ADME of Recombinant Proteins Flashcards
Why do protein drugs generally have poor oral systemic absorption?
Poor protein stability due to pH and digestive enzymes
Poor permeability
Innate cellular system laying ambush ready to attack
List some of the challenges protein drugs require to go through resulting in its poor permeability
Mucus layer lining the entire GIT
Intestinal epithelium has overall negative charge
Tight junction between epithelial cells make it tougher for absorption
How are protein molecules absorbed SC? Describe their mechanism
Diffusion: movement of single particles from high to low concentration
Convection: collective bulk movement of large mass of particles into a fluid
What is the relationship between absorption and MW size of particles?
Diffusion is inversely related to MW size of particles
Convection has no limit unless particles are excessively big
How do larger proteins get absorbed into the body?
Slow movement across the capillary membrane
Proceeds into the lymphatic system to be drained into lymph nodes and larger vessels
Enters circulatory system
How do smaller proteins get absorbed into the body?
Both circulatory and lymphatic system at the same time
What are some factors to consider about rate of absorption?
Perfusion: varies with site of injection or patient’s poor peripheral perfusion
Immune cells in hypodermis
- can cause protein degradation
Basement membrane of lymph vessels
- can be porous
Define the rate limiting factors of absorption
Interstitial fluid transport rate
Lymphatic transport rate
What is interstitial fluid transport rate dependent on?
Charge
Hydrostatic pressure
Oncotic pressure
Presence of fibrous tissue
Anatomical issues influencing drug transport
What influences lymphatic transport rate?
Depends on patient’s lymphatic system and whether he has cardiac problems
Define the two way model
Movement of protein from circulation into interstitial fluid and then tissues
Define what the two pore model means
The two pore model is used to describe trans vascular movement of protein drugs of various size.
Movement is typically reversible where proteins move out of into tissue into interstitial fluid; drained into lymphatic flow and recycled back to systemic circulation.
What are the key characteristics of the two pore model?
Endosomal space: Porous tissue microvascular endothelium
Big pore and small pore represent how fluid can pass through both pores and recirculate from plasma space to interstitial space.
This is done by diffusion and fluid phase convection
Does metabolism occur for recombinant proteins? Why?
No.
Proteolysis occurs with the help of proteolysis enzymes
Where do proteolysis occur?
Interstitial fluid in tissue and organs
Cell surfaces
Intracellularly
How is neonatal FcRn transported to the baby?
Transport mother’s IgG through placenta to fetus and mother’s milk
What are the key roles neonatal FcRn plays?
IgG homeostasis mediating antibody recycling
Recycling of serum albumin
How does FcRn increase half life of IgG and albumin?
IgG and albumin dissolved in blood and taken up by endothelial cells by pinocytosis forming early endosomes
Acidic endosomes containing internalized FcRn fuse with endosomes containing IgG and albumin; forming a complex
Complex formed is recycled to cell surface to undergo exocytosis of IgG and albumin
Neutral pH of blood then disfavours FcRn binding to IgG and hence, allow IgG and albumin to dissociate from complex into blood
How does FcRn allow transport of IgG and albumin?
At apical site of mucosal epithelial cells, acidic pH allow binding of FcRn to ligands
Binding between FcRn and ligand occur within acidified endosomes and is further processed to complexes
Endosomes then fuse into basolateral site of epithelial cells and expose complex to neutral pH in interstitium, thus releasing IgG and and albumin
How can elimination of recombinant proteins be done?
Proteolytic degradation
Renal filtration
What are the factors affecting renal excretion?
Cut off MW
Charge: negative charged basement membrane
Shape rigidity
Tubular reabsorption:
Negatively charged hence those positively charged are reabsorbed
What are the 3 strategies to improve PK profile of recombinant proteins?
Glycosylation
PEGylation
Increasing size by fusing proteins
Define glycosylation
Addition of glycans to specific amino acids in proteins
How does glycosylation help with improving PK profile of recombinant proteins?
Enhances receptor binding
Increase half life of proteins
What are some example of traditional glycosylation and glycoengineering?
Traditional: N-linked glycosylation
Glycoengineering:
- Removal of fucose
- Addition of high mannose glycans
Describe what PEG is
Addition of amphiphilic, chemically inert polymers made of repeating units of ethylene oxide
How does PEG help to increase half lives of recombinant proteins?
Increase size of conjugated proteins help retard the glomerular filtration
Decreases elimination by proteolysis by forming a protective layer
Decreases elimination by action of antibodies and activated immune cells also by forming a protective layer
How is an increase in protein size done by fusion?
Done by fusing proteins with a Fc domain of antibody or albumin
How does albumin help to increase protein size?
Albumin naturally has 3 domains with each domain containing binding pockets
Altering the Fc domain through fusion with albumin helps by allowing the protein to attract binding of smaller molecules and making it bigger
This compromises FcRn binding and FcRn mediated recycling
