enzymes as drugs.pdf

The enzyme as drug: application of enzymes as pharmaceuticalsMichel Vellard

Enzymes as drugs have two important features that distinguish

them from all 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 desired products. These two features 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.

AddressesDepartment of Cellular Genetics, BioMarin Pharmaceutical Inc.,

46 Galli Drive, Novato, CA 94949, USA

e-mail: [email protected]

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

AbbreviationsADA 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

IntroductionThe 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, Activase1

(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, Adagen1, 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 Adagen1 (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 Activase1 (alteplase) and Adagen1

(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 therapyAdagen1 (pegadamase bovine), used for the treatment

of SCID, represents the first successful application of an

enzyme therapy for an inherited disease [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 ADA in carrier erythrocytes

also improves substantially the half-life of the enzyme.

Ceredase1 (alglucerase injection) for the treatment of

Gaucher disease, a lysosomal storage disease (LSD), was

444

Current Opinion in Biotechnology 2003, 14:444–450 www.current-opinion.com

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 (Ceredase1). Recombinant DNA

technology subsequently allowed the more efficient pro-

duction of a glucocerobrosidase, Ceredase1 (imiglucer-

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.

Trade

name

Generic name Year

designed

Year

approved

Indication Sponsor

Adagen1 Pegademase

bovine

1984 1990 For enzyme replacement therapy

for ADA in patients with SCID

Enzon, Inc.

Ceredase1 Alglucerase

injection

1985 1991 For replacement therapy in patients

with Gaucher’s disease type I

Genzyme Corporation

Pulmozyme1 Dornase a 1991 1993 To reduce mucous viscosity and enable the

clearance of airway secretions in patients with CF

Genentech, Inc.

Cerezyme1 Imiglucerase 1991 1994 Replacement therapy in patients with types I,

II, and III Gaucher’s disease

Genzyme Corporation

Oncaspar1 Pegaspargase 1989 1994 Treatment of acute lymphocytic leukemia Enzon, Inc.

Sucraid Sacrosidase 1993 1998 Treatment of congenital

sucrase-isomaltase deficiency

Orphan Medical, Inc.

Elitek1 Rasburicase 2000 2002 Treatment of malignancy-associated or

chemotherapy-induced hyperuricemia

Sanofi-Synthelabo Research

Fabrazyme1 Agalsidase beta 1988 2003 Treatment of Fabry’s disease Genzyme Corporation

Aldurazyme1 Laronidase 1997 2003 Treatment of patients with MPS I BioMarin Pharmaceutical, Inc.

ReplagalTM a-Galactosidase A 1998 2003? Long-term enzyme replacement therapy

for the treatment of Fabry’s disease

Transkaryotic Therapies, Inc.

Figure 1

MPS VI

Genetic diseases

Burndebridement

Infectious diseases

Cancer

Miscellany

Clotting

Therapeutic enzymes

Antiprotozoa

Antifungi Antibacteria

Antineoplasticenzymes

Prodrug activatorenzymes

AnticoagulantsProcoagulants

Current Opinion in Biotechnology

SCIDGaucher MPS IFabry Pompe PKUCF

Therapeutic enzymes are used in the treatment of a variety of disorders and diseases.

The application of enzymes as pharmaceuticals Vellard 445

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:444–450

Table 2

Enzymes designated as orphan drugs under investigation in the USA.

Trade

name

Generic name Year

designated

Indication Sponsor

Superoxide dismutase (human) 1985 Protection of donor organ tissue from damage or injury mediated by

oxygen-derived free radicals that are generated during the necessary

periods of ischemia (hypoxia, anoxia), and especially reperfusion

Pharmacia-Chiron Partnership

Erwinia L-asparaginase 1985 Used as an alternative to E. coli asparaginase in those situations where repeat

courses of asparaginase therapy for acute lymphoblastic leukemia are required or

where allergic reactions force the discontinuance of the E. coli preparation

Lyphomed, Inc.

