Bench to Bedside

Question 1

What are the advantages of large biomacromolecular drugs?

Answer 1

Large surface area for binding and high specificity.

Question 2

What are the drawbacks of large biomacromolecular drugs?

Answer 2

Not accessible by chemical synthesis, not membrane permeable and antigenic

Question 3

What are the main steps in protein production by recombinant DNA technology?

Answer 3

• Identification, amplification and isolation of the target gene
• Integration of target gene into a cloning vector e.g. plasmid
• Introduction of the vector into a host cell
• Growth of the cell, in vitro
• Identification of cells containing the target protein.
• Isolation and purification of the target protein

Question 4

Possible host organisms?

Answer 4

Microorganisms, yeast, animal cell lines, insect cells, plants

Question 5

What are the steps in the creation of a functional protien?

Answer 5

1. Translation of mRNA sequence into amino acid sequence on the ribosome.
2. Completed polypeptide must: fold correctly into 3D conformation, bind any necessary cofactors, assemble with its partner protein chains (if any).
3. The above are non-covalent bond formations.
4. Post translational modifications (PTM) are covalent modifications of selected amino acids.
5. Glycosylation and phosphorylation are the most common.

Question 6

Types of PTMs

Answer 6

Glycosylation - increases solubility, alters biological half life and activity.
Phosphorylation - regulates the activity of many polypeptide hormones.

Question 7

What are post-translational modifications (PTMs)?

Answer 7

A covalent process where proteins are changed after protein biosynthesis. The host cell MUST be a eukaryote as they possess the machinery to do post-translational modifications.

Question 8

What is N-linked glycosylation?

Answer 8

The addition of a oligosaccharide (a big sugar molecule) to a nitrogen atom on a protein surface (Asparagine-XXX-Serine or Threonine OR Asparagine-XXX-Ser where XXX is any amino acid except Proline).

Question 9

Why is N-linked glycosylation helpful?

Answer 9

It is thought to aid in protein folding and transport through the cell.

Question 10

What is O-linked glycosylation?

Answer 10

The addition of an oligosaccharide to an oxygen on the protein surface (The oxygen is attached a Serine or Threonine residue).

Question 11

What is a glycoform?

Answer 11

These are different proteins with variations in the patterns of glycosylation.

Question 12

What can changes in glycosylation patterns give rise to?

Answer 12

Differences in stability, solubility, serum half-life, biological activity and immunogenicity.

Question 13

Why does recombinant protein production need to be done in eukaryotes?

Answer 13

Prokaryotic cells do not have the necessary intracellular machinery to perform glycosylation.

Question 14

Name some examples of cells that can be used to create recombinant proteins.

Answer 14

Chinese Hamster Ovary cells (CHO cells) or Human Embryonic Kidney cells (HEK cells)

Question 15

Describe how hybridoma production works for mAb production.

Answer 15

1. Mice are immunised with specific antigens.
2. Plasma B-cells producing antibodies are isolated.
3. Plasma B-cells are mixed with immortal myelomas and fused with PEG.
4. The cells are then grown in a medium which only allows fused cells to grow.
5. Fused cells showing high antibody production are expanded and grown in large numbers.

Question 16

What is a hybridoma?

Answer 16

A cell combination of a plasma B-cell and an immortal myeloma cancer cell fused with PEG.

Question 17

How are recombinant proteins made, isolated and purified?

Answer 17

1. Inoculate a liquid bacterial culture broth with bacteria harbouring recombinant gene required.
2. Grow eukaryotic cells that carry a plasmid coding for the protein of interest.
3. If protein needs to be recovered from a producing organism, chemical treatment, sound energy agitation, or mechanical agitation can be used.
4. If the protein is found in the cell, chromatographic purification can be used.

Question 18

What methods of chromatographic purification can be used to obtain recombinant proteins?

Answer 18

Affinity chromatography, ion exchange chromatography, gel filtration chromatography.

Question 19

What is a biosimilar?

Answer 19

A biologic drug that has the same sequence but may have different properties (i.e. PTMs, so it may fold differently).

Question 20

What is a liposome?

Answer 20

A vesicle formed of a bilayer of phospholipids, which are amphipathic (both hydrophilic and hydrophobic). The bilayer consists of phospholipids with polar (hydrophilic) heads and 2 non-polar tails (hydrophobic).

Question 21

Why is cholesterol added to liposomes?

Answer 21

It reduces bilayer permeability, and therefore increases drug retention inside the liposome.

Question 22

What does PEGylation do to a liposome?

Answer 22

It helps to disguise the liposome from blood opsonins which work to remove foreign material from the bloodstream, as well as this the PEG helps to stabilise the liposome by acting as a non-ionic head group.

Question 23

Why does PEG stop opsonins from recognising and removing liposomes?

Answer 23

PEG is inert and non-immunogenic so opsonins do not recognise it as foreign and a threat.

Question 24

Why is log P of drugs important in the design of liposomes?

Answer 24

The log P of a drug depends where in the liposome it will sit. Drugs with a log P of <1.7 will be incorporated into the aqueous compartment, and drugs with a log P of >5 will be retained within the lipid bilayer.

Question 25

Why is a drug with intermediate log P (~3) values bad?

Answer 25

Drugs with a log P of ~3 can be difficult to incorporate as they can partition between the bilayer and the aqueous compartment and can become lost.

Question 26

Why is drug loading important in the design of liposomes?

Answer 26

To stop drugs with poor pharmacokinetic properties (i.e. log P) from partitioning between layers in the liposome.

Question 27

How can remote drug loading be achieved and how does it work?

Answer 27

The liposome can be preformed with a gradient of e.g. pH or ionic strength, within the aqueous compartment which forces the drug to follow the gradient and then become trapped by a trapping agent, keeping it in a complex in the compartment.

Question 28

What trapping agents can be used in remote drug loading?

Answer 28

Ammonium sulfate, ammonium citrate or gels

Question 29

What are the clinical applications of liposomes in cancer treatment (where PEGylation isn’t used)?

Answer 29

1. Injected IV liposomes interact with blood opsonins
2. Opsonised liposomes enter the mononuclear phagocytic system (MPS).
3. This results in a build-up of drug-containing liposomes at these sites, creating an ‘MPS depot.
4. These depots facilitate the slow release of drug into circulation, mimicking slow transfusion.

Question 30

What are the clinical applications of liposomes in cancer treatment (where PEGylation is used)?

Answer 30

1. Where MPS deposition is not beneficial, opsonisation must be avoided.
2. Stability, clearance rates and tissue distribution of liposomes is now important.
3. Surface modification (e.g. PEGylation) creates hydroPHILIC surfaces which repel opsonins and maintain liposomes in circulation.
4. This enhances the opportunities for liposomes to accumulate at pathogenic sites by Enhanced Permeability and Retention (EPR).

Question 31

How does anti-cancer agent Myocet (Doxorubicin) work in the body

Answer 31

This drug is placed inside liposomes WITHOUT a PEGylated surface which causes it to form MPS depots and be released slowly into circulation. This is known as passive targeting.

Question 32

How does anti-cancer agent Caelyx (Doxorubicin) work in the body?

Answer 32

This drug is placed inside a liposome WITH a PEGylated surface, making it hydrophilic, so therefore undetectable by opsonins. The liposome then accumulates in tumours via the EPR effect.

Question 33

How does anti-cancer agent Abraxane (Paclitaxel) work in the body?

Answer 33

This drug is bound to human protein, albumin. As albumin is an endogenous component of the blood, use in drug delivery circumvents many issues associated with liposomes. There is also no need for PEGylation.

Question 34

How do solid lipid nanoparticles work inside the body?

Answer 34

A self-emulsifying drug delivery system (SEDDS) is used within these nanoparticles which can solubilise poorly soluble drugs and also it work to protect drugs, proteins and genetic materials from degradation by harsh biological conditions.

Question 35

What is the determining factor for renal clearance?

Answer 35

Size - this is very important in renal clearance, as substances between 6-8nm are able to be cleared by glomerulus.

Question 36

What happens to larger molecules which bypass renal clearance due to size?

Answer 36

These molecules will be targeted by opsonins and be taken up by the mononuclear phagocytic system and remain there for months/years.

Question 37

What happens to molecules which are too large for renal clearance and avoid the MPS?

Answer 37

These will be hepatically cleared and excreted through bile and then faeces.

Question 38

What is the Enhanced Permeability and Retention (EPR) effect?

Answer 38

Within the tumour microvasculature, lymphatic drainage is compromised which causes enhanced retention and because the tumour cells and the vascular system combine for increased perfusion during angiogenesis, there is also enhanced permeability.

Question 39

What are the 5 mechanisms of absorption across mucosae?

Answer 39

1. Transcellular - passive diffusion
2. Paracellular - diffuse through tight junctions
3. Transceullular - via active transport
4. Lipid mediated absorption via micelles/bile salts
5. Particulate absorption via GALT - gut-associated lymphatic transport