Bench to Bedside Flashcards
What are biomolecular (biologic) drugs?
Large biological molecules that have some therapeutic effect (mw>2KD)
What are the advantages of using biologics?
Large SA good for binding
Exquisite specifity- stronger/specific binding then small molecule techniques
What are the disadvantages of using biologics?
Not accessible by chemical synthesis
Not membrane permeable
Antigenic
Give 3 examples of biologics:
Insulin
mAbs
Erthroporetin
How are mAbs manufactured and what are the disadvantages of this?
In a unique living cell line, similar but not identical copies can be made
Difficult to fully characterise
Higher potential for immunogenicity
Describe recombinant production of therapeutic protein and give another name for it:
Heterologous expression of recombinant proteins
Introduction of a gene or cDNA coding for a protein of interest into a suitable producer organism
Heterologous because the protein of interest doesn’t occur naturally in the cell
What are the main steps in protein production by recombinant DNA technology?
Identification- amplification and isolation of the target gene
Introduction- of the vector into a host cell
Growth of the cell in vitro
Identification of cells containing the target protein
Isolation of purification of the target protein
What is the advantages of protein production by recombinant DNA technology?
Cleaner and efficient
Give examples of some suitable host organisms:
Microorganisms (Ecoli)
Yeast (S Cerevisiae)
Animal cell lines
What are the advantages of Ecoli being a host?
Molecular biology well characterised (well understood)
High expression levels of heterologous proteins are possible
Quick and cheap
Possible to scale up to large fermentation culture
What are the disatvantages of Ecoli being a host?
Heterologous proteins are intracellular (need to lyse cell and purify)
Inability to undertake post translational modifications (PTM)
Presence of LPS on E coli surface (pyrogenic)
Formation of inclusion bodies (insoluble aggregates of partially soluble heterologous proteins)
What is post translational modification (PTM) and give a major example?
Any covalent modification of peptide sequence that occurs after the peptide chain has been synthesised
Glycosylation
Describe what glycosylation is important for and its effects?
Is an important part of eukaryotic protein production, especially extracellular and cell surface proteins (glycocalyx)
Some proteins are unaffected by removal of glycosylated groups
IgG has one glycosylation site that strongly affects biological activity
What affect can glycosylation have for some proteins?
Increase solubility
Alter biological half life and activity
What are the 2 types of glycosolation?
N- linked glycosylation (most common)
O-linked glycosylation
Describe the steps in N-linked glycosylation:
Starts with a transfer of 14-merogliosaccharide donor to protein as it emerges from rough ER
Ogliosaccharide is anchored in ER membrane by dolichol by high energy phosphate bond
Transfer reaction is catalysed by oligosaccharyl transferase
Which sites can transfer occur in N-linked glycosylation?
Asn (N)-xxx-Ser/Thr (T/S) OR
Asn-xxx-Ser
Where xxx is any a.a except Pro
What occurs in N- linked glycosylation once the glycosyl chain has been added to peptide?
Other enzymes can trim bind off in different patterns, common trimming:
-3 glucose and 1 mannose are later removed in the ER by glucosidases
-Leaves (Man)3(GIcNAc)2 protein
Further saccharides are added and removed according but a core motif is always retained
This is thought to aid protein folding and transport through the cell
Describe O-linked glycosylation:
Occurs post-translationally in ER/Golgi (common in mucins)
Ser/Thr residues
Up to 8 different core structures (more differences)
Describe the assembly of ogliosaccharide chains:
Glycosylation requires a sugar donor (sugar-nucleotide e.g UDP-glucose) and an acceptor e.g nascent protein or ogliosaccharide
Glycosylation is not template driven
Proceeds according to donor/ enzyme availability
What are glycoforms?
Variations in glycosylation patterns
What can different glycoforms of one protein causes differences?
Stability
Solubility
Serum half life
Biological activity
Immunogenicity
When wouldn’t it be ideal to use recombinant protein production (using Ecoli)?
Prokaryotes do not have the necessary glycosylation machinery
Need to produce proteins for eukaryotes
Need to control production process- maintain consistent glycosylation
What alternative host can be used instead of Ecoli and why?
Chinese Hamster Ovary cells (CHO)
They have all the enzymes necessary
Describe the process of hybridoma technology to produce mAbs:
Immunise mouse with specific antigen
IS of mouse produces Abs which produce plasma B cells (isolation of these)
Plasma cells mixed with immortal myeloma and fused with PEG
Cells grown in HAT medium, which only allows fused cells to grow
Cells are diluted to one cell per well
Fused cells showing high Ab production are expanded and grown in large number for Ab production
What are the problems and therefore solution with using hybridoma technologies?
Human anti-mouse antibody (HAMA) response
Solution is to use humanised and full human antibodies
Describe the large scale process of production of recombinant proteins:
Innoculate a liquid bacterial culture broth (thousands of litres) with bacteria harbouring recombinant gene of interest
Grow eukaryotic cells (1000L+) that carry a plasmid coding for the protein of interest
Describe the isolation process:
Treatment with chemicals (e.g detergent) or alkaline conditions
Sonication/ homogenisation
Agitation in the presence of abrasives
Describe the types of chromatographic purifaciton:
Affinity chromatography
Ion exchange chromatography
Gel filtration chromatography
How long is a patent for a biologic?
Around 20 years
Why can’t a biosimilar be generic?
As the new protein has the same sequence but may have different properties e.g different folding
What are liposomes?
Composition of primarily phospholipids
Describe the method of preparation of liposomes:
Lipid components are added to an organic solvent and freeze dried- forms a lipid cake
To make water soluble, lipid cake is added to aq solution
Then hydrated and mixed (agitation) with extra water to form large MLV (have multiple lipid bilayers within structure)
To get down to individual lipids with single layer, need sonication, extrusion, homogenisation= unilamella
Name the different classes of liposomes:
SUV- small unilamella vesicles
LUV- large unilamella vesicles
MLM- multilamella vesicles
Describe SUVs:
Single lipid bilayer enclosing aqueous component
25-100nm diameter
Describe LUVs:
100nm-1µm diameter
Describe MLMs:
More than 1µm diameter
What does it mean that phospholipids are amphiphatic?
They have a polar head and non polar tail
What is and describe the packing shape of a phospholipid?
Compare the size of head group to tail group
Cone (spherical molecule)= Less than 1/3
Cylinder (planar bilayer)= 1
Truncated cone (vesicles/liposomes)= 1/3-1
What is the function of cholesterol addition to phospholipids?
Extended planar group-hydrophobic
Occupies tail part of bilayer so decrease permeability (stiffen and rigidify) so increase drug retention inside liposome
What is the function of PEGulation of liposomes?
Polyethylene glycol
Disguise the liposome- as recognises PEG, stops liposome from coagulating
Stops repulsions
Describe which drugs can be placed where in a liposome:
Drug characteristics also determine which are suitable for liposomal formulation
Drugs with log P less than 1.7 (hydrophilic) can be incorporated into the aq compartment of liposome
Drugs with lop P more than 5 will be retained in the lipid bilayer
Drugs with intermediate log P ( between 1.7-5) can be difficult to incorporate as they partition between bilayer and compartment can be lost
What can be the resolution for drugs that have an intermediate log P that need to go into the liposome?
Use remote drug loading, rather than the older method of mixing drug and lipids
Describe active remote drug loading:
