The enzyme as drug: application of enzymes as pharmaceuticals


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The enzyme as drug:application of enzymes as pharmaceuticals
Michel Vellard
Enzymes as drugs have two important features that distinguish
themfromall other types of drugs.First,enzymes often bind and
act on their targets with great affinity and specificity.Second,
enzymes are catalytic and convert multiple target molecules to
the desiredproducts.These twofeatures make enzymes specific
and potent drugs that can accomplish therapeutic biochemistry
in the body that small molecules cannot.These characteristics
have resulted in the development of many enzyme drugs for a
wide range of disorders.
Department of Cellular Genetics,BioMarin Pharmaceutical Inc.,
46 Galli Drive,Novato,CA 94949,USA
Current Opinion in Biotechnology 2003,14:444–450
This review comes from a themed issue on
Protein technologies and commercial enzymes
Edited by Gjalt Huisman and Stephen Sligar
0958-1669/$ – see front matter
￿ 2003 Elsevier Science Ltd.All rights reserved.
DOI 10.1016/S0958-1669(03)00092-2
ADA adenosine deaminase
CF cystic fibrosis
FDA Food and Drug Administration
HIV human immunodeficiency virus
LSD lysosomal storage disease
MPS mucopolysaccharide
PAL phenylalanine ammonia lyase
PEG polyethylene glycol
SCID severe combined immunodeficiency disease
The application of enzyme technologies to pharmaceu-
tical research,development and manufacturing is a grow-
ing field and is the subject of many articles,reviews and
books.We will limit the scope of this review to recent
articles on the use of enzymes as drugs.
The concept of the therapeutic enzyme has been around
for at least 40 years.For example,a therapeutic enzyme
was described as part of replacement therapies for genetic
deficiencies in the 1960s by de Duve [1].
In 1987,the first recombinant enzyme drug,Activase
(alteplase;recombinant human tissue plasminogen acti-
vator),was approved by the Food and Drug Administra-
tion (FDA).This ‘clot-buster’ enzyme is used for the
treatment of heart attacks caused by the blockage of a
coronary artery by a clot.This was the second recombi-
nant protein drug to be marketed (the first genetically
engineered drug was insulin in 1982).Several other
enzymes used as anticoagulant or coagulant agents have
since been approved by the FDA.
In 1990,Adagen
,a form of bovine adenosine deami-
nase (ADA) treated with polyethylene glycol (PEG) was
approved to treat patients afflicted with a type of severe
combined immunodeficiency disease (SCID),which is
caused by the chronic deficiency of ADA.Of particular
note is that Adagen
(pegadamase bovine) was the first
therapeutic enzyme approved by the FDA under the
Orphan Drug Act.The Orphan Drug Act was passed in
1983 in the United States to encourage pharmaceutical
companies to develop treatments for diseases affecting
only small numbers of people (less than 200 000).Among
the many provisions and incentives,drugs given Orphan
drug status receive seven years of market exclusivity.In
Europe and Australia,there is comparable legislation
that provides similar protection and incentives (Tables 1
and 2).
The approval of Activase
(alteplase) and Adagen
(pegadamase bovine) marked the beginning of a new
era for enzymes,taking them from the status of ‘holistic’
supplement to that of approved therapeutic drug.
Because their biological action hinges on catalysis,a
property that enhances potency,it is not surprising to
see that therapeutic enzymes now cover a wide range of
diseases and conditions (Figure 1).
Enzyme therapy
(pegadamase bovine),used for the treatment
of SCID,represents the first successful application of an
enzyme therapy for aninheriteddisease [2].The enzyme
ADA cleaves the excess adenosine present in the circu-
lation of these patients and reduces the toxicity to the
immune system of the elevated adenosine levels.The
success of the treatment [3] depends upon the modifica-
tion of ADA with PEG.PEG enhances the half-life of
the enzyme (originally less than 30 min) and reduces
the possibility of immunological reactions due to the
bovine origin of the drug (for reviews on PEGylation see
[4] and [5
As a side note,studies by Bax et al.[6] have shown that the
efficient entrapment of native ADAin carrier erythrocytes
also improves substantially the half-life of the enzyme.
(alglucerase injection) for the treatment of
Gaucher disease,a lysosomal storage disease (LSD),was
Current Opinion in Biotechnology 2003,14:444–450
the first enzyme replacement therapy in which an exo-
genous enzyme was targeted to its correct compartment
within the body.The effort to replace the missing glu-
cocerebrosidase in Gaucher patients was initiated by
Brady and colleagues [7],utilizing modified placental
glucocerebrosidase (Ceredase
).Recombinant DNA
technology subsequently allowed the more efficient pro-
duction of a glucocerobrosidase,Ceredase
ase),which was approved in 1994 (Table 1).This medical,
as well as financial,success has paved the way for other
enzyme therapies,in particularly those for other LSDs.
Another LSD that has attracted the interest of pharma-
ceutical companies is Fabry’s disease,a fat (glycolipid)
storage disorder caused by a deficiency in a-galactosidase.
The disease primarily affects the vasculature and results
in renal failure,pain,and corneal clouding.Two compa-
nies are currently seeking FDA approval (and exclusivity
under the orphan drug status) after having completed
Phase III clinical trials (reviewed in [8] see also Update).
One company has a recombinant a-galactosidase
expressed in Chinese hamster ovary cells [9] and the
other has the same enzyme expressed in human cells
Table 1
Approved enzymes designated as orphan drugs in the USA.
Generic name Year
Indication Sponsor
1984 1990 For enzyme replacement therapy
for ADA in patients with SCID
1985 1991 For replacement therapy in patients
with Gaucher’s disease type I
Genzyme Corporation
Dornase a 1991 1993 To reduce mucous viscosity and enable the
clearance of airway secretions in patients with CF
Imiglucerase 1991 1994 Replacement therapy in patients with types I,
II,and III Gaucher’s disease
Genzyme Corporation
Pegaspargase 1989 1994 Treatment of acute lymphocytic leukemia Enzon,Inc.
Sucraid Sacrosidase 1993 1998 Treatment of congenital
sucrase-isomaltase deficiency
Orphan Medical,Inc.
Rasburicase 2000 2002 Treatment of malignancy-associated or
chemotherapy-induced hyperuricemia
Sanofi-Synthelabo Research
Agalsidase beta 1988 2003 Treatment of Fabry’s disease Genzyme Corporation
Laronidase 1997 2003 Treatment of patients with MPS I BioMarin Pharmaceutical,Inc.
a-Galactosidase A 1998 2003?Long-term enzyme replacement therapy
for the treatment of Fabry’s disease
Transkaryotic Therapies,Inc.
Figure 1
Genetic diseases
Infectious diseases
Therapeutic enzymes
Prodrug activator
Current Opinion in Biotechnology
SCIDGaucher MPS IFabry Pompe PKU
Therapeutic enzymes are used in the treatment of a variety of disorders and diseases.
The application of enzymes as pharmaceuticals Vellard 445 Current Opinion in Biotechnology 2003,14:444–450
446 Protein technologies and commercial enzymes
Current Opinion in Biotechnology 2003,14:444–450
[10].A third a-galactosidase product,expressed in plants,
was designated as an orphan drug by the FDA in early
2003 (Table 2).
Enzyme replacement therapies for at least three muco-
polysaccharide (MPS) storage disorders (a subgroup of
LSD) are currently under investigation [11
].APhase III
clinical trial of Aldurazyme
(laronidase),an enzyme
replacement therapy for MPS I,was recently completed
[12] and is awaiting approval in the US and Europe (see
Update).This LSD is characterized by a deficit in a-
iduronidase.Likewise,a Phase II clinical trial of Ary-
(recombinant human N-acetylgalactosamine-4-
sulfatase),an enzyme replacement therapy for Maroteaux-
Lamy syndrome (MPS VI),was recently successfully com-
pleted.The treatment was assessed for safety,efficacy
and potential end-points for later trials.Finally,a Phase
I/II clinical trial for an enzyme replacement therapy for
Hunter’s disease (MPS II),an LSD resulting from a
deficiency in iduronate 2-sulfatase,was completed in
2002.The data show a dose-dependent reduction in urin-
ary glycosaminoglycan (GAG).
Pompe’s disease,or glycogen storage disorder type II
(GSDII),results from a deficiency in a-glucosidase and
is the subject of several clinical trials.Pompe’s disease
primarily affects muscle.To date,the published preli-
minary results using recombinant enzymes [13,14] appear
promising.High doses are necessary for successful treat-
ment in this devastating disorder and immune responses
may interfere with therapy in some patients.Pompe’s
disease may be the first muscle disorder to be treated by
enzyme replacement therapy [15
Oral and inhalable enzyme therapies
In contrast to the treatments reviewed thus far,there are
some diseases that do not require intravenous injection
of an enzyme of human origin.Updating the age-old
application of enzymes as digestive aids,several diseases
have yielded to oral enzyme formulations.Congenital
sucrase-isomaltase deficiency (CSID),for example,is
treatable with sacrosidase (Table 1) —a b-fructofurano-
side fructohydrolase from Saccharomyces cerevisiae that
can be taken orally.CSID patients are unable to use
the disaccharide sucrose.The drug hydrolyses sucrose,
allowing the consumption of a more normal diet and is
particularly useful for young patients where strict com-
pliance with a sucrose-free,low-starch diet is proble-
matic [16].Phenylketonuria (PKU) is another genetic
disorder requiring strict compliance with a specialized
diet.PKU is caused by low or non-existent phenylal-
anine hydroxylase activity,which catalyzes the con-
version of phenylalanine to tyrosine.An oral treatment,
,is being developed based on the use of
recombinant yeast phenylalanine ammonia lyase (PAL).
