Therapeutic enzyme


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Therapeutic enzymes : 

Therapeutic enzymes U. C. BanerjeeProfessor Department of Pharmaceutical Technology (Biotechnology)National Institute of Pharmaceutical Education & Research Sector- 67, S.A.S Nagar Mohali 160062E-mail:- us:-

Differences between enzyme as a drug and other drugs : 

Differences between enzyme as a drug and other drugs Enzymes often bind and act on their targets with great affinity and specificity Enzymes convert multiple target molecules to the desired products proteinaceous in nature thus can’t be administered orally, need to be specially formulated as enteric coated if intended for oral therapy Need to be of very high purity, inferior preparations have shown to elicit severe immune response

Sources of therapeutic enzymes : 

Sources of therapeutic enzymes Animal sources Plant sources Microbial sources Bacterial sources Fungal sources

Examples of animal sources : 

Examples of animal sources Trypsin (Ox bile) Chymotripsin (Ox bile and pancreas) Lipase ( Pancreas) Alteplase ( Chinese hamster) Urokinase ( Human urine) Lysozyme ( Hen eggs) Adenosine deaminase ( Bovine intestine) Pepsin (Hog pancreas) Ribonuclease Dornase α (recombinant human cells) Urokinase (human urine) Reptilase (snakevenom, borthrops atrox)

Examples of plant sources : 

Examples of plant sources Papain (Carica papaya) Bromelain (Ananas comosus) Amylase (Malted barley-Hordeum vulgare) Nattokinase ( Natto)

Examples of microbial sources : 

Bacterial sources L- asparginase (E.coli) Collagenase (Closteridium histolyticum) Streptokinase (beta haemolytic streptococci) Glutaminase (E. coli) Rhodanse (Sulfobacillus sibiricus) Staphylokinase (Staplococci sp.) Beta lactamase (Staphylococci sp.) Serratiopeptidase (Serratia sp.) Amylase (Bacillus sp.) Fungal sources Protease (Serratia spp.) Brinase ( Aspergillus oryzae) Lipase (Aspergillus oryzae) Amylase ( Aspergillus spp.) Uricase (Candida utilis) Examples of microbial sources

Choice of enzyme sources : 

Choice of enzyme sources Microbes are preferred because They are generally cheaper to produce Their enzyme content is more predictable and controllable Reliable supplies of raw material of constant composition Plant and animal sources contain more harmful material than microbes which include phenolic compounds (from plants), endogenous enzyme inhibitors and proteases

Classification of therapeutic enzymes : 

Classification of therapeutic enzymes

Lysosomal Storage Diseases (LSDs) : 

Lysosomal Storage Diseases (LSDs) These are a group of genetic diseases passed down in the family It effects 1 in 7700 live births Each results from different defects in the genes. Some common LSDs are: Fabry Disease Hurler’s Disease Gaucher’s Disease Hunter’s Disease

Fabry Disease : 

Fabry Disease It results due to accumulation of a fatty substance called globotrianosylceremide (Gb3) in the kidneys, heart, nerves, blood vessel. Treated with Recombinant human galctosidase A It prevents Gb3 accumulation by breaking it down

Gaucher Disease : 

Gaucher Disease It results due to the deficiency of an enzyme Glucocerebrosidase Causes lipid accumulation in the spleen and liver and their subsequent swelling The enzyme Glucocerebrosidase is used to treat the disease, it is derived from human tissue, purified and then chemically altered in order to target the cell with lipid buildup

Thrombolytic Drugs (Fibrinolytics) : 

Thrombolytic Drugs (Fibrinolytics) These drugs are used to lyse thrombi/clot to recanalyse occluded blood vessels They are curative rather than prophylactic and act by activating the natural fibrinolytic system plasminogen plasmin Fibrin (insoluble) Fibrin fragments (Soluble) Activators—Streptokinase, Urokinase, Altepase (rt-PA)

Streptokinase : 

