Current Status of Enzyme Therapy in the Management of Chronic Disorders

Enzymes:
Enzymes are present in organisms as single units or in multienzyme complexes. Posttranslational modifications of amino acid residues take place after peptide assembly on the ribosome; hydroxylation, phosphorylation, sulfation, N-terminal acetylation, and glycosylation are a few examples. The nature of posttranslational modifications and the hydrophobicity of amino acid side chains determine whether the enzyme is free or membrane-bound.1, 2

Enzymes catalyze biochemical reactions in living systems which would otherwise proceed too slowly at physiological temperature and pH to sustain life. Specificity and high catalytic power are two special attributes of enzymes which distinguish them from ordinary chemical catalysts.3

Clinical and pharmaceutical importance of enzymes

A variety of diseases can be detected through altered body fluid levels of specific enzymes.

Table 1 Disease detection via enzymes


Enzyme Disease
Aspartate aminotransferase Liver disease
Alanine aminotransferase Liver disease
Acid phosphatase Prostate carcinoma
Alkaline phosphatase Bone disease, Hepatobiliary disease
Creatine kinase Myocardial infarction, Muscle disease
Lactate dehydrogenase Myocardial infarction, Liver disease
Cholinesterase Organophosphate poisoning
Pancreas enzymes Pancreatic diseases.
glutamyltranspeptidase Liver disease, Alcoholism

Elevated plasma and urine lysozyme levels are typical for lymphocytic leukemia's and degenerative kidney diseases with glomerular and proximal tubular damage. Normalization of lysozyme plasma levels and disappearance of lysozyme in the urine are of prognostic value in successful kidney transplants.
Various types of cancer are associated with a general increase in plasma proteinases. The presence of various plasminogen activators and their inhibitors in many malignancies suggests that the fibrinolytic system is involved in the regulation of tumor growth and metastatis. Local changes in fibrinolytic activity such as reduced tPA and increased urokinase levels in biopsies of the intestinal mucosa are characteristic in inflammatory and (pre) malignant processes in the colon.4
Replacement therapy in enzyme dysfunction or as adjusting agents in biochemical processes that have gone awry. Examples include, Fibrinolytic enzymes in thrombotic disorders; Proteolytic enzymes in wound healing; Amino acid degrading enzymes and Dimeric ribonuclease in cancer therapy; Digestive enzymes; Hyaluronidase and superoxide dismutase in inflammations, and many others.

Enzymes of nonhuman origin sometimes are potent immunogens or allergens. Their delivery systems must protect them from inactivation before the target side is reached, and yet allow the enzymes to be released at the target site, eventually with their specific cofactors if required. Nonetheless enzymes are attractive drugs because of their specificity and efficiency.5

Enzyme inhibitors:

Natural and synthetic enzyme inhibitors have become increasingly important in medicine, and have developed into a separate class of drugs. These inhibitors form tight noncovalent or irreversible covalent complexes with their target enzymes.6,7

Thrombin is a key enzyme in clot formation, acting as a catalyst of conversion of fibrinogen to fibrin. Hirudin, a potent thrombin inhibitor from leech extracts, is available as a recombinant polypeptide and is currently under investigation in various clinical trials.8,9
The bovine basic pancreatic proteinase inhibitor aprotinin inactivates kallikrein and is used successfully in supplementary treatment of acute pancreatitis and shock.10Rennin inhibitors such as pepstain are currently under investigation to lower blood pressure.11,12

Enzyme in therapy:

Genetic defects 13, 14

Metabolic diseases are caused by specific enzyme defects, in which the enzyme is not expressed or is dysfunctional due to a sequence mutation or posttranslational inactivation. Some of these diseases can be treated with controlled diets. For example, phenylketonuria caused by phenylalanine-hydroxylase deficiency requires a phenylalanine-free diet. More often replacement therapy is needed, requiring the targeting of a replacement enzyme toward specific organs or tissues.

Other well-known "inborn errors of metabolism" are Pompe's disease or type II glycogen-storage disease in which deficient ± -1,4-glucosidase results in excessive accumulation of glycogen in liver and muscle cell lysosomes, Alcaptonuria (deficienct homogentisate 1,2 -dioxygenase), Hemophilia B (factor IXa), Galactosemia (UDPG-hexose-1-phosphate transferase), Gaucher's disease (²-glucocerebrosidase), Von Gierke's disease (glucose-6-phosphatase), Pentosuria (xylulose reductase), Nieman- Pick disease (Sphingomyelin phosphodiesterase), and the Lesch-Nyhan syndrome in which the absence of hypoxanthine-guanine phosphoribose transferase causes impaired nucleotide metabolism in brain cells and results in a severe neurological disorder.

