kD (PEG-170) after iv administration to mice. Similar
to other polymers such as dextrans (4, 5), the plasma concentrations (Fig.
2) and area under the plasma concentration-time curves (AUCs) (Table
1) of higher MW PEGs were substantially greater than those of the
lower MW polymers. Additionally, the half life of the polymers
progressively increased as the MW increased from 6 kD to 170 kD (Table
1); the relationship between the half and the MW of PEGs is sigmoidal
(Figure 3), which appears to be one of the characteristics of the
kinetics of macromolecules.
2. Blood radioactivity-time courses after iv administration of PEG
with different molecular weights. Key: (s)
PEG-170; (∆) PEG-50; (l)
PEG-6. From Ref. (2).
1. Mean ± SD of AUC and terminal half life of PEGs with
different MWs after iv administration to micea
With regard to the site of
injection, PEG-50 is retained at the injection site longer than PEG-6
after im and sc injections (3), suggesting that the absorption of PEG
from im and sc sites is MW dependent. However, after the ip
administration, the injection site disappearance profiles of both MWs
were very similar (3).
3. Relationship between the plasma half life of PEG and its molecular
weight. From data presented in Table 1, Ref. (2).
The differences among the plasma
concentration-time courses of PEGs with different MWs (Fig.
mostly due to the size of these PEGs in relation to the pore sizes of
the vascular beds in kidneys. Chang et al. (6) reported that, in rats,
renal elimination of another linear polymer, neutral dextrans, with a MW
of ~ 10 kD occurred without any molecular restriction. However, the
renal clearance of dextrans of larger MWs progressively decreased and
approached zero at a MW of ~40 kD. This is in agreement with a study (2)
in mice using radiolabeled PEG, demonstrating a sigmoidal relationship
between the renal clearance and the log MW of PEGs. This type of
sigmoidal relationship (2) agrees well with the theoretical models of
renal excretion of macromolecules based on the pore sizes of the
glomerular capillary wall.
The relatively limited
information on the metabolism of PEG in the body (7, 8) indicates that
PEG undergoes cytochrome P-450 oxidation, resulting in the formation of
ketone, ester, and aldehyde groups (8). Additionally, smaller MW PEGs
are excreted into bile (7).
In terms of tissue distribution,
it appears that PEGs with MWs between 6 kD to 170 kD distribute
insignificantly to tissues such as heart, lung, liver, spleen, kidney,
and thyroid gland (2). However, the distribution of PEGs to
gastrointestinal tract and feces is relatively substantial (2).
Additionally, no clear MW dependency is observed for the accumulation of
PEG in tissues (2).
During the last three decades,
PEG has been investigated extensively for delivery of various proteins
via parenteral routes. Some examples are listed below.
Generally, polymers have been
most widely used for the delivery of both traditional (small molecule)
drugs and proteins/enzymes in the treatment of cancer. However, PEGs
have been specifically investigated for the delivery of anticancer
proteins/enzymes as discussed below.
One of the major problems for the use of xenogenic monoclonal and
polyclonal antibodies for the treatment of tumors is their
immunogenicity which results in a rapid removal of the antibodies from
the body and the possibility of allergic reactions after multiple
administration. Kitamura et al. (9) conjugated the F(ab')2 fraction of
the murine monoclonal antibody A7 to PEG 5 kD and studied the tumor
accumulation and the kinetics of the conjugate in mice. The conjugate
had a longer plasma half life and higher tumor accumulation, compared
with the free F(ab')2 fraction. However, the tumor: blood ratio of the
free F(ab')2 fraction was higher than that for the conjugate (9).
Takashina et al. (10) studied the
pharmacokinetics and dynamics of conjugates of monoclonal antibody A7 to
PEG 5 kD and dextran 70 kD. In vitro studies showed that the conjugates
retained the antigen binding activity of the antibody. Additionally,
after the iv administration of the conjugated and free antibody, the PEG
conjugate had a plasma half life twice of that for the free antibody
(10). On the other hand, the dextran conjugate showed higher clearance
and shorter half life, compared with the free and PEG conjugated
antibody. Additionally, the tumor accumulation of dextran-antibody
conjugate was less than those for the free and PEG conjugated antibody.