Erwinase1 Erwinia L-asparaginase 1986 Treatment of acute lymphocytic leukemia Porton International, Inc.

Superoxide dismutase (recombinant human) 1988 Prevention of reperfusion injury to donor organ tissue Bio-Technology General Corp.

Oxsodrol1 T4 endonuclease V, liposome-encapsulated 1989 Prevention of cutaneous neoplasms and other skin abnormalities in xeroderma pigmentosum AGI Dermatics

Fabrase a-Galactosidase A 1990 Treatment of Fabry’s disease Desnick, Robert J. M.D. The

Mount Sinai School Of Medicine

Oxsodrol1 Recombinant human superoxide dismutase 1991 Prevention of bronchopulmonary dysplasia in premature neonates weighing less than 1500 g Bio-Technology General Corp.

CC-galactosidase a-Galactosidase A 1991 Treatment of a-galactosidase A deficiency (Fabry’s disease) Orphan Medical, Inc.

Lysodase PEG-glucocerebrosidase 1992 Chronic enzyme replacement therapy in patients with Gaucher’s disease

who are deficient in glucocerebrosidase

National Institute of Mental Health,

Vianain1 Ananain, comosain 1992 Enzymatic debridement of severe burns Genzyme Corporation

Butyrylcholinesterase 1992 Reduction and clearance of toxic blood levels of cocaine encountered during a drug overdose Shire Laboratories Inc.

Butyrylcholinesterase 1992 Treatment of post-surgical apnea Shire Laboratories Inc.

PhenylaseTM Phenylalanine ammonia-lyase 1995 Treatment of hyperphenylalaninemia Ibex Technologies, Inc.

c/o BioMarin Pharmaceutical

Chondroitinase 1995 Treatment of patients undergoing vitrectomy Bausch & Lomb Pharmaceuticals

Alglucerase injection 1995 Replacement therapy in patients with Type II and III Gaucher’s disease Genzyme Corporation

Clostridial collagenase 1996 Treatment of advanced (involutional or residual stage) Dupuytren’s disease Hurst, LMD and M Badalamente

(PhD) University of New York

PlaquaseTM Collagenase (lyophilized) for injection 1996 Treatment of Peyronie’s disease Advance Biofactures Corporation

Pompase1 Human acid precursor a-glucosidase,

recombinant

1996 Treatment of glycogen storage disease type II Pharming/Genzyme LLC

c/o Genzyme Corporation

MyozymeTM Recombinant human acid a-glucosidase 1997 Treatment of glycogen storage disease type II Genzyme Corporation

Zurase lPEG-modified uricase 1998 Treatment of tumor lysis syndrome in cancer patients undergoing chemotherapy Phoenix Pharmacologics, Inc.

Wobe-Mugos1 Papain, trypsin and chymotrypsin 1998 Treatment of multiple myeloma Marlyn Nutraceuticals, Inc.

Zurase PEG-modified uricase 1999 Prophylaxis of hyperuricemia in cancer patients prone to develop tumor lysis

syndrome during chemotherapy

Phoenix Pharmacologics, Inc.

AryplaseTM N-Acetylgalactosamine-4-sulfatase,

recombinant human

1999 Treatment of MPS VI (Maroteaux-Lamy syndrome) BioMarin Pharmaceutical, Inc.

Melanocid PEGylated arginine deiminase 1999 Treatment of invasive malignant melanoma Phoenix Pharmacologics, Inc.

Hepacid PEGylated arginine deiminase 1999 Treatment of hepatocellular carcinoma Phoenix Pharmacologics, Inc.

TBD Recombinant human highly phosphorylated

acid a-glucosidase

2000 Enzyme replacement therapy in patients with all subtypes of glycogen

storage disease type II (GSDII, Pompe’s disease)

Novazyme Pharmaceuticals, Inc.

Fasturtec1 Recombinant urate oxidase 2000 Prophylaxis of chemotherapy-induced hyperuricemia Sanofi-Synthelabo Research

I2S Iduronate-2-sulfatase 2001 Long-term enzyme replacement therapy for patients with MPS II (Hunter’s Syndrome) Transkaryotic Therapies Inc.