PAL has been shown to degrade phenylalanine in the
gastrointestinal tract [17].
A mixture of pancreatic enzymes,including lipases,pro-
teases and amylases,has been shown to be useful in the
treatment of fat malabsorption in patients with human
immunodeficiency virus (HIV) [18].These enzymes are
also used to treat pancreatic insufficiency,a condition
affecting most cystic fibrosis (CF) patients [19].Interest-
ingly,the lipases developed for this use have a transgenic
corn origin.Another mixture of pancreatic enzymes,with
the trade name TheraCLEC Total
and protease mix) uses enzyme crystallization and cross-
linking methods for its formulation and was designated as
an orphan drug in 2002 (Table 2).This novel formulation
allows for lower doses and higher efficacy.
It is possible to imagine enzyme treatments for several
other diseases that primarily affect digestion.For exam-
ple,oral peptidase supplement therapy could be used for
the treatment of Celiac Sprue,also know as Celiac dis-
ease,a widely prevalent disorder of the small intestine
caused by an immune reaction to the gliadin protein in
ingested wheat products [20
Inhalable enzyme formulations have found application in
the treatment of CF.Pulmozyme
(Dornase a),a DNase,
received one of the fastest approvals by the FDA under
the orphan drug status.Dornase a liquefies accumulated
mucus in the lung [21].The use of Dornase a in CF
patients can also diminish pulmonary tissue destruction
by lowering the level of matrix metalloproteinases in the
bronchoalveolar lavage fluid [22].
Proteolytic and glycolytic enzymes for
treating damaged tissue
In the past,a large number of proteolytic enzymes of
plant and bacterial origin have been studied as a replace-
ment for the mechanical debridement (removal of dead
skin) of burns.Unfortunately,the results have been
variable due,perhaps,to the poor quality of the enzymes
used [23,24].
Several products of higher quality and purity,owing in
some to their recombinant origin,are nowin clinical trials.
Debrase gel dressing,comprising a mixture of enzymes
extracted from pineapple,received clearance in 2002
from the US FDA for a Phase II clinical trial for the
treatment of partial-thickness and full-thickness burns.
This product also received orphan drug status in Europe.
(recombinant vibriolysin),a proteolytic
enzyme from the marine microorganism Vibrio proteolyti-
cus,has been shown to have efficacy against denatured
proteins such as those found in burned skin.A Phase I
clinical trial was recently initiated to evaluate the safety
and tolerability of this topically applied enzyme for
debridement of burns (
encouraging results have been obtained recently from a
study with the collagenase clostridiopeptidase in children
with partial-thickness burns [25].
The application of enzymes as pharmaceuticals Vellard 447 Current Opinion in Biotechnology 2003,14:444–450
Chondroitinases couldbe usedfor the treatment of spinal
injuries where they have been demonstrated to promote
regeneration of injured spinal cord.The enzyme acts by
removing,in the glial scar,the accumulated chondroitin
sulfate that inhibits axon growth [26
has a similar hydrolytic activity on chondroitin sulfate
and may also help in the regeneration of damaged nerve
tissue [27].
Enzymes for the treatment of infectious
Lysozyme has been used as a naturally occurring anti-
bacterial agent in many foods and consumer products,
because of its ability to break carbohydrate chains in the
cell wall of bacteria.Lysozyme has also been shown to
possess activity against HIV,as has RNase A and urinary
RNase U,which selectively degrade viral RNA [28]
opening some exciting possibilities for the treatment
of HIV infection.Other naturally occurring antimicro-
bial agents are the chitinases.As an element of the cell
wall of various pathogenic organisms,including fungi,
protozoa and helminths,chitin is a good target for anti-
microbials [29].The cell walls of Streptococcus pneumonia,
Bacillus anthracis and Clostridium perfringens have been
targeted with the use of bacteriophage-derived lytic
enzymes [30,31
,32].The use of lytic bacteriophages
themselves as a treatment for infections is also being
developed and could prove useful against new drug-
resistant bacterial strains.