This study (10) suggests that the kinetics of polymer-monoclonal
antibody A7 are significantly dependent on the structure of the polymer.
A PEG 5 kD conjugate of arginase retained 65% of the activity of the
enzyme and prolonged its plasma half life in mice after multiple dose
therapy (11); 30 days after the start of the treatment, the half life of
the native enzyme was 1 hr, while the half life of the conjugate was 12
hr. In terms of effects, the conjugate increased the survival time in
mice with Taper liver tumor. However, the free enzyme did not show any
improvement in the survival time (12). With regard to the effects of the
enzyme against L5178Y mouse leukemia cells, whereas the conjugate was
more effective than the native enzyme in vitro, neither was able to stop
the growth of tumor in vivo (12).
Asparaginase, isolated from Escherichia Coli and Erwinia Carotovora,
metabolizes asparagine, a necessary nutrient for sensitive tumors.
However, after multiple injection of the enzyme, antibodies raised
against the enzyme would quickly remove the enzyme from the circulation,
and also significant immunogenicity may be observed. Several studies
(13-19) have documented the usefulness of a conjugate of asparaginase
with PEG for the treatment of various cancers in both humans and
animals. Ho and his colleagues (15, 17) showed that the conjugate would
alter the pharmacokinetics of the enzyme drastically in both humans and
rabbits. In humans (15), conjugation resulted in an increase in the
plasma half life from 20 hr (for native enzyme) to 357 hr (for the
conjugate). In rabbits (17), the half life values of the free and
conjugated asparaginase were 20 and 144 hr, respectively. The increase
in the plasma half lives in both species was due to a significant
decrease in the clearance of the enzyme (15, 17). The alterations in the
kinetics of the enzyme by PEG conjugation also resulted in significant
improvements in the toxicity and efficacy profile of the enzyme after in
vivo administrations to animals (14, 16, 18) and humans (13, 19). A
conjugate of asparaginase and PEG (pegaspargase) was marketed (OncasparÒ)
in 1994 for the treatment of acute lymphoblastic leukemia (ALL) in
patients who are hypersensitive to native forms of L-asparaginase.
is marketed by Rhône-Poulenc Rorer Pharmaceuticals, Inc. in the U.S.
It is known that all the tumor cells have elevated requirement for
methionine. Therefore, methioninase may be used in cancer therapy.
However, the recombinant enzyme, obtained from bacteria, has a short
plasma half life and may be immunogenic upon multiple dose
administration. Very recently, Tan et al. (20) demonstrated the
potential of a conjugate of methioninase and PEG 5 kD in cancer therapy.
In vitro tests demonstrated that the conjugate retained 70% activity of
the enzyme. Additionally, in rats, the plasma half life of the enzyme
was increased by a factor of 2 when it was conjugated to PEG 5 kD (20).
Further, the effects of the conjugate lasted for 8 hr, as opposed to 2
hr for the free enzyme. In vitro studies in human lung and kidney cancer
cells showed identical IC50 values for the conjugated and free
methioninase, demonstrating the effectiveness of the enzyme in the
conjugated from. Also, after the injection of the conjugate to
tumor-bearing mice, the tumor: blood enzyme ratio was higher for the
conjugate (1:6), compared with the free enzyme (1:10) administration
(20). More studies are needed to confirm these promising findings.
A deficiency of the enzyme adenosine deaminase (ADA) results in combined
immunodeficiency disease (CID). For several years, conjugates of PEG and
ADA have been used successfully for enzyme replacement in the treatment
of CID in children (21-23). A conjugate of PEG and ADA, which is also
named pegademase, was marketed (AdagenÒ)
by Enzon, Inc. (Piscataway, NJ) in the US in 1990. The outcome of
therapy with the conjugate appears to be better than red blood cell
transfusion (23), which is another treatment for ADA deficiency. Studies
(21-23) have shown that weekly intramuscular injections of the conjugate
of PEG with bovine ADA would reverse the symptoms of ADA deficiency in
most cases without substantial toxicity or hypersensitivity. The
conjugate appears to have a very long half life of 48-72 hr in children
(21). From a historic perspective, the PEG-ADA conjugate served as one
of the earliest examples of polymer conjugates marketed in the US and
prompted more research interest in this area.
is an enzyme which converts uric acid to allantoin and is lacking in
humans. When the enzyme is administered to humans, it causes a
significant reduction in the plasma and urine levels of uric acid.
