ADME of Macromolecules Flashcards
Describe the differences b/w chemical-based drugs and biologics/biopharmaceutical products
Chemical-based
- MW <1000Da
- Chemically synthesized (or biosynthesized)
- Purified to homogeneity
- Chemical modifications can change activity drastically
- May have more off-target effects/side effects
Biologics
- MW in kDa
- Derived from living sources (e.g., proteins => require protein purification process)
- Unable to extract 100%
- Modifications by differing AA residue
- Behave more predictably, lesser side effects
What are the benefits of protein-based pharmaceutical products?
- Proteins control diverse cellular and physiological processes (metabolism, immune response, memory and learning)
- More naatural way of treating diseases
- Provide basis for targeted therapy/personalized medicine
Define biopharmaceuticals
Explain how clinical devices and diagnostics may make use of biopharmaceuticals
Biopharmaceuticals - recombinant proteins, monoclonal antibodies, nucleic acid-based products etc. (MACROmolecules)
Clinical devices and diagnostics may incorporate biopharmaceuticals (e.g., enzymes in glucose test strips, antibodies in immunoassays ELISA)
List the challenges of using biopharmaceuticals
- Immunogenicity
- Host cell contaminants (CHO cells etc.)
- Due to excipients (that may cause allergic reaction)
- Proteins are susceptible to denaturation and protease degradation (EXTRAcellular)
- Proteins >200kDa can be recognised by immune cells in the ECF and phagocytosized
- Proteases can also be released by activated immune cells
*Also note that protein subunits can aggregate tgt, may not exist as monomers hence they become large and easily recognized
- Proteins are susceptible to degradation by degradation systems (INTRAcellular)
- Lysosomal degradation (after endocytosis)
- Ubiquitin-proteasomal degradation
- Other activated intracellular proteases in the cytoplasm
*Intracellular half-life can only be determined by clinical trials
- Distribution of proteins (macromolecules) to tissues limited by permeability (porosity) of vasculatures
[ABSORPTION]
Proteins generally have ______ oral systemic absorption. Why?
Proteins generally have poor oral systemic absorption
- Poor protein stability
- e.g., acidic gastric fluid, digestive enzymes - Poor permeability
- e.g., through mucus layer lining the GIT (high viscosity, impeded speed of protein drug permeation)
- e.g., intestinal epithelium (-ve charge and tight junctions - restrict absorption of hydrophilic proteins) - Innate immune response
[ABSORPTION]
How does the innate immune response play a role in impeding protein drug absorption?
Mucosal epithelia lining serves as an external barrier + possess internal defenses (cellular + humoral)
- Mucosal epithelia have immune cells lying in ambush, ready to recognize and phagocytosize foreign particles (esp >200kDa), eventually degrade the proteins
[ABSORPTION]
How might proteins aggregate tgt, larger MW => more likely recognized
Partially unfolded proteins may have exposed hydrophobic surfaces, hence proteins may aggregate tgt via hydrophobic interactions
[ABSORPTION after SC administration]
Proteins are delivered to the subcutaneous tissues (aka hypodermis)
What does the hypodermis consist of?
Hypodermis:
- Adipose tissue
- Extracellular matrix: collagen (tensile strength)
- Nerves
- Blood capillaries
- Lymphatic capillaries
- Also take note of presence of immune cells that may target proteins and degrade them
[ABSORPTION after SC administration]
_____ is a major barrier that controls the rate of drug absorption into the blood capillaries
Protein drugs move through ____ via 2 transport mechanisms to reach blood or lymphatic capillaries
What are the 2 transport mechanisms?
Extracellular matrix (ECM)
2 transport mechanism to move through ECM:
1. Diffusion
2. Convection
[ABSORPTION after SC administration]
Describe diffusion (transport mechanism)
Also describe what might limit absorption via diffusion
Diffusion is the movement of single particles from high to low concentrations
It is inversely related to MW/size of proteins (smaller proteins move down conc. gradient via diffusion better)
Absorption by diffusion can be limited by:
- ECM: scarring, presence of lots of collagens and fibrous tissue
- Adipose tissue: thicker in fatter people
[ABSORPTION after SC administration]
Describe convection (transport mechanism)
Also describe what might limit absorption via convection
Convection is the collective bulk movement of large mass of particles in interstitial fluid (the flux is fluid-drive)
It is not limited by MW, though enormous proteins can get trapped in the ECM
Convection can be affected by:
- Hydrostatic pressure (higher at arterial ends, push proteins into interstitial)
- Oncotic pressure (higher at venous ends, pull interstitial fluid into blood capillaries via convection)
Absorption by convection can be limited by:
- Steric hindrance (-ve charge of ECM): negative proteins will repel the ECM and move into the capillaries faster VS positive proteins that will stay in the ECM
*Absorption by convection is LESS affected by collagen in ECM and fats in adipose tissue since it is driven by the motion of the bulk fluid
[ABSORPTION after SC administration]
Explain the absorption of larger proteins (>16-20kDa), how do they enter the circulatory system?
Larger proteins (>16-20kDa)
- Slow movement across capillary membrane
- Absorption occurs mostly via lymphatic system before draining into circulatory system
=> Protein in hypodermis can permeate the leaky endothelial membrane (as there are celfts that exist b/w the endothelial cells), and enter the lymphatic circulation
=> Lymphatic capillaries lack well-defined basement membrane, hence facilitating the permeation of the large molecules into the lymphatic circulation
*Take note that lymph nodes and lymphatics may contain lymphocytes that can attack foreign protein particles
[ABSORPTION after SC administration]
Explain the absorption of smaller proteins (<16-20kDa), how do they enter the circulatory system?
