Intro2u.net > Reference > Enzyme Technology

Development of medical applications for enzymes have been at least as extensive as those for industrial applications, reflecting the magnitude of the potential rewards: for example, pancreatic enzymes have been in use since the nineteenth century for the treatment of digestive disorders. The variety of enzymes and their potential therapeutic applications are considerable. A selection of those enzymes which have realised this potential to become important therapeutic agents is shown in Table 4.4. At present, the most successful applications are extracellular: purely topical uses, the removal c toxic substances and the treatment of life-threatening disorders within the blood circulation.

Table 4.4 Some important therapeutic enzymes

Enzyme	EC number	Reaction	Use
Asparaginase	3.5.1.1	L-Asparagine H2O L-aspartate + NH3	Leukaemia
Collagenase	3.4.24.3	Collagen hydrolysis	Skin ulcers
Glutaminase	3.5.1.2	L-Glutamine H2O L-glutamate + NH3	Leukaemia
Hyaluronidasea	3.2.1.35	Hyaluronate hydrolysis	Heart attack
Lysozyme	3.2.1.17	Bacterial cell wall hydrolysis	Antibiotic
Rhodanaseb	2.8.1.1	S2O32- + CN- SO32- + SCN-	Cyanide poisoning
Ribonuclease	3.1.26.4	RNA hydrolysis	Antiviral
b-Lactamase	3.5.2.6	Penicillin penicilloate	Penicillin allergy
Streptokinasec	3.4.22.10	Plasminogen plasmin	Blood clots
Trypsin	3.4.21.4	Protein hydrolysis	Inflammation
Uricased	1.7.3.3	Urate + O2   allantoin	Gout
Urokinasee	3.4.21.31	Plasminogen plasmin	Blood clots

^a Hyaluronoglucosaminidase
^b thiosulphate sulfurtransferase
^c streptococcal cysteine proteinase
^d urate oxidase
^e plasminogen activator

As enzymes are specific biological catalysts, they should make the most desirable therapeutic agents for the treatment of metabolic diseases. Unfortunately a number of factors severely reduces this potential utility:

1. They are too large to be distributed simply within the body's cells. This is the major reason why enzymes have not yet been successful applied to the large number of human genetic diseases. A number of methods are being developed in order to overcome this by targeting enzymes; as examples, enzymes with covalently attached external b-galactose residues are targeted at hepatocytes and enzymes covalently coupled to target-specific monoclonal antibodies are being used to avoid non-specific side-reactions.
2. Being generally foreign proteins to the body, they are antigenic and can elicit an immune response which may cause severe and life-threatening allergic reactions, particularly .on continued use. It has proved possible to circumvent this problem, in some cases, by disguising the enzyme as an apparently non-proteinaceous molecule by covalent modification. Asparaginase, modified by covalent attachment of polyethylene glycol, has been shown to retain its anti-tumour effect whilst possessing no immunogenicity. Clearly the presence of toxins, pyrogens and other harmful materials within a therapeutic enzyme preparation is totally forbidden. Effectively, this encourages the use of animal enzymes, in spite of their high cost, relative to those of microbial origin.
3. Their effective lifetime within the circulation may be only a matter of minutes. This has proved easier than the immunological problem to combat, by disguise using covalent modification. Other methods have also been shown to be successful, particularly those involving entrapment of the enzyme within artificial liposomes, synthetic microspheres and red blood cell ghosts. However, although these methods are efficacious at extending the circulatory lifetime of the enzymes, they often cause increased immunological response and additionally may cause blood clots.

In contrast to the industrial use of enzymes, therapeutically useful enzymes are required in relatively tiny amounts but at a very high degree of purity and (generally) specificity. The favoured kinetic properties of these enzymes are low K_m and high V_max in order to be maximally efficient even at very low enzyme and substrate concentrations. Thus the sources of such enzymes are chosen with care to avoid any possibility of unwanted contamination by incompatible material and to enable ready purification. Therapeutic enzyme preparations are generally offered for sale as lyophilised pure preparations with only biocompatible buffering salts and mannitol diluent added. The costs of such enzymes may be quite high but still comparable to those of competing therapeutic agents or treatments. As an example, urokinase (a serine protease, see Table 4.4) is prepared from human urine (some genetically engineered preparations are being developed) and used to dissolve blood clots. The cost of the enzyme is about £100 mg^-1, with the cost of treatment in a case of lung embolism being about £10000 for the enzyme alone. In spite of this, the market for the enzyme is worth about £70M year^-1.

A major potential therapeutic application of enzymes is in the treatment of cancer. Asparaginase has proved to be particularly promising for the treatment of acute lymphocytic leukaemia. Its action depends upon the fact that tumour cells are deficient in aspartate-ammonia ligase activity, which restricts their ability to synthesise the normally non-essential amino acid L-asparagine. Therefore, they are forced to extract it from body fluids. The action of the asparaginase does not affect the functioning of normal cells which are able to synthesise enough for their own requirements, but reduce the free exogenous concentration and so induces a state of fatal starvation in the susceptible tumour cells. A 60% incidence of complete remission has been reported in a study of almost 6000 cases of acute lymphocytic leukaemia. The enzyme is administered intravenously. It is only effective in reducing asparagine levels within the bloodstream, showing a half-life of about a day (in a dog). This half-life may be increased 20-fold by use of polyethylene glycol-modified asparaginase.