Therefore, it can be effective in the treatment of gout and other
diseases related to high levels of urate. However, after multiple
administration, the antibodies against this enzyme would deactivate it
very rapidly. Several conjugates of uricase with PEGs (24-28) have been
investigated to overcome this problem. Yasuda et al. (28) reported that
conjugation of uricase with PEG
resulted in a decrease in antibody production and reactivity towards
uricase. When administered intravenously to rats, the enzymatic activity
half life of the PEG conjugate (~ 7 hr) was almost 10 times of that for
the parent enzyme (0.6 hr) (28).
4. Relationship between the percentage of amino groups of uricase
modified with dextran 10 kD (m)
or PEG 10 kD (l)
and the percentage of remaining enzymatic activity
of uricase. From Ref. (28).
The enzymatic activity of the
polymer conjugated uricase is shown to be dependent on the degree of
modification of the amino groups of the enzyme during the conjugation
process (28). An increase in the modification would result in a decrease
in the enzymatic activity of uricase for both dextran and PEG conjugates
(Fig. 4). However, the decrease in the activity is more pronounced for
the PEG conjugate, compared with dextran conjugation (Figure 4) (28).
to superoxide dismutase (SOD), catalse is an antioxidant enzyme, and
several studies (29-32) have investigated the effects of PEG conjugates
of SOD and catalse on the same animal model. The PEG-catalse conjugate
was first prepared by Abuchowski et al. (33) using both PEG 1900 and 5
kD. These investigators (33) demonstrated that both conjugates retained
significant (>90%) activity of the enzyme and were resistant to
digestion by trypsin, chymotrypsin, and protease. Further, the half life
of the conjugates was long even after their repeated administration to
mice (33). A later study (34) using subcutaneous osmotic pumps
delivering the conjugate showed the conjugate’s beneficial effects in
a rat model of lung injury due to asbestosis. Despite promising effects
of the PEG-catalase conjugate, recent work in this area has concentrated
more on a conjugate of SOD and PEG described below.
Among the conjugates of PEG, SOD is the
most widely studied. Superoxide dismutase is an antioxidant enzyme which
eliminates superoxide anion, reducing tissue injury. After its iv
administration in animals, the plasma half life of the enzyme is very
short (5-10 min). Several investigators have reported the effects of
conjugation of SOD with PEG on the pharmacokinetics and dynamics of the
enzyme, some of which are summarized in Table 2 (29-32, 35-47). Although
some of these studies have compared the effects of PEG-SOD with those of
the free enzyme, most of the studies have concentrated on the effects of
PEG-SOD without a comparison with the free SOD (Table 2). There is
little doubt that conjugation of SOD with PEG increases its plasma half
life (35) and reduces its immunogenicity (29, 35). However, conflicting
reports (30-32, 36-40, 43, 45-47) exist with regard to the effects of
PEG-SOD in various animal models of injury. Additionally, the results of
clinical trials (42, 44) with PEG-SOD have not been unequivocal.
2. Some of studies on the conjugates of PEG and SOD.
In vitro and
in vivo kinetics and dynamics in rats
PEG 5 kD
conjugate retained 51% enzyme activity; plasma half life of
conjugate was longer than the native SOD after repeated dosing;
anti-inflammatory effect of the conjugate was higher than SOD.
immunogenicity in mice
immunogenicity; antibody titer to the conjugate was 0.03%-0.07%
of that observed with SOD.
effects in endotoxemia in pigs
effects in a dog model of ischemia/ reperfusion
results: both no effect (37) or a reduction (36) in heart injury
associated with reperfusion have been reported.
effects in a rat model of brain ischemia
of PEG-SOD before induction of focal cerebral ischemia resulted
in a reduction in brain injury.
effects in a rabbit model of ischemia/ reperfusion
No effect in
heart injury associated with reperfusion.
distribution into brain of piglets
of PEG-SOD did not increase the enzyme level in the brain in
control piglets and in animals subjected to global
effects in hemorrhagic shock in rats
of a PEG-SOD conjugate to a rat model increased survival from
25% to 67%.