Puricase1 PEG-uricase 2001 Control of the clinical consequences of hyperuricemia in patients with severe

gout in whom conventional therapy is contraindicated or has been ineffective

Bio-Technology General

Corporation

TheraCLEC-TotalTM Lipase, amylase and protease 2002 Treatment of pancreatic insufficiency Altus Biologics, Inc.

Recombinant human porphobilinogen deaminase 2002 Treatment of acute intermittent porphyria attacks HemeBiotech A/S

Plant-produced human a-galactosidase A 2003 Treatment of Fabry’s disease Large scale Biology

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[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�]. A Phase III

clinical trial of Aldurazyme1 (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-L-

iduronidase. Likewise, a Phase II clinical trial of Ary-

plaseTM (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 therapiesIn 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,

PhenylaseTM, 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 TotalTM (lipase, amylase

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. Pulmozyme1 (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 fortreating damaged tissueIn 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 now in 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.

VibrilaseTM (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 (http://www.bmrn.com). Some

encouraging results have been obtained recently from a

study with the collagenase clostridiopeptidase in children

with partial-thickness burns [25].



http://www.bmrn.com

Chondroitinases could be used for 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��]. Hyaluronidase

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 infectiousdiseasesLysozyme 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 cancerThe 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, Oncaspar1

(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�,37,38].

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 enzymesSuperoxide 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].

ConclusionsAdvancements 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


has had a beneficial effect on the development of treat-

ments for both rare and common diseases.

UpdateIn April 2003 Fabrazyme1 (agalsidase beta; Table 1) was

approved by the FDA for the treatment of Fabry’s dis-

ease. Six days later, Aldurazyme1 (laronidase; Table 1)

was approved by the FDA for the treatment of MPS I.

AcknowledgementsThanks to my colleagues at Biomarin Pharmaceutical, particularly ToddZankel and Emil Kakkis for critically reading the manuscript and helpfulsuggestions.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest��of outstanding interest

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2. Aiuti A: Advances in gene therapy for ADA-deficient SCID.Curr Opin Mol Ther 2002, 4:515-522.

3. Hershfield M: PEG-ADA replacement therapy for adenosinedeaminase deficiency: an update after 8.5 years. Clin ImmunolImmunopathol 1995, 76:228-232.

4. Greenwald RB: PEG drugs: an overview. J Control Release 2001,74:159-171.

5.�

Roberts MJ, Bentley MD, Harris JM: Chemistry for peptide andprotein PEGylation. Adv Drug Deliv Rev 2002, 54:459-476.

In combination with [3] the authors describe and compare the first andsecond generation of PEG–protein therapeutics and the chemistryinvolved. The second generation addresses mainly the issues of deac-tivated PEG impurities, unstable linkages and the absence of selectivity inmodification, raising the potential of this technique which has knownsome ups and downs in the past.

6. Bax BE, Bain MD, Fairbanks LD, Webster AD, Chalmers RA: In vitroand in vivo studies with human carrier erythrocytes loaded withpolyethylene glycol-conjugated and native adenosinedeaminase. Br J Haemtol 2000, 109:549-554.

7. Barton NW, Brady RO, Dambrosia JM, Bisceglie AM, Doppelt SH,Hill SC, Mamkin HJ, Murray GJ, Parker RI, Argoff CE et al.:Replacement therapy for inherited enzyme deficiency-macrophage-targeted glucocerebrosidase for Gaucher’disease. N Engl J Med 1991, 324:1464-1470.

8. Germain DP: Fabry disease: recent advances in enzymereplacement therapy. Expert Opin Investig Drugs 2002,11:1467-1476.

9. Eng CM, Guffon N, Wilcox WR, Germain DP, Lee P, Waldek S,Caplan L, Linthorst GE, Desnick RJ: International collaborativeFabry disease study group: safety and efficacy of recombinanthuman a-galactosidase: replacement therapy in fabry disease.N Engl J Med 2001, 345:9-16.

