Advanced drug delivery 3 - PPC and PEGylated Proteins

Question 1

What is a polymer protein conjugate? (PPC)

Answer 1

Drug delivery system where there is a polymer covalently conjugated to a protein
- The protein is the API
- Single protein has one or more polymeric chains attached (usually multiple).

Question 2

Polymer drug conjugate (PDC)

Answer 2

Drug delivery system where there is a polymer covalent conjugated to the drug
- The drug is the API
- API is typically low MW molecules like traditional anticancer drugs e.g. dox, paclitaxel
- Each polymeric chain carries several drug molecules

Question 3

Similarity between PDC and PPC

Answer 3

Both have covalent conjugation between API and polymer

Question 4

Problems with using proteins as therapeutic agents

Answer 4

• Rapid renal excretion
• Proteolytic degradation
• Immunogenicity
• Aggregation
• Solubility
• Difficulty in formulation

Question 5

Rapid renal excretion

Answer 5

• Smaller proteins are rapidly excreted
• Too quick = insufficient therapeutic action

Question 6

Aggregation

Answer 6

Occurs before or after administration

Question 7

Solubility

Answer 7

Needs to be soluble in blood - some proteins arent.

Question 8

Advantages of PPC

Answer 8

• Protection
• Increased hydrodynamic volume, therefore reduced renal clearance.
• Reduced protein aggregation

Question 9

PPC: Protection

Answer 9

• Polymeric chains protect proteins from proteolytic degradation
• Also protects the body from unwanted characteristics of the protein e.g. immunogeneicity

Question 10

Increased hydrodynamic volume and reduced renal clearance

Answer 10

Conjugated protein vs Native protein:
Conjugated protein = increased hydrodyanmic volume = slower renal excretion because of the larger size

Question 11

Reduced protein aggregation

Answer 11

• Attaching polymeric chains prevents protein aggregation
• Because of steric hindrence.
• Conjugate polymer in areas that are not key to the binding site to prevent interaction with molecular target

Question 12

General structure of protein

Answer 12

• Amine group
• Alpha carbon
• Variable R group (side chain)
• Carboxylic acid

Question 13

What does the polymer bind to in a protien

Answer 13

• The side chain: either C terminus or N terminius

Question 14

Lysine

Answer 14

• Use lysine with a polymer with an activate carboxyl group
• Side chain of lysine has an amino group that the polymer can bind to
• Available in 2 forms: NH2, NH3+

Question 15

Lysine group

Answer 15

• Use lysine with a polymer with an activated carboxyl group
• Side chain of lysine has an amino group that the polymer can bind to
• Available in 2 forms: NH2, NH3+

Question 16

pH when conjugating lysine

Answer 16

• Must be used at a pH above pKa (>9.3-9.5) so that it is in the unprotonated form
• The unprotonated (NH2) form is suitable for conjugation
• Participates in a nucleophilic attack reaction, lone pair reacts with COOH
• pH ~10, not too high to denature protein

Question 17

Monofunctional polymer

Answer 17

Polymer with only one reactive group per monomer unit

Question 18

Polyfunctional polymer

Answer 18

More than one reactive group in side chain. So it has multiple sites for reaction

Question 19

Which type of polymer is best for PPC

Answer 19

• Monofunctional
• Polyfunctional can end up cross-linking with several proteins and form a very heterogeneous mixture

Question 20

Which type of polymer is best for PDC

Answer 20

• Polyfunctional

Question 21

Why multifunctional polymers are not used in PPC

Answer 21

• High variability
• Too large conjugates form cross linking with several proteins = very homogenous mixture
• Difficult characterisation

Question 22

What enzyme is ideal for protein conjugation

Answer 22

• PEG, in its methoxy form (mPEG)

Question 23

Why should PEG be in its methoxy form?

Answer 23

• Only one reactive group (OH) - monofunctional
• Water soluble
• Soluble in some organic solvents
• Non-toxic
• Broadly non-immunogenic (although research is now indicating some immunogenecity)

Question 24

Which enzyme is most stable

Answer 24

1. mPEG2-trypsin
   - It is PEGylated with branched PEG
2. mPEG-trypsin
3. Native trypsin
   - Not stable
   - Very fast degdradation

Question 25

Example of PEGylated protein for cancer

Answer 25

Pegaspargase (Oncospar)
- Acute lymphoblastic leukemia
- Increases half life of L-Asparginase in body

Question 26

3 catgeories of proteins used for therapeutic applications

Answer 26

• Enzymes
• Singalling proteins
• Antibodies

Question 27

What is the advantage of using branched PEG compared to linear PEG?

Answer 27

More protection of the protein from enzyme degradation

Question 28

Mention one point in the protein where you can have polymer-conjugation

Answer 28

Amino terminus, carboxylic acid terminus

Question 29

What is pKa

Answer 29

Shows the strength of an acid