In vivo brain
distribution in rats
concentrations of PEG-SOD in the brain and CSF of normal rats
were low; brain and CSF concentrations were higher after
hypertensive brain injury
effects in piglets with hypoxic brain injury
of PEG-SOD 5 min after reoxygenation did not have any positive
clinical trial study in severe head injury
outcome at 3 and 6 months after the treatment with PEG-SOD
(10,000 U/kg), compared with placebo.
In vivo study
in rats with oxygen toxicity
of PEG-SOD increased survival time, in comparison with both
placebo and free SOD.
in severe head injury
patients in a vegetative state or dead at 3 and 6 months
postinjury was lower after the conjugate, compared with placebo.
In vivo effect
in a rat model of ischemic renal failure
was more effective than an equivalent dose of free SOD.
effects in a rat model of warm renal ischemia
conjugates were more protective, compared with free SOD.
effects in a rat model of ischemia/reperfusion
to PEG showed a superior effect over that conjugated to
Rajagopalan et al. (48) conjugated streptokinase to PEG 2 kD, 4 kD, and 5
kD, and investigated the thrombolytic activity and antigenicity of the
conjugates. In vitro studies demonstrated comparable activity for the
conjugates and the free enzyme. However, the binding of the conjugates to
antibodies against streptokinase was reduced by 95% (48). In vivo studies
in mice (48, 49) revealed low clearance of the conjugates attached to
plasmin, resulting in a half life of 200 min for the conjugate, compared
to a half life of 15 min for streptokinase itself. These studies (48, 49)
demonstrate that PEG conjugation of streptokinase retains the activity of
the enzyme, prolongs its plasma circulation by blocking plasmin
degradation, and reduces the antigenicity of the enzyme.
dogs, a conjugate of urokinase, a thrombolytic agent, with PEG 5 kD was
shown (50) to have longer activity and more activation of fibrinolysis,
compared with the native enzyme. Also, a polypropylene glycol-PEG
conjugate of urokinase showed a decreased activity on plasminogen and had
a longer plasma half life in rabbits, compared with the native enzyme
(51). Later (52), it was shown that this conjugate blocked autolysis of
the enzyme at 370C. Unfortunately, these early positive results
have not been followed by more extensive in vivo studies.
Several studies have examined the
feasibility of the conjugation of hemoglobin to PEG for use as a blood
substitute. Hemoglobin binds to oxygen and can be used as an oxygen
carrier. However, because of its rapid elimination, the plasma half life
of the protein is very short. Additionally, the affinity of hemoglobin to
oxygen is too high for release of oxygen in the tissues. A conjugate of
PEG with pyridoxylated hemoglobin has been shown (53, 54) to have longer
plasma half life and better therapeutic effects in rats, compared with the
free hemoglobin. The benefits of PEG-hemoglobin conjugates as a blood
substitute have been shown in several animal models, including a
hemorrhagic hypotension pig model (55) and in partial exchange transfusion
and top-loaded rat models (56). Additionally, a PEG-hemoglobin conjugate
has been used (57) for an increase in the sensitivity of tumors to
radiation by increasing oxygen delivery to the tumor. These studies point
to the potential of hemoglobin conjugated to PEG for manipulation of the
oxygen levels in normal and malignant tissues.
and Hematopoietic Growth Factors
Both animal and clinical studies have been
conducted using PEG conjugates of IL-2. Earlier studies in animals (58,
59) and humans (60) showed that PEG conjugation would increase stability,
decrease clearance, and increase plasma half life (> 20 fold) of IL-2.
Further, these studies (58-61) suggested promising effects for the
PEG-IL-2 conjugate in the treatment of various cancers. However, more
recent data (62-64), mostly in patients, have failed to clearly
demonstrate an advantage for PEG-IL-2, compared with free IL-2, in terms
of therapeutic or toxic end points for the treatment of cancer. On the
other hand, it appears that recent interest in the PEG-IL-2 conjugate
revolves around its potential beneficial effects in patients with human
immunodeficiency virus (65-69). Recent studies (65-69) in patients with
HIV show that low dose PEG-IL-2, alone or in combination with zidovudine,
would increase the immune response by increasing the number of CD4 T cells
without significant toxicity. Additional clinical studies, comparing free
and PEG conjugated IL-2 will shed more light on these exciting results.