10. Schiffmann R, Kopp JB, Austin HA III, Sabnis S, Moore DF, WeibelT, Balow JE, Brady RO: Enzyme replacement therapy in fabrydisease: a randomized controlled trial. J Am Med Assoc 2001,285:2743-2749.

11.�

Kakkis E: Enzyme replacement therapy for themucopolysaccharidoses storage disorders. Expert Opin InvestigDrugs 2002, 11:675-685.

The author gives a detailed description of several preclinical and clinicaltrials initiated to treat mucopolysaccharide storage disorders. Hedescribes the challenges of these diseases.

12. Kakkis ED, Muenzer J, Tiller GE, Waber L, Belmont J, Passage M,Izykowski B, Phillips J, Doroshow R, Walot I et al.: Enzyme-replacement therapy in mucopolysaccharidosis I. N Engl J Med2001, 344:182-188.

13. Van den Hout JM, Reuser AJ, de Klerk JB, Arts WF, Smeiting JA,Van der Ploeg AT: Enzyme therapy for Pompe disease withrecombinant human a-glucosidase from rabbit milk. J InheritMetab Dis 2001, 24:266-274.

14. Amalfito A, Bengur AR, Morse RP, Majure JM, Case LE,Veerling DL, Mackey J, Kishnani P, Smith W, McVie-Wylie A et al.:Recombinant human acid a-glucosidase enzyme therapy forinfantile glycogen disease type II: results of a phase I/II clinicaltrial. Genet Med 2001, 3:132-138.

15.��

Raben N, Plotz P, Byrne B: Acid a-glucosidase deficiency(Glycogenosis type II, Pompe disease). Curr Mol Med 2002,2:145-166.

This complete and recent review on Pompe’s disease has a very infor-mative section on the different therapies in use or in clinical trials, inparticular gene therapy and enzyme replacement therapy. The authorspoint out the large doses needed during the enzyme replacement therapyclinical trials, in particular for the enzyme from transgenic rabbit, and theneed for a larger number of treated patients to judge the efficacy of thetreatment. Even if the results are promising, the discrepancies betweenthe different trials [7,8] at the level of the immune reactions of the patientsneeds to be explored in more detail.

16. Treem WR, McAdams L, Stanford L, Kastoff G, Justinich C,Hyams J: Sacrosidase therapy for congenital sucrase-isomaltase deficiency. J Pediatr Gastroenterol Nutr 1999,28:137-142.

17. Sarkissian CN, Shao Z, Blain F, Peevers R, Su H, Heft R, Chang TM,Scriver CR: A different approach to treatment ofphenylketonuria: phenylalanine degradation with recombinantphenylalanine ammonia lyase. Proc Natl Acad Sci USA 1999,96:2339-2344.

18. Carroccio A, Guarino A, Zuin G, Verghi R, Berni-Canani R,Fontana M, Bruzzese E, Montalto G, Notarbatolo A: Efficacy of oralpancreatic therapy for the treatment of fat malabsorption inHIV-infected patients. Aliment Pharmacol Ther 2001,15:1619-1625.

19. Schibli S, Durie PR, Tullis ED: Proper usage of pancreaticenzymes. Curr Opin Pulm Med 2002, 8:542-546.

20.��

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM,Khosla C: Structural basis for gluten intolerance in celiac Sprue.Science 2002, 297:2275-2278.

The authors demonstrate the link between this autoimmine diseaseand the existence of a 33 amino acid peptide derived from gliadins, themajor toxic component of wheat gluten. This peptide is resistant to thedigestion of the small intestinal brush-border membrane enzymes andcontains several patient-specific T-cell epitopes already identified.This proline-rich peptide is broken down by a bacterial propyl endo-nuclease, making peptidase therapy a potential treatment for thisdisease.

21. Robinson PJ: Dornase a in early cystic fibrosis lung disease.Pediatr Pulmonol 2002, 34:237-241.

22. Ratjen F, Hartog CM, Paul K, Wermelt J, Braun J: Matrixmetalloprotease in BAL fluid of patients with cystic fibrosis andtheir modulation by treatment with dornase alpha. Thorax 2002,57:930-934.

23. Klasen HJ: A review on the nonoperative removal of necrotictissue from burn wounds. Burns 2000, 26:207-222.

24. Rutter PM, Carpenter B, Hil SS, Locke IC: Varidase: the sciencebehind the medicament. J Wound Care 2000, 9:223-226.

25. Ozcan C, Ergun O, Celik A, Corduk N, Ozok G: Enzymaticdebridement of burn wound with collagenase in children withpartial-thickness burns. Burns 2002, 28:791-794.

26.��

Bradbury E, Moon L, Popat R, King VR, Bennett GS, Patel PN,Fawcett JW, McMahon SB: Chondroitinase ABC promotesfunctional recovery after spinal cord injury. Nature 2002,416:636-640.

Some elegant experiments in vivo (anatomical, electrophysiological andbehavioural) demonstrate that intrathecal injection of chondroitinase ABChelps greatly the regeneration of spinal cord injury, even if at the anato-mical level the regeneration was not complete.

27. Moon L, Asher R, Fawcett J: Limited growth of severed CNSaxons after treatment of adult rat brain with hyaluronidase.J Neurosci Res 2003, 71:21-37.



28. Lee-huang S, Huang PL, Sun Y, Kung HF, Blithe DL, Chen HC:Lysozyme and RNases as anti-HIV components in beta-corepreparations of human chorionic gonadotrophin. Proc NatlAcad Sci USA 1999, 96:2678-2681.

29. Fusetti F, von Moeller H, Houston D, Rozeboom HJ, Dijkstra BW,Boot RG, Aerts JM, Aalten DM: Structure of humanchitotriosidase. Implications for specific inhibitor design andfunction of mammalian chitinase-like lectins. J Biol Chem 2002,227:2537-2544.

30. Loeffler JM, Nelson D, Fischetti VA: Rapid killing ofStreptococcus pneumoniae with a bacteriophage cell wallhydrolase. Science 2001, 294:2170-2172.

31.�

Schuch R, Nelson D, Fischetti VA: A bacteriolytic agent thatdetects and kills Bacillus anthracis. Nature 2002, 418:884-889.

The authors describe the identification and purification of a lysin isolatedfrom a phage of B. anthracis. The lysin has high specificity and activitytowards B. anthracis. As in [26��], the same group demonstrates theadvantage of lytic enzymes isolated from phages as an alternative toantibiotics.

32. Zimmer M, Vukov N, Scherer S, Loessner M: The mureinhydrolase of the bacteriophage /3626 lysis system is activeagainst all tested Clostridium perfringens strains. Appl EnvironMicrobiol 2002, 68:5311-5317.

33. Ensor CM, Bomalaski JS, Clark MA: PEGylated argininedeiminase (ADI-SS PEG20,000 mw) inhibits human melanomasand hepatocellular carcinomas in vitro and in vivo. Cancer Res2002, 62:5443-5450.

34. Avrami VI, Sencer S, Periclou AP, Bostrom BC, Cohen LJ, EttinjerAG, Ettinjer LJ, Franklin J, Gaynon PS: A randomized comparisonof native Escherichia coli asparaginase and polyethylene glycolconjugated asparaginase for treatment of children with newlydiagnosed standard-risk acute lymphoblastic leukemia: achildren’s cancer group study. Blood 2002, 99:1986-1994.

35. Kurre HA, Ettinger AG, Veenstra DL, Gaynon PS, Franklin J, SencerSF, Reaman GH, Lanje BJ, Holcenberg JS: A pharmacoeconomicanalysis of pegaspargase versus native Escherichia coliL-asparginase for the treatment of children with standard-risk,acute lymphoblastic leukemia: the children’s cancer groupstudy (CCG-1962). J Pediatr Hematol Oncol 2002, 24:175-181.