Cancer therapy:
L-Asparaginase is used as an anticancer drug. Certain tumor cell types lack asparagines synthetase activity and need this amino acid as an essential nutrient, in contrast to normal cells. Asparaginase selectively kills the tumor cells by depleting the circulating level of asparagines. It has also been suggested that L-aspartate as a metabolite may be toxic toward neoplastic cells. 15
Acute lymphocytic leukemia treatment with asparaginase is relatively successful; some studies report complete remission in up to 60% of the treated patients.16 Patients subject to prolonged treatment with the enzyme often develop a resistance due to the high titer of their neutralizing antibodies. Nevertheless, the therapeutic index of asparaginase compares very favorably to other antileukemic drugs.

It was speculated that certain types of cancer cells might also lack other particular pathways for amino acid synthesis and thus have amino acid requirements that are masked because of the presence of these amino acids in the diet. Enzyme therapy depleting the required amino acid would lead to the selective killing of these cells. Glutamine, Cysteine, and Arginine have been the subject of studies for possible enzyme-depletion therapy. In addition to the E.coli and Erwinia asparaginase, two types of glutaminase-asparaginase (PGA and AGA) might be suitable therapeutic enzymes since both have antitumor activity in experimental animal models.17 Allergic reactions and some times neurotoxicity were the most serious side effects . The use of polymerized enzyme is preferred, since the unmodified enzyme has a plasma half-life of only 80 min.18
Of particular interest in cancer therapy is Carboxypeptidase G which hydrolyzes the terminal aspartate and glutamate moieties in oligopeptides and the glutamate moiety in reduced and nonreduced folates. Purified carboxypeptidase G from various Pseudomonas strains prevents methotrexate toxicity in humans.19

The antineoplastic effect of bovine pancreas ribonuclease has been reported in chronic myelocytic leukemia patients. However, limited information is available from clinical trials. Dimeric ribonuclease displays selective toxicity in animal tumors and may be a promising candidate as therapeutic enzyme.

Bacteriolytic, antiviral, and anti-inflammatory enzymes:
Lysozyme is present as an antibacterial agent in body fluids and cavities in direct contact with the external environment. It is available as a pharmaceutical preparation in tablets, ointments, powders, and infusions and is used as an antibacterial, antiviral, and anti-inflammatory drug. The commercial preparations contain hen egg-white lysozyme which is easily isolated and purified from egg whites in large-scale projects. This enzyme is nontoxic and only weakly antigenic and can be administered internally in large doses without significant side effects. Lysozyme has chitinase, muramidase, and transglycosidase activity and acts upon bacteria in many ways. The proteoglycan layer in cell walls is the natural substrate for this enzyme.

Lysozyme has a distinct antiviral activity against herpes labialis, zoster, and simplex I and II types in humans, as well as against some oncogenic viruses in animal studies. Lysozyme stimulates phagocytosis and favors wound healing and regression of degenerative and necrotic processes.20 Lysozyme is administered in intramuscular or parenteral injections in herpes zoster and viral hepatitis, and in ointments for the treatment of herpetic keratitis, burns, and wounds and gynecological infections.

Corticoids and antibiotics have a synergestic action and are combined with lysozyme in aerosols for the treatment of bronchopulmonary diseases. Proteolytic enzymes and antiseptics are frequently used as adjuvant agents for dermatological applications. Bovine pancreatic ribonuclease appears to be an effective antiviral enzyme against tick-borne encephalitis. It has no side effects and results in more rapid temperature normalization and regression of meningeal symptoms than antiencephalitic gamma globulin.21

Commercial hyaluronidase preparations contain the bovine-testicular type (hyaluronate-4-glycanohydrolase) or the leech-type enzyme (hyaluronate-3-glycanohydrolase). They are used in the treatment of keloids, ligneous conjunctivitis, and connective-tissue inflammation and as adjuvant in cancer therapy to facilitate transport and resorption of cytostatic agents.22 Animal studies revealed that hyaluronidase also acts as a cardiac lymphagogue, thereby reducing myocardial infarction after coronary artery occlusion.23