Recombinant human granulocyte
colony-stimulating factor (rhG-CSF):
This is a 156 amino acid glycoprotein which is produced by Escherichia
Coli and increases production and phagocytic and cytotoxic activities of
neutrophils (70). The plasma half life of rhG-CSF is short (3.5 hr) (70),
requiring daily injections to sustain the neutrophil levels in situations
like cancer chemotherapy. In 1991, Tanaka et al. (71) reported that a
conjugate of rhG-CSF with PEG
increased the plasma half life of the growth factor from 1.8 hr (native
factor) to 7 hr (conjugated factor) in mice. The increase in half life was
associated with an increase in both the intensity and duration of the
effect of the drug on the neutrophil count (71). These results were later
(72) confirmed in mice made neutropenic by the administration of
anticancer agents cyclophosphamide and fluorouracil. Recent studies
(73-75) demonstrated that the in vivo activity of the conjugate is
dependent on both the MW of PEG (73, 74) and the total number of PEG units
attached to rhG-CSF (73, 75); there
was a positive relationship between the total mass of the conjugate and
the intensity and duration of the effect of rhG-CSF. Future studies should
be conducted to determine whether these positive results in animals can be
extended to humans.
granulocyte/macrophage colony-stimulating factor (rhGM-CSF):
This is a 127 amino acid glycoprotein produced in yeast which acts similar
to rhG-CSF to increase neutrophils, with a broader action on monocytes,
macrophages, and eosinophils (70). Similar to rhG-CSF, the plasma half
life of rhGM-CSF is short (2-3 hr) (70), requiring daily injections to
sustain the neutrophil levels in patients undergoing bone marrow
transplantation or intensive chemotherapy. Compared with rhG-CSF, the
studies on the conjugates of PEG with rhGM-CSF are scarce (76, 77). The
limited information indicates that similar to rhG-CSF, PEG conjugation
increases the plasma half life (76) and some biological activities of
Table 3 lists the use of PEGs for
delivery of some other therapeutic agents
(78-86) which are not discussed in detail in this review. These
studies (Table 3) show that polymer conjugation could result in altered
pharmacokinetics, decreased affinity of the conjugate to bind to the
protein receptor, and/or a decrease in antigenicity of proteins.
The examples provided in this
review clearly point to the potential advantages of polyethylene glycols
for parenteral delivery of proteins. Despite significant promise of
protein therapeutics in cell culture and other in vitro studies, optimal
delivery of these agents in humans is very challenging. This is mostly
because of relatively high clearance and short plasma half life of these
agents, especially after multiple administration which results in
activation of the immune system and faster elimination of the proteins.
The available studies on the use of PEG for delivery of proteins indicate
that these polymers will continue to have a significant role in the
delivery of proteins in the future.
3. Additional studies on the conjugates of PEGs with proteins
study in man showed that a 5 kD conjugate may be useful for the
immunotherapy of ragweed hay fever (78).
A 10 kD
conjugate retained the activity of the enzyme while losing its
ability to bind to anti-batroxobin antibodies in dogs (79).
In a rat model
of jaundice, the conjugate reduced the blood and liver levels of
bilirubin, but, did not improve the liver function tests (80).
In a clinical
study, a 5.7 kD conjugate showed lower systemic reactions during
immunotherapy and less efficacy against honeybee sting (81).
In humans, the
half life of the conjugate was twice as long as that of free
protein; however, this did not result in a substantial reduction
in the frequency of the protein administration (82).
A 5 kD
conjugate had activity similar to that of free protein but with a
reduced binding affinity; the plasma half life of the conjugate
was significantly longer than that of free protein in rats (83).
A 12 kD
conjugate showed significantly higher thrombopoietic effects
(increase in the platelet counts), compared with free IL-6 in mice
In mice, the
half life of radioactivity after the injection of the radiolabeled
conjugate with 5 kD and 20 kD was long; however, the effect
disappeared much faster (85).
A 5 kD
conjugate was resistant to anti-trypsin antibody precipitation and
retained some of the activities of trypsin to varying degrees
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Sciences Center, 1300 Coulter, Amarillo, TX 79106
Published by the Canadian Society for
Copyright © 1998 by the Canadian Society for Pharmaceutical Sciences.