Therapeutic enzymes

U. C. Banerjee
Professor
Department of Pharmaceutical Technology (Biotechnology)
National Institute of Pharmaceutical Education & Research
Sector- 67, S.A.S Nagar
Mohali 160062
E-mail:- ucbanerjee@niper.ac.in
Visit us:- www.niper.gov.in
 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 cant 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

Animal sources
Plant sources
Microbial sources
Bacterial sources
Fungal 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 (recombinant human cells)
Urokinase (human urine)
Reptilase (snakevenom, borthrops atrox)
 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)
 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
 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:
1. Fabry Disease
2. Hurlers Disease
3. Gauchers Disease
4. Hunters 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
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)
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
ActivatorsStreptokinase, Fibrin (insoluble)
Urokinase, Altepase (rt-PA)

plasminogen plasmin

Fibrin fragments
(Soluble)
 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)
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

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 bodys production of plasmin
Taken orally
Low dose of 100 mg per day
 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
Sarcosidase:
it is a -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
Pulmozyme (Dornase ) 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)
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
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..
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
pH-dependence of the recombinant L-asparaginases Vmax
(a) and Km (b) values for L-Asn
 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
 Some important therapeutic enzymes

S.No Enzymes Reaction Uses

1 Asparginase L-Asperginase + H2O Leukemia

L-Aspartate + NH3
2 Collagenase Collagen hydrolysis Skin ulcers
3 Hyaluronidase Hyaluronate hydrolysis Heart attack
4 Lysozymes Bacterial cell wall hydrolysis Antibiotics
5 Ribonuclease RNA hydrolysis Antiviral
6 Streptokinase Plasminogen Plasmin Blood clots

7 Trypsin Protein hydrolysis Inflammation

8 Uricase Urate + O2 allantoin Gout

9 Urikinase Plasminogen Plasmin Blood clots

 Proteolytic and glycolytic enzymes for
treating 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
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
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
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
Trauma Surgery
Surgery
Plastic Surgery
Respiratory Medicine
Infections
Male Genital Infection
Dermatology
Dentistry
Obstetrics & Gynaecology
 Uricase
Urate oxidase is obtainied from Candida
utilis
it is used in the treatment of hyperuricemia
and gout
Urate + H2O Allantoin + H2O2 + CO2
 Ribonuclease
Base

It is used as an 5' CH
2 O

antiviral agent to
H H
hydrolyse the viral
Ribonuclease 1 O OH

{
RNA
phospho HO P O
Ribonuclease 1 diester
O
from pancreas linkage Base

Ribonuclease T1 Ribonuclease T1 5' CH2

O
from molds
H H

OH OH
 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
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
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
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
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, Crohns
disease and AIDS
These enzymes reduce the no. of
circulating immune complexes which are
responsible for the disease process
 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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