Superoxide dismutase (SOD) acts as an oxygen radical scavenger; under inflammatory conditions its levels are increased. Depending on the nature of the disease, SOD is administered in injections, encapsulated in liposomes as a copper-zinc-SOD complex, or externally in creams.24 the enzyme is effective in the treatment of rheumatoid arthritis, crohn's disease, progressive systemic sclerosis, dermatitis herpetiformis, and mucocutaneous lymph-node syndrome. It also prevents myocardial injury as a consequence of chemotherapy in neoplstic diseases.25

Proteolytic enzymes:
Enzymes hydrolyzing peptide bonds are not only important in food digestion, but also play essential roles in biological processes such as coagulation and hemostasis, complement activation, peptide hormone release, wound healing, and control of protein metabolism.25,26

Trypsin and chymotrypsin are classical examples of proteinases used in wound healing. They facilitate the removal of necrotic tissue and scab material trapping bacteria inside the wound. These enzymes are frequently combined with antiseptics or antibiotics in ointments and bandages. Recently a new enzyme extract from Antarctic krill (E.superba) has been tested as a possible candidate preparation for the debridement of ulcerative lesions.28

Antithrombotic therapy:

An imbalance between coagulation and fibrinolysis leading to excessive fibrin deposition can be approached either by the reduction of the coagulation potency or by an increase of the fibrinolytic potency. Various aspects of fibrinolytic enhancement are still under development. Thrombolytic thearpy is used in the initial management of patients with deep venous thrombosis and pulmonary embolism.
Streptokinase and urokinase have been used extensively in the treatment of venous thromboembolism. They are more potent than free circulating plasmin, which is rapidly inactivated by circulating alpha 2-antiplasmin 30, 29. Randomized studies have demonstrated that intravenous tissue plasminogen activator is more efficient than streptokinase in the treatment of coronary occlusion in acute myocardial infarction 31, 32 . Commercial recombinant tissue plasminogen activator for the treatment of acute myocardial infarct is mainly obtained from Chinese hamster ovary cells (CHO).

Chemonucleolysis:
Chymopapain, an oxidation-sensitive cysteine ​​proteinase from Carica papaya has been proposed for the treatment of herniated lumbar discs. Intradiscal injection of chymopapin results in dissolution of the mucopolysaccharide – protein complex of an extruding nucleus pulposus, the centre cushioning of gelatinous mass lying within the intervertebral disc, thereby relieving the pain associated with a pressurized nerve. Large-scale follow-up studies indicate that chemonucleolysis is as successful a procedure as surgical discectomy, with a 76-80% success rate in both groups 33, 34, 35. Some pathological peculiarities associated with herniated discs might be unfit for treatment by chemonucleolysis , for example, discs extruding nucleus pulposus through the annulus, a case in which the risk of damage to the spinal cord is predominant .36

Pancreas enzymes:
Digestion of food is facilitated by the pancrease enzyme trypsin, chymotrypsin and elastase, carboxypeptidase A and B, phospholipase A – 2 and lipase and amylase. The main lipid component in food is long-chain triacylglycerol, which is hydrolyzed into fatty acids and sn-2-monoacylglycerol. Both products are readily absorbed in the intestine. This hydrolysis is catalyzed sequentially by gastric lipase secreted by the chief cells of the stomach and by colipase-dependent pancrease 37.

Pancreatic lipases, proteases and amylases are prescribed as replacement therapy in pancreatic insufficiency where the enzyme output has fallen below 10%. Pancreatin of mammal origin is used in commercial preparations for substitution treatment 38, 40. Pharmaceutical formulations show considerable variation in enzyme activity and bile salt content. Cellulose is sometimes added as adjuvant enzyme. Since the extent of pancreas malfunction varies significantly among patients, individualization of the treatment is indicated in determining the optimal enzyme dosage, formulation type, time of administration with respect to meals and frequency of administration.

Conclusion and future trends:
The elucidation on a molecular level of disease-related biochemical processes enables the definition of the type and specific target area of ​​key enzymes or inhibitors necessary for restoring normal physiological conditions. Enzymes are attractive drug candidates because of their reaction specificity and catalytic efficiency. However, their protein nature imposes some limitations on their use in therapy. Organ specific targeted enzymes require parenteral administration and proteins of non-human origin are often allergenic or immunogenic. Considerable progress has been made in the refinement of isolation procedures to reduce or eliminate toxic contaminants, and the development of specific chemical modification and targeting techniques offers new possibilities for prolonging half life and improving enzyme bioavailability. The most promising results are undoubtedly to be expected from the field of genetic engineering and site specific mutagenesis. Mutant enzymes with altered or improved specificity, enhanced stability, and reduced immunogenicity will become available at an affordable cost.

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