Streptokinase It is obtained from beta haemolytic Streptococci group C Inactive as such: combines with the circulating plasminogen to form an activator complex which then causes limited proteolysis of other plasminogen molecules to plasmin It is antigenic, can cause hypersensitivity reactions and anaphylaxis Urokinase Isolated from human urine, now prepared by cultured human kidney cells Activates plasminogen directly Non antigenic

Altepase (recombinant tissue Plasminogen Activator-rtPA) : 

Altepase (recombinant tissue Plasminogen Activator-rtPA) It is produced by recombinant DNA technology using human tissue culture It specifically activates gel phase plasminogen already bound to fibrin It is a serine protease of 527 amino acid residues Binds to fibrin via lysine binding sites which are in a finger domain It is a rather expensive product

Nattokinase : 

Nattokinase Extracted from a traditional Japanese food called Natto (cheese like food) Natto is produced by a fermentation proces adding Bacillus natto to boiled soyabeans It dissolves fibrin directly Also enhances body’s production of plasmin Taken orally Low dose of 100 mg per day

Nattokinase : 

Nattokinase It produces a prolonged action Dual action of preventing coagulation of blood and dissolving existing thrombus Efficacy and prolonged action determined by measuring in vivo parameters like Euglobin fibrinolytic activity (EFA) Fibrin degradation products. (FDP) Both get elevated as fibrin gets dissolved

Oral and inhalable therapies : 

Oral and inhalable therapies Sarcosidase: it is a β-fructofuranoside fructohydrolase from Sacchromyces cerevesiae Taken orally to treat Congenital sucrase-isomaltase deficiency (CSID) It hydrolyses sucrose allowing the consumption of a more normal diet Phenylase: It is a recombinant yeast phenylalanine ammonia lyase Used for the treatment of Phenylketonuria (PKU) cause due to the deficiency of phenylalanine hydroxylase which converts Phenylalanine to tyrosine

Inhalable therapies : 

Inhalable therapies Pulmozyme® (Dornase α) is a DNase It is used in the treatment of cystic fibroses in which the pulmonary fluid thickens up and contains a high concentration of DNA It liquefies accumulated mucus in the lung It also diminishes pulmonary tissue destruction by lowering the level of matrix metalloproteinases in the bronchoalveolar lavage fluid

L- Asparaginase (Oncolytic) : 

L- Asparaginase (Oncolytic) Mechanism of action Normal tissues synthesize l-asparagine in sufficient quantities for protein synthesis Most neoplastic tissues require its exogenous supply from circulation L-asparaginase breakdowns circulating asparagine to l-aspartate and ammonia, prevents protein synthesis of neoplasms It produces cell death through activation of apoptosis

Production of l-Asparaginase : 

Production of l-Asparaginase Produced from Erwenia chrysanthemi and Recombinant E. coli. Aspergillus, Penicillium and Fusarium Recombinant asparaginase production: Recombinant E.coli grown in LB media Inducer– IPTG Grown for 16– 20 h Average level of l-asparaginase expression -- 30- 40 iu/ml

L-Asparaginase production cont.. : 

L-Asparaginase production cont.. Cells harvested by centrifugation, stored at -20 deg C The asparaginase produced to be released by sonication or french press The soluble portion of the cell free extract purified by gel filtration chromatography using sepharose column One component low salt elution used with phosphate buffer

Slide 23: 

pH-dependence of the recombinant L-asparaginase’s Vmax (a) and Km (b) values for L-Asn

Other potential candidates for cancer treatment : 

Other potential candidates for cancer treatment PEGylated arginine deaminase – arginine degrading enzyme, can inhibit human melanoma and hepatocellular carcinomas, which are auxotrophic for arginine owing to a lack of arginosuccinate synthetase Chondroitinase AC – acts by removal of chondroitin sulfate proteoglycans thus prevents proliferation, neovasculrization and metastasis ADEPT– antibody dependent enzyme prodrug therapy – a monoclonal Ab carries an enzyme to the cancer site and activates the drug at the site

Slide 25: 

Some important therapeutic enzymes

Proteolytic and glycolytic enzymes fortreating damaged tissue : 