36.�

Denholm E, Lin Y, Silver P: Anti-tumor activities of chondroitinaseAC and chondroitinase B: inhibition of angiogenesis,proliferation and invasion. Eur J Pharmacol 2001, 416:213-221.

Chondroitinase AC, which digests chondroitin sulfate A and chondroitinsulfate C, is shown, in vitro, to be more effective in inhibiting angiogenesisand invasion than chondroitinase B (specific of the dermatan sulfate).Whereas proliferation decreases and apoptosis increases with chondroi-tinase AC, chondoritinase B only inhibits proliferation and has no effecton apoptosis. The activities of these enzymes in vivo will be interestingto study.

37. Su H, Shao Z, Tkalec L, Blain F, Zimmermann J: Development of agenetic system for transfer of DNA into Flavobacteriumheparinum. Microbiology 2001, 147:581-599.

38. Blain F, Tkalec L, Shao Z, Poulin C, Pedneault M, Gu K,Eggimann B, Zimmermann J, Su H: Expression system for highlevels of GAG lyase gene expression and study of the hepAupstream region in Flavobacterium heparinum. J Bacteriol2002, 184:3242-3252.

39. Xu G, McLeod H: Strategies for enzyme/prodrug cancertherapy. Clin Cancer Res 2001, 7:3314-3324.

40. Jung M: Antibody directed enzyme prodrug therapy (ADEPT)and related approaches for cancer therapy. Mini Rev Med Chem2001, 1:399-407.

41. Genencor International website. URL:http://www.genencor.com/wt/gcor/adv_therapeutics.

42. Pui CH: Rasburicase: a potent uricolytic agent. Expert OpinPharmacother 2002, 3:433-452.

43. Cairo MS: Prevention and treatment of hyperuricemia inhematological malignancies. Clin Lymphoma 2002, 1:26-31.

44. Bomalaski J, Holtsberg F, Ensor CM, Clark MA: Uricaseformulated with polyethylene glycol (Uricase PEG20):biochemical rationale and preclinical studies. J Rheumatol2002, 29:1942-1947.

45.�

Veronese F, Calceti P, Schiavon O, Sergi M: Polyethylene glycol-superoxide dismutase, a conjugate in search of exploitation.Adv Drug Deliv Rev 2002, 54:587-606.

The authors show the huge amount of work carried out on the PEGylatedform of superoxide dismutase: the chemical aspects, the biopharmaceu-tical properties as well as the therapeutic activities. This enzyme wasshown to have therapeutic activity in the blood vessels, the heart, thelung, the brain, the liver and the kidney, but despite this the potential drugnever received approval in human therapy.

46. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE,Wallace DC, Malfroy B, Doctrow SR, Lithgow GJ: Extension oflife-span with superoxide dismutase/catalase mimetics.Science 2000, 289:1567-1569.

47. Izumi M, McDonald MC, Sharpe MA, Chatterjee PK, Thiermann C:Superoxide dismutase mimetics with catalase activity reducethe organ injury in hemorrhagic shock. Shock 2002,18:230-235.

48. Duysen EG, Bartels CF, Lockridge O: Wild-type and A328Wmutant human butyrylcholinesterase tetramers expressed inChinese hamster ovary cells have a 16-hour half-life in thecirculation and protect mice from cocaine toxicity. J PharmacolExp Ther 2002, 302:751-758.

49. Sun H, Pang YP, Lockridge O, Brimijoin S: Re-engineeringbutyrylcholinesterase as a cocaine hydrolase. Mol Pharmacol2002, 62:220-224.

50. Applied molecular evolution. URL:http://www.amevolution.com/.

51. Huisman G, Gray D: Towards novel processes for the fine-chemical and pharmaceutical industries. Curr Opin Biotechnol2002, 13:352-358.

450 Protein technologies and commercial enzymes


http://www.genencor.com/wt/gcor/adv_therapeutics

http://www.amevolution.com/

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