ADME Flashcards
2 pathways proteins are degraded in mammal cells
Lysosomal degradation
Proteasome degradation
protein degradation depends on
§ Structural protein = long (no need high turnover)
§ Regulatory protein = short (after respond to environment change, not needed)
protein degradation impt
§ Proper regulation of cell signalling pathways (maintain normal protein turnover)
§ To remove misfolded (not native conformation) and damaged (wear and tear) proteins that can lead to abnormal cellular activities
- Prevent no activity/ abnormal activity: DISEASED STATE in body
HIF-1 a
Transcription factor
□ For oxygen homeostasis during hypoxic conditions
□ Induce expression of genes involved in:
- ANGIOGENESIS
- Cell migration
- Glycolytic pathway
Gene VHL:
□ Code protein pVHL
□ Substrate-recognizing component of multimeric E3 ligase
○ Ubiquitin-proteasome system
○HIF1a (become substrate) for ubiquitination (degrade)
normal HIF-1a — normoxic condition
- HIF-1a maintained at low lvls (undetectable)
- Half-life 5-8min
- Hydroxylated (Pro402, Pro564)
- By oxygen-dependent propylhydroxylase
○ Targeted for ubiquitination - Ubiquited HIF1a degraded by 26S proteasome
normal HIF-1a — hypoxic condition
- HIF1a used to incr expression of target genes
○ Incr (MMP) matrix metalloproteinase
○ Incr vascular endothelial GF (VEGF)
- Incr invasion, metastasis, angiogenesis
Von Hippel-Lindau (VHL) disease pathophysiology
- Hereditary disease, autosomal dominant mutation in allele of gene VHL
1) Mutated pVHL lead to failure to degrade HIF1a
2) Accumulated
3) Incr HIF1a transcriptional activity
4) incr expression of target genes (MMP, VEGF)
Von Hippel-Lindau (VHL) disease effects
- predispose for tumour types
(pheochromocytomas, hemangioblastomas CNS, clear-cell renal carcinoma, retinal capillary angiomas) – high vascularisation
Cells act like they are at hypoxic condition, so incr blood vessel formation everywhere
- formation of tumours everywhere
Lysosomal degradation OCCURS AT
- Occur in lysosome
- Mem-bound organelles
- Acidic interior pH4.5
- 10-20% of proteins
lysosomal degradation _____ process
effect on what proteins?
- Non-specific process
- As long as in lysosome
- Mem associated proteins, alien proteins INTERNALISED (endocytosis – phagocytosis, pinocytosis, receptor-mediated endocytosis)
*Not intracellular proteins
Phagocytosis
a. Cell eating
b. Large solid particles
i. Cell debris, dead cells, protein aggregates, pathogenic microorganism (bact), dust, particulate non-living matter
c. Phagocytosed into PHAGOSOMES
Pinocytosis
a. Cell drinking of extracellular fluid
b. Fluid: solute dissolved, ingested by budding of small vesicles from cell mem
c. non-specific
Receptor-mediated endocytosis
a. Molecules taken up are LIGANDS recognised by receptors expressed on cell mem of cells
b. Specific molecules:
i. hormone, metabolites, proteins, some virus
c. Internalised into coated vesicles
d. Fuse with endosomes
e. Sent to lysosomes for degradation// RECYCLE to plasma mem
Proteasomal degradation effect is on ____
- 80-90% of intracellular proteins
- Degrade by 26S proteasome
- Specific process
- Ubiquitinated proteins
Some non-ubiquitinated
- Ubiquitinated proteins
proteasome size
- Large cylindrical particle
- At least 33 subunits
- ~2.5MDa
- Different variants exist in cells
BUT 26S proteasome is found in ALL CELLS
26s proteasome function
- Specific degradation of regulatory proteins
- Remove damaged proteins
26s proteasome structure
○ 20S core particle
- 4 heptameric rings
- 2 outer (a subunit)
- 2 inner (b subunit)
□ Inner ring house central cavity with proteolytic active sites
○ Cap: 19s regulatory particle (both ends)
20S core particle function
- Degradation chamber reached through channel running along LONG AXIS
1) NARROW Entrance: folded protein, partially unfolds to translocate into 20s core protein
2) Protein unfolds
a. Stretch along channel
b. Hydrolysed to short pepetides (3-25aa)
3) Released from opp end of channel
19s regulatory particle (lid, base) function
a. Has ATPase subunits
b. Substrate recognition
c. Unfolding
d.Translocation into the core
steps of degradation inside 26s proteasome
- Gate of 20s core particle closed
- Proteolysis is selective
- Polyubiquitin chain formed on protein (proteasome recognises chain)
1) Ubiquitin tag CLEAVED by deubiquitinating enzymes (DUBs)
a. Into monomers
2) Monomers escape from proteasomes
a. Recycled to label other protein substrates
3) Proteasome engage protein substrate
a. Pp unfolds, translocate into core for degradation
b. Narrow core – regulate entry
4) 19s cap hydrolyse ATP to provide energy, drive:
a. removal of Ub
b. Protein unfolding
c. Transfer of unfolded protein to inner chamber of proteasome
i.Released as short peptide fragment (other end 19s)
Delivery to proteasome
Substrate bring to proteasome
a) Interact with 19s regulatory particle subunit
b) By adaptor protein
- Bind both proteasome & polyubiquitination chain
- Deliver for degradation
c) Some degraded w/o ubiquinated
Ubiquitin (Ub)
○ 76 residue pp
-Contain 7 lysine (6,11,27,29,33,48,63)
○ Attaches to substrate protein through
-Isopeptide bond
- c-terminal Gly of ub + amino grp Lys in substrate
○ Minimal 4 Ub monomers linked through Lys 48 to be functional
Monoubiquitination
○ Attach 1 Ub protein
○ Predominant regulatory modification (post-translational mod)
monoubi of histones & TF
□ Incr transcription
monoUb of surface cell receptors
□ Signal for endocytosis
□ Degradation in lysosome
Traditional chemical based drugs
<1000Da
Chemically synthesized, purified to homogeneity
Chemical modification lead to drastic changes in activity. New drugs for new uses
MAY have off target effects
Biologics/ biopharm drugs characteristics
* Recombinant proteins * MAB (monoclonal Ab) * Nucleic acid-based products Also have clinical devices, diagnostics
Large, kDa
Derived from living source
(human, animal tissue, cells, microorg)
Not easily characterized
Not easily refined
Called by same name despite modification in 1/ more aa residues
More predictable, less SE
Targeted therapy/ personalised (since disease are affected by the proteins it is made from)
Other than immunogenicity
Challenges of biopharmaceuticals:
immunogenicity
degradation
distribution of macromolecule –> tissue
Immunogenicity
a. Contaminants of whole cells, CHO cell, bact cells
b. Poor purification
c. Excipients (solvent)
Lead to rash, anaphylaxis etc
Degradation
a. Protease degradation in biological fluids (ECF) upon admin
i. When MW > 200 kDa: phagocytosis involved
b. Degraded intracellularly
i. Lysosomal
ii. Intracellular proteases
iii. Ubiquitin-proteasomal degradation
Distribution of MACROMOLE —> tissue
limited by permeability (Porosity of vasculatures)
MAB KDa size
2 light chain (25kDa each), 2 heavy chain (55 kDa each) = 160 kDa
Compare to insulin 5-6kDa
IL, GF, cytokines 5-10kDa
- Protein usually have poor systematic absorption
PO: F =2%
○ Poor protein stability
-Acid pH of gastric fluids (2-5.5)
- Digestive enzymes present (pepsin, chymotrypsin, trypsin, bile salts)
○ Poor permeability
-Mucus layer lining GIT (viscous layer, affect diffusion rate)
-Intestinal epithelium carry -ve charge
- Tight junctions restrict absorb of hydrophilic peptides/ proteins
absorption from SC/ IM inj STEPS:
Absorption varies in diff animal species
1) proteins delivered to hypodermis (sc tissue)
- Has adipose tissues, network of ECM (collagen, tensile strength acts as barrier),
- nerves, blood cap, lymphatic cap
2) Proteins move through ECM via diffusion & convection
- Reach blood, lymphatic capillaries
DIFFUSION
Movement of single particles from high to low conc
Inversely related to MW/ size of proteins
(poorer for large prot)
convection
Collective bulk movement of large mass of particles in fluid
(influenced by oncotic/ hydrostatic P art end)
Not limited by MW
* unless protein molecules are enormously large. Trapped in ECM
* Steric hindrance
* Charge interactions (both -ve, repel move faster
Larger (>16-20kDa) movement is
○ Movement across cap mem is SLOW
○ Absorption –> lymphatic system
○ Drain into lymph nodes –> vessels –> circ system
- More permeable:
□ No basement mem. Have clefts (seep through)
Smaller proteins (<16-20kDa)
○ Absorption can be by BOTH circ & lymphatic system
○ Perfusion (blood flow through tissue) influences absorption
affected by rate limiting factors/ innate immune
Immune cells (innate system)
located in hypodermis
Degrade proteins injected
Rate limiting factors that change absorption rates
1) Interstitial fluid transport rate
§ Depends if diffusion or convection
□ ECM, fibrous layer
□ Charges
□ Capillary network (hydro > oncotic, push fluid out to interstitial fluid)
2) Lymphatic transport rate
§ Blockage/ congestive HF affects rate