Enzymes for the treatment of cancer
The field of cancer research has some good examples of
the use of enzyme therapeutics.Recent studies have
shown that PEGylated arginine deaminase,an argi-
nine-degrading enzyme,can inhibit human melanoma
and hepatocellular carcinomas,which are auxotrophic
for arginine owing to a lack of arginosuccinate synthetase
activity [33].
Recently,another PEGylated enzyme,Oncaspar
(pegaspargase),already in use in the clinic,has shown
better results for the treatment of children with newly
diagnosed standard-risk acute lymphoblastic leukemia
than the native,bacterial asparaginase [34].Whereas
normal cells are able to synthesize asparagine,cancer
cells are not and die in the presence of this asparagine-
degrading enzyme.In spite of the higher pharmacy cost of
PEG-asparaginase,the overall cost of the treatment is
very similar to the one with the native enzyme [35].
Asparaginase and PEG-asparaginase are effective
adjuncts to standard chemotherapy.
Another characteristic of the process of oncogenesis is
proliferation.It has been shown that the removal of
chondroitin sulfate proteoglycans by chondroitinase AC
and,to a lesser extent,by chondroitinase B,inhibits tumor
growth,neovascularization and metastasis [36
Antibody-directed enzyme prodrug therapy (ADEPT)
illustrates a further application of enzymes as therapeutic
agents in cancer.A monoclonal antibody carries an
enzyme specifically to cancer cells where the enzyme
activates a prodrug,destroying cancer cells but not normal
cells [39,40].This approach is being utilized to discover
and develop a class of cancer therapeutics based on
tumor-targeted enzymes that activate prodrugs.The tar-
geted enzyme prodrug therapy (TEPT) platform,involv-
ing enzymes with antibody-like targeting domains,will
also be used in this effort [41].
One of the side-effects of cancer chemotherapy is hyper-
uricemia,a build-up of uric acid that results in gouty
arthritis and chronic renal disease.Urate oxidase is able to
degrade the poorly soluble uric acid.Interestingly,the
gene for this enzyme is present in humans,but possesses a
nonsense codon.In recent years,five drugs using this
enzyme were granted orphan drugs status by the FDA
(Tables 1 and 2).Recombinant Rasburicase is safe and
effective as a uricolytic agent [42,43] and the PEGylated
form of the enzyme diminishes its immunogenicity and
increases its half-life [44].
Prospective therapeutic enzymes
Superoxide dismutase,the most important detoxifying
enzyme present in cells,transforms the highly toxic
superoxide anion to moderately toxic hydrogen peroxide.
This enzyme,which has been of interest to the pharma-
ceutical industry for some time,has never fulfilled its
promise —even in the PEGylated form[45
].The same
is true for catalase,another anti-oxidant that converts
hydrogen peroxide to water and oxygen.Surprisingly,
these enzymes have been shown to prolong the life of
(the nematode) Caenorhabditis elegans and this effect may
translate to mammals [46].Future versions of these
enzymes may help to reduce organ injury in hemorrhagic
shock [47].
Human butyrilcholinesterase,a naturally occurring serum
detoxification enzyme,acts to break down acetylcholine.
It could be useful for the treatment of cocaine overdose,
as demonstrated by recent results [48].Structure-based
re-engineering of the enzyme has resulted in higher
activity toward cocaine [49].Directed evolution has also
resulted in even more efficient optimization of butyril-
cholinesterase [50];directed evolution promises to be the
most powerful tool yet in the development of enzyme
drugs [51].
Advancements in biotechnology over the past ten years
have allowed pharmaceutical companies to produce safer,
cheaper enzymes with enhanced potency and specificity.
Along with these advances,changes in orphan drug laws
and new initiatives by the FDA have been effective in
facilitating efforts to develop enzyme drugs.This synergy
448 Protein technologies and commercial enzymes
Current Opinion in Biotechnology 2003,14:444–450
has had a beneficial effect on the development of treat-
ments for both rare and common diseases.
In April 2003 Fabrazyme
(agalsidase beta;Table 1) was
approved by the FDA for the treatment of Fabry’s dis-
ease.Six days later,Aldurazyme
(laronidase;Table 1)
was approved by the FDA for the treatment of MPS I.
Thanks to my colleagues at Biomarin Pharmaceutical,particularly Todd
Zankel and Emil Kakkis for critically reading the manuscript and helpful
References and recommended reading
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