Therapeutic Enzymes

Question 1

What is the function of asparaginase?

Answer 1

It’s an enzyme that catalyzes the hydrolysis of L-asparagine, producing aspartic acid and ammonia.

Question 2

How was the effectiveness of asparaginase discovered?

Answer 2

• In the late 1970s
• Serum from healthy guinea pigs transferred into mice with leukemia inhibited the proliferation of leukemia cells.
• The active agent was asparaginase.

Question 3

Why is asparaginase used in treating certain forms of human leukemia?

Answer 3

Many transformed cells, including leukemia cells, lose the ability to synthesize asparagine.
Asparaginase:

• deprives these cells of asparagine
• by hydrolyzing plasma asparagine
• making it an effective treatment for leukemia.

Question 4

What is an example of a specific form of asparaginase used in the treatment of childhood acute lymphoblastic leukemia?

Answer 4

PEG-L-asparaginase

Has been approved for the treatment of refractory childhood acute lymphoblastic leukemia.

Question 5

What is the importance of high substrate affinity in therapeutically useful asparaginases?

Answer 5

• The plasma concentration of asparagine is typically low,
• Therapeutically useful asparaginases must have a high substrate affinity
• To effectively deplete asparagine in the body.

Question 6

Which organisms have been studied extensively for their asparaginase activity?

Answer 6

• E. coli
• Erwinia
• Pseudomonas
• Acinetobacter

shown to be effective in inhibiting the growth of various leukemias and other transformed cell lines.

Question 7

What are some common side effects associated with asparaginase treatment?

Answer 7

• severe nausea
• vomiting
• diarrhea,
• compromised liver and kidney function

due to transient asparaginase deficiency in various tissues.

Question 8

Why is the lung tissue in cystic fibrosis patients susceptible to frequent and recurrent microbial infections?

Answer 8

• Due to the pathophysiological changes induced in the cystic fibrosis lung
• Particularly by Pseudomonas species.

Question 9

What happens as a result of the presence of microbes in the lung?

Answer 9

• Phagocytic neutrophils digest microbes in the cystic fibrosis lung
• They release large quantities of DNA,
• Significantly increases the viscosity of the respiratory mucus.

Question 10

Why is it beneficial to break down DNA and reduce the viscosity of respiratory mucus in cystic fibrosis?

Answer 10

Breaking down DNA and reducing the viscosity of respiratory mucus in cystic fibrosis has obvious clinical benefits as
* it helps in improving airway clearance
* reducing the risk of respiratory complications.

Question 11

What experiments were undertaken for the treatment of cystic fibrosis?

Answer 11

Since 1950s
Experiments entailing inhalation of DNase-enriched extracts of bovine pancreas

Question 12

What were the issues associated with early experiments for cystic fibrosis treatment?

Answer 12

Raised concerns about:
Product safety

• Damage to underlying lung tissue by trypsin and other contaminants

Efficacy issues

• Due to neutralization of bovine DNase by the host immune system.

Question 13

What was the solution to the safety and efficacy issues with DNase treatment for cystic fibrosis?

Answer 13

• Highly purified recombinant human DNase (rhDNase) has been used since the end of 1993
• It proved safe and effective
• Leading to marketing authorization under the tradename Pulmozyme.

Question 14

How is Pulmozyme administered for cystic fibrosis treatment?

Answer 14

• Produced by an engineered CHO cell line
• Administered directly into the lungs by inhalation of an aerosol mist
• Generated by a compressed-air-based nebulizer system.

Question 15

What did in vitro studies show regarding the use of rhDNase in cystic fibrosis treatment?

Answer 15

• That incubation of the rhDNase enzyme with sputum derived from a cystic fibrosis patient resulted in a significant reduction in sputum viscosity
• indicating its potential efficacy in improving airway clearance.

Question 16

What is the approved use of α-galactosidase?

Answer 16

Approved for long-term enzyme replacement therapy in patients with Fabry disease

Question 17

What is Fabry disease?

Answer 17

a genetic disease of lipid metabolism characterized by little or no liposomal α-galactosidase activity.

Question 18

How does Fabry disease affect patients?

Answer 18

Affects patients by causing complex clinical manifestations that impact the nervous system, vascular endothelial cells, and major organs.

Question 19

What is the consequence for untreated sufferers of Fabry disease?

Answer 19

Untreated sufferers of Fabry disease typically have a reduced life expectancy and usually die in their 40s or 50s

Question 20

What are the names of the two recombinant α-galactosidases available on the market?

Answer 20

Fabrazyme - produced by Genzyme,
Replagal - produced by TKT Europe.

Question 21

What are the reactive molecules generated by the incomplete reduction of oxygen?

Answer 21

• superoxide radical (O2-)
• hydrogen peroxide (H2O2)
• hydroxyl radical (OH-)

Question 22

How do the superoxide and hydroxyl radicals impact cellular components?

Answer 22

Are highly reactive and can attack:

• membrane components
• Nucleic acids
• Other cellular macromolecules
  leading to their destruction or modification.

Question 23

What are some examples of pathological conditions associated with increased generation of superoxide or reduced levels of superoxide dismutase (SOD)?

Answer 23

Increased generation of superoxide (O2-) or reduced SOD levels have been implicated in various pathological conditions, including:
aging,
asthma,
accelerated tumor growth,
neurodegenerative diseases
inflammatory tissue necrosis.

Question 24

What are the catalytic reactions catalyzed by SOD and catalase or glutathione peroxidase (GSH-px)?

Answer 24

SOD catalyzes the reaction:
O2- + O2- + 2H+ -> H2O2 + O2
catalase or GSH-px catalyzes the reaction:
H2O2 + H2O2 -> 2H2O + O2.

Question 25

How has SOD been used medically?

Answer 25

SOD isolated from bovine liver or erythrocytes has been used as an anti-inflammatory agent in medical applications.

Question 26

What is being evaluated regarding human SOD in relation to tissue damage prevention?

Answer 26

Human SOD expressed in recombinant systems is currently being evaluated to assess its ability to prevent tissue damage induced by exposure to excessively oxygen-rich blood.

Question 27

What is debridement?

Answer 27

The process of cleaning a wound by removing foreign material and dead tissue.

Question 28

Why is debridement necessary for wound healing?

Answer 28

Formation of a clot and scab on a wound surface can trap bacteria, leading to the multiplication of bacteria and slowed healing. Debridement helps remove bacteria and promote the healing process.

Question 29

How can debridement be achieved?

Answer 29

Through physical means, such as cutting away dead tissue and cleaning the wound, or by using proteolytic enzymes to facilitate the process.

Question 30

What are some common microbial enzymes used as debriding agents?

Answer 30

• collagenase (in Collagenase Santyl ointment)
• papain (in Gladase-C ointment)
• trypsin (in Granulex V spray)

Question 31

How is collagenase obtained for therapeutic use?

Answer 31

Collagenase used therapeutically is usually obtained from cell fermentation supernatants of Clostridium histolyticum, although it can also be produced by animal cell culture.

Question 32

Where is papain derived from for its use as a debriding agent?

Answer 32

Papain, a cysteine protease, is isolated from the latex of the immature fruit and leaves of the plant Carica papaya.

Question 33

How is trypsin obtained for medical use as a debriding agent?

Answer 33

• By enzymatic activation of trypsinogen,
• Extracted from the pancreatic tissue of slaughterhouse animals.

Question 34

Besides enzyme-based debridement, what other method has been historically used to clean wounds?

Answer 34

• By the application of protease-containing maggot saliva
• The use of maggots themselves directly onto wound tissue.