Smaller proteins (<16-20kDa)
- Absorption can occur via circulatory and lymphatic systems
=> Smaller proteins can permeate the tight endothelial membrane that lead into the blood circulation
[ABSORPTION after SC administration]
What are the considerations for absorption into the lymphatic or blood circulation?
- Perfusion of blood affects/influences capillary absorption (BLOOD CIRCULATION)
- Site of injection => tissues with high perfusion (e.g., liver, kidney)
- Patient’s peripheral perfusion (is it poor or good)
- Presence of fibrous tissue in ECM affects absorption via DIFFUSION (into LYMPHATIC or BLOOD CIRCULATION)
- Permeability of ECM from interstitial fluid in hypodermis into lymphatic or blood circulation
What are the MW of:
- Recombinant human insulin
- Recombinant cytokines
- Full-length monoclonal antibody
- Recombinant human insulin: 5-6kDa
- Recombinant cytokines: 10-12kDa
- Full-length monoclonal antibody: 160kDa (heavy chain 55kDa, light chain 25kDa)
[ABSORPTION after SC and IM administration]
Rate limiting factors can cause changes to absorption rate (into the blood circulation)
What are the two transport rate limiting factors?
- Interstitial fluid transport rate (affect rate of absorption into blood circulation)
- Lymphatic transport rate (affect rate of absorption into blood circulation)
[ABSORPTION after SC and IM administration]
How might interstitial fluid transport rate affect rate of absorption into blood circulation?
Interstitial fluid transport rate affects whether drug is transported via diffusion or convection
Interstitial fluid embeds the ECM
- Fibrous tissue: affects permeability of ECM (scarring, collagen etc. dcr interstitial transport rate)
- Negative charge: repels -ve proteins, incr interstitial transport rate
- Hydrostatic pressure: arterial vs venous end
[ABSORPTION after SC and IM administration]
How might lymphatic transport rate affect rate of absorption into blood circulation?
Lymphatic transport rate can be affected by:
- Lymphatic vessel blockage
- Low drainage into blood circulation
- Congestive heart failure: heart cannot pump well, ECF buildup, does not drain well into blood circulation
[DISTRIBUTION]
Distribution occurs once protein drug reaches systemic circulation
Describe tissue distribution of protein drugs (where does it go?)
Movement of protein from circulation into interstitial fluid of tissues, and then into tissues
*Note this movement is bidirectional
[DISTRIBUTION]
Movement of proteins across vascular barrier into interstitial fluid of tissues can occur either ________ or ___________
Either by movement across endothelial cells or between endothelial cells
*Distribution of proteins (macromolecules) across endothelial cells into tissues is limited by permeability (porosity) of vasculatures
[DISTRIBUTION]
Distribution of protein drug occurs via _______ movement, by which transport mechanism?
Passive movement
By both convection and diffusion pathway of trans-capillary transportation of proteins
*Size of protein no longer matters
[DISTRIBUTION]
How does plasma protein binding affect distribution of protein drugs?
- Improves circulation half-life
- Longer time for drug to dissociate from protein carrier
- Protection from proteases and degradation
- More efficient delivery to target tissues
- Higher hydrostatic pressure at arterial end can push proteins out of vasculature into interstitial of target tissues
[DISTRIBUTION]
The 2 pore model describes the trans vascular/cellular movement of protein drugs of various sizes
Describe the 2 pore model
2 pore model:
- Plasma space (blood), endosomal space (porous tissue microvascular endothelium), interstitial space (tissue)
- Two pores exist in the endosomal layer: small and large
- Fluids passing through the pores can recirculate
- Passive movement of protein molecules via diffusion AND convection through the pores
- Extent of movement and distribution is related to MW/size of protein as well as porosity of vasculatures => larger proteins more limited and slower distribution
[METABOLISM]
Do protein drugs undergo metabolism in the liver?
NO, they are not substrates of CYP enzymes
[METABOLISM]
How do protein drugs get metabolized?
Via proteolysis (protein degradation)
[METABOLISM]
Explain the extracellular proteolytic actions that might occur
In interstitial fluid/ECF present in tissues and organs:
- ECF contain proteases released by activated immune cells and other cell types, involved in proteolysis
- Immune cells lying in ambush in the ECF can take part in phagocytosis and proteolysis
[METABOLISM]
Explain the intracellular proteolytic actions that might occur
- (not intracellular) On cell surface - many proteases
- Lysosomal degradation
- Ubiquitin-proteasomal degradation
- Activated intracellular proteases (in the cytoplasm)
- These proteases in the cytoplasm exist in the pro/inactivated form, they only get activated under apoptotic conditions to degrade proteins and kill the cell
- In normal circumstances, only a small amount is in activated form, insufficient to degrade natural intracellular protein, but may degrade foreign protein drugs
[ELIMINATION]
Besides being eliminated via proteolytic degradation (extra and intracellularly), protein drugs can also be eliminated via _____
Renal excretion, mainly glomerular filtration
[ELIMINATION]
What are the factors affecting renal excretion of proteins
- Cut-off MW
- Proteins >50kDa are NOT renally eliminated
- Charge of protein
- Positively charged proteins have higher renal filtration due to negative charges on glomerular basement membrane
- Shape and rigidity of protein
- Can affect how well they undergo glomerular filtration
- Tubular reabsorption
- Tubular epithelium has negative charge, positively charged protein gets more reabsorbed