distribution
Once protein drug reach systemic circ –> tissue circ
- Protein binding in circ
○ Improve circ half life of drug
§ Escape degradation
○ Efficient delivery to target tissue - Tissue distribution of protein drugs
○ Move proteins from circ –> interstitial fluid of tissues –> tissues
§ 2 way (in/ out of vasculature) - Movement of proteins across vascular barrier
○ Into interstitial fluid of tissue occurs by movement ACROSS/ BETWEEN endothelial cells - Passive movement of protein drugs
○ Convection// diffusion pathway of trans-capillary transportation of proteins
○ No energy needed
Between endothelial cells vs lymphatic
Between endothelial cells
□ Less leaky < lymphatic
□ > leaky mucosal lining
Lymphatic > endo > mucosa
2 pore model – characterise tissue lvl protein disposition
describe trans vascular/ cellular movement of protein drugs
MOVE out of tissue –> interstitial fluid –> drained to lymphatic flow
MOVE in plasma –> endosomal space –> interstitial space
2 pore model factors
○ Endosomal space: porous tissue microvascular endothelium (line vascular walls)
○ 2 type of pores in endosomal layer = SMALL, LARGE PORES
○ Fluid passes through BOTH small, large pores. RE-CIRCULATE
○ Passive movement of proteins molecules from vascular (plasma) space To interstitial space
passive movement
passive movement from vascular (plasma) –> To interstitial space
- Use both small, large pores via diffusion (PS) or fluid phase convection
□ Extent of movement and distribution related to MW/ SIZE
□ Vs absorption: large = convection
- Larger proteins: limited distribution, slower movement IN/OUT
- Smaller proteins faster (go through small & large pores)
Mathematical equation ultilise n.o. of derived parameters to predict PK profile of proteins of diff size
METABOLISM
- No metabolism of protein drugs by LIVER
- Poor substrate of CYP enzymes
- Metabolism of protein drugs via PROTEOLYSIS (proteolytic enzymes)
○ Activated protease
○Peptide bond cleaved
metabolism occurs in
- Interstitial fluid (ECF) in tissue/ organs
○ Protease released by activated immune cells
○ Immune cells lying in ambush in ECF (phagocytosis + proteolysis) - Cell surfaces (proteases on surface)
- Intracellularly after protein drug taken up into cells
○ Lysosomal degradation (from endocytosis)
Neonatal Fc receptor (FcRn) - mediated recycling of IgG & serum albumin
FcRn is IgG binding Fc receptor
- FcRn: transport maternal IgG from mother –> neonatal offspring
○ Across placenta to foetus
○ Mother’s milk to intestine of newborn
○ PO protein drugs development
IgG Fc receptor
6 binding Fc receptors on effector cells: macrophage, NK cells, Neutrophils
for Fc domain of IgG/ albumin (2 domains) to bind to
When IgG binds to FcRn expressed on effector cells
if pH at cell surface is appropriate
1) trigger activation of effector cells
2) regulating IgG turnover (incr t1/2 of IgG)
Lines the intestinal wall for: Transcytosis, uptake of Ab
FcRn found:
○ on effector cells (activation when pH environment suitable)
○ effector cell FcRn has Binding sites to IgG and serum albumin (diff binding sites)
- mediate Ab recycling (intracellular traffic Ab, escape degradation in lysosomes)
- Recycle serum albumin (intracellular traffic mechanism as Ab)
Cellular recycling of IgG and albumin
1) Pinocytosis, albumin (plasma protein) taken up since it is dissolved in bloodstream (pH7.4) –> endosomes
2) Early endosomes with soluble Ab and albumin + fuse + endosome w/ FcRn
- In acidic environment pH 5-6 catalyse reactions
- FcRn will bind to Fc domain of Ig = FcRn-Ig G complexes/ albumin complex
3) FcRn-Ig G complexes // FcRn-albumin complex –> recycled to cell surface by exocytosis
4) Neutral pH 7.4: Dissociation of complexes IgG and albumin release to blood
a) Release back to plasma or back onto cell surface = RECYCLE
□ Long half life of protein plasmas
□ Since it has many roles
□ By recycle, extend half life
b) FcRn recycled reexpressed on cell surface
5) Proteins not bound to Fc sorted to LYSOSOMES for degradation
Transcytosis of IgG and albumin –> transport
○ Epithelial cells usually hard to pass § Mucosal lining Tight junctions
1) Apical side (mucosal epithelial cell)
a) Acidic pH allow bind of FcRn (surface) – ligands (IgG, albumin)
b) Binding b. FcRn-ligand also within acidified endosomes (case A)
2) Further processing of FcRn-IgG/ FcRn-albumin complex
3) Endosomes fuse with basolateral side of epithelial (TRANSCYTOSIS)
a) Lead to exposure of complex to neutral pH in interstitium
b) Release IgG and albumin
c) FcRn recycled, reexpressed on cell surface
IMPROVE PK profile of protein therapeutics:
1) glycoslyation of proteins
2) PEGylation of proteins
3) incr MW fusion proteins
1) glycoslyation of proteins
a. Add glycans (carbs) to specific aa in prot
i. Diff types of glycan – straight, branched chain
ii. Bind to diff aa
b. Pattern and amt of glycosylation affects activity
i. Enhanced receptor binding
ii. Incr half-life of proteins eg: N-linked glycosylation
□ large size > 50kDa, limits glomerular filtration (modify binding to glycoprotein receptors - STERIC HINDRANCE)
□ poorer substrate to proteolysis
disadv of glycosylation
i. Decr affinity to Fc receptor if fucosylated
□ N-linked glycans at Asn297 Fc domain of IgG
ii. Decr half-life
□ If high mannose glycans rapidly eliminated
□ Bind to mannose & asialoglycoprotein receptors rapid removal of Ab
□ Pattern recognition receptors, recognises pathogens
2) PEGylation of proteins
a. Amphiphilic, chemically inert polymers made up of repeating units of ethylene oxide
b. PEG conjugation:
i. Reactive functional grp of activated PEG/ mPEG attached to sites
ii. Usually amino grp: lysine, sulfhydryl -SH grp in cysteine, nucleophilic grp on aa
□ Ensure conjugation doesn’t affect receptor binding affinity of drug
types of peglyation
2 types of configurations:
i. LINEAR
ii. BRANCHED (effective, but for large prot not significant)
PEG/mPEG polymers conjugated to protein drugs = diff extended half-life
2 types:
i. PEG w/ free -OH (hydroxyl) at both ends
ii. Methoxylated PEG (mPEG) w/ (-OH) at 1/ 2 ends methoxylated
Incr half-life by peglyation
1) Incr size of conjugated proteins
□ Glomerular filtration not for >50kDa
2) Decr elimination by proteolysis
□ PEG protective layer on surface. Decr chance for PROTEOLYTIC enzymes
- Interact and break down proteins
3) Decr elimination by action of Ab and activated immune cells
□ PEG molecules form PROTECTIVE layer on surface of protein mole
- Decr recognition by Ab/ activated immune cells (macro, dendritic, NK,)
□ REDUCE IMMUNOGENICITY
- Maybe not for vaccine, used to irritate immune system
3) Incr size (MW) by fusion proteins
a. Larger prot slower CL (longer half-life)
b. Fusion proteins with Fc domain of Ab/ albumin fused to therapeutic protein
i. Ultilise FcRn-mediated recycling (incr half-life of therapeutic protein) = RECYCLE
- Enhance circulation half-life
disadv: Fc domain may trigger unwanted effector functions –> unwanted immune (inflamm) response
Fc domain of Ab or albumin
fused to therapeutic protein (FcRn-mediated recycling)
□ Albumin is plasma prot (has 3 domains – which has binding pockets for small endo// exogenous sub – drugs, FA, metal ions)
-Albumin-FcRn binding involves 2 albumin domains
- May attract binding of small moles/ FA in interstitial fluid & plasma
- Compromise FcRn binding and FcRn-mediated recycling
Current recombinant DNA (rDNA) technology overcomes ______
1) Overcomes limitations regarding source availability
a) Natural sources often rare, expensive
b) Yield can be low due to limited amts present in natural sources
2) Allow production of safer biopharmacauticals
a) Eliminate transmission of blood-borne pathogens (HIV, Hep B virus)
b) If pdt is directly isolated from infected sources
3) Provide alternative way to obtain protein-based pdts other than
a) Direct extraction from inappropriate source material
i) Urine, placenta
4) Gene of interest can be synthetic = opportunity for scientist to design desirable mutations
a) Produce engineered protein-based biopharm pdts that possess advantages
b) Greater clinical efficacy, greater protein stability for longer half-life
c) Short/ longer circ half-life
- Cheaper, safer, abundant supply of protein-based biopharmaceuticals
Application of rDNA technology
usually upstream processing in manufacturing
§ Host cells (bact/ mammalian cell) successfully transfected w/ recombinant DNA
□ Each transfected cell is diff from each other
□ Differ in terms of:
- Number of copies of plasmids being transfected
◊ Higher copy of plasmid = higher amt of proteins expressed
in recombinant protein making selection of __
1 transfected cell that possesses the best cell growth properties and highest protein yield
used to develop a master cell line
master line for recombinant cells
1) Gene of interest + vector
2) Transfect cells for chromosomal integration
3) Cloning
a) Selection process: low lvl of MTX, absence of glycine, hypoxanthine, thymidine —> Cells not integrated with vector killed
b) Recovery and expansion = Cells presumed to express protein of interest are recovered
c) Amplification
i) Surviving cells exposed to high conc of MTX –> incr selection P
◊ Force CHO cells genomic rearrangement and amplification of locus of DNA integration
◊ Incr copy number of protein of interest
d) Screening
i) Serial dilution of each cell clone, 1 cell in multi-well plate
ii) Grow into colony
◊ Screened for recombinant protein productivity
◊ Isolated if high yield
e) Expansion & evaluation
i) Chosen clones expanded through subculturing in lab-scale bioreactors under conditions of large scale production facilities
f) Cell banking
i) MASTER CELL LINE for downstream processes of recombinant protein making
4) Select 1 transfected cell — develop master cell line
a) Best cell growth properties
b) Highest protein yield
TYPES OF HOST CELLS USED:
e coli (bact)
CHO (mammalian cells)
Yeast - Saccharomyces cerevisiae
Transgenic - Plants, animals
Downstream processing in recombinant protein making
□ Centrifugation/ filtration
- Cell harvesting
□ Ppt and/or lq-lq extraction
□ Chromatography
Downstream processing is needed to
Ensure impurities and contaminants excluded from final biopharm:
1. Protein isolation
2. Concentration
3. purification
4. Viral inactivation steps
Biosimilars
○ Biologic that is almost an identical alternative version of original biologic (innovator/ reference) manufactured by a different company
§ Follow-on biologic that uses innovator/ reference **crucial for regulatory authority approval
§ Each biological pdt displays variability even between diff batches of same pdt
□ Variability of biological expression system
□ Manufacturing process
- Downstream, upstream processing influence nature of final pdt
Final characteristics of biologic influenced by:
-Manufacturing process
□ Type of host cells
□ Development of genetically modified cell for production
□ Cell culture system
□ Production process
□ Purification process
□ Formulation of recombinant protein
chemical drugs
production of generic drugs not an issue
analytical criteria based on chemical compositions
for biologics of relatively low MW
erythropoietin, isulin, human growth hormone
- compared to MABS, development is more straightforward, more biosimilars approved
- short half-life, less PK effects
biosimilars of MABS larger proteins with post-translational modifications
CANNOT BE 100% identical to innovator
Dependent on cell production system (host cell, culture, conditions)
* Pattern of glycosylation
* Amt of glycosylation
○ Affect half-life
○ Affect how drug works
Glycosylation at Fc domain of Ab
§ Pattern of glycosylation influence structure and function of Fc domain of therapeutic Ab
§ amount of glycosylation
what affects glycosylation
dependent on cell production system
- type of host cells used for culture
- cell culture conditions
eg biosimilar difficulty: Glycosylation at Fc domain of Ab
□ Control: IVIG-Fc (donor)
-Glycan profile of polyclonal IgG from pooled plasma of human donors
-Glycans from Fc fragment of IgG labeled with fluorescent probes
◊ MULTIPLE peaks = highly heterogenous pattern of glycosylation among polyclonal IgG
□ MAB (nivolumab, bevacizumab, mogamulizumab)
- Less heterogenous, less peaks corresponds
- Pattern of glycosylation differs b. CHO & human cells
◊ Some mutant CHO cells: don’t allow for fucosylation
◊ Sialylated glycans (sialic acid) almost absent in MABS produced in CHO cell (they lack enzyme)
Approval of biosimilars
○ SG authority similar to US FDA = stricter regulations
§ The methods/ assays used to establish comparability of biosimilars to reference pdt
§ Sufficiently SPECIFIC (cross reactivity), SENSITIVE (qty needed to test) to detect difference b. 2 pdts
1) Extensive in vitro studies (demonstrate similarity to reference pdt
2) Non-clinal (animal) and clinical studies demonstrate
- comparable PK, clinical efficacy, safety, immunogenicity
rapid insulin
poor association, fast onset
intermediate insulin
co-crystallised, cloudy susp
long acting
binds to albumin/ crystal formed in sc, prolonged dissociation