Proteolytic and glycolytic enzymes fortreating damaged tissue Used for the mechanical debridement of damaged tissue from burns Debrase Gel Dressing – mixture of enzymes from pineapple extracts in Phase 2 Clinical Trials VibrilaseTM (recombinant vibriolysin), a proteolytic enzyme from the marine microorganism Vibrio proteolyticus is used for the treatment of denatured proteins such as those found in burned skin. In Phase I clinical trials

Proteolytic enzymes : 

Proteolytic enzymes Sutilains Produced by fermentation of Bacillus subtilis Purified by filtration, salt and solvent precipitation and lyophilization Debridement of 2nd and 3rd degree burns, incisional, traumatic and pyrogenic wounds It digests denatured proteins found in necrotic tissues Moist environment essential for optimal activity

Enzymes for infectious diseases : 

Enzymes for infectious diseases Lysozyme, RNAse A, Urinary RNAse U— antiHIV Chitinase-- Antifungals, antiprotozoans, anthelminthics: acts by disintegrating the cell wall Bacteriophage derived lytic enzymes– used against Streptococcus pneumonia, Bacillus anthracis and Clostridium perfringens

Serratiopeptidase : 

Serratiopeptidase Obtained from Serratia sp. The purified enzyme has antiinflammatory and sputum liquefying properties It neutralizes bradykinin and proinflammatory eicosinoids It eliminates inflammatory edema and swelling, accelerates liquefaction of pus and sputum, and enhances the action of antibiotics

Serratiopeptidase– therapeutic indications : 

Serratiopeptidase– therapeutic indications Trauma Surgery Surgery Plastic Surgery Respiratory Medicine Infections Male Genital Infection Dermatology Dentistry Obstetrics & Gynaecology

Uricase : 

Uricase Urate oxidase is obtainied from Candida utilis it is used in the treatment of hyperuricemia and gout Urate + H2O Allantoin + H2O2 + CO2

Ribonuclease : 

Ribonuclease It is used as an antiviral agent to hydrolyse the viral RNA Ribonuclease 1 from pancreas Ribonuclease T1 from molds

Hyaluronidase : 

Hyaluronidase Obtained from bovine testicular tissue extracts It breaks down hyaluronic acid in connective tissue, tempprarily decreases the viscosity of cellular cement and thus increasing tissue permeability Also enhances the diffusion of subcutaneously injected agents Typically used as an adjunctive agent with local anaesthetics, here it increases the speed of onset of action

Hyaluronidase : 

Hyaluronidase In cataract surgery, it is given by retrobulbar injection to increase the hypotonic effect of local anesthetics Used to treat extravasations caused by antineoplastics, vinca alkaloids (vincristine, vinblastine, vinorelbine) The prepration has the potential for contamination with bovine pathogens

Papain : 

Papain A protein cleaving enzyme derived from papaya Therapeutic indications include: Defibrination of wounds in hospitals Prevention of cornea scar deformation Treatment of jellyfish and insect stings Treatment of edemas, inflammatory processes, acceleration of wound healings Ingredient of cleaning solutions of soft contact lenses

Pancreatic Enzymes– Digestive aids : 

Pancreatic Enzymes– Digestive aids Lipases– digestion of fats. Deficiency leads to malabsorption of fats and fat- soluble vitamins Amylases– break down starch molecules into smaller sugars. Secreted by salivary glands and the pancreas Proteases– (trypsin, chymotrypsin, carboxypeptidases) break down proteins to amino acids Commercially available in combination packs, indicated in food alergies

Pacreatic enzymes– other uses : 

Pacreatic enzymes– other uses Anti-inflammatory agent in cases of trauma, inflammation, thrombophlebitis In autoimmune conditions such as Rheumatoid arthritis, systemic lupus erythmatosus, scleroderma and multiple sclerosis, ulcerative colitis, Crohn’s disease and AIDS These enzymes reduce the no. of circulating immune complexes which are responsible for the disease process

Conclusions. : 

Conclusions. The onus in on biotechnology to produce safer and cheaper enzymes with enhanced potency and specificity Treatment of rare diseases should be sought Oral administration, confirmed efficacy and prolonged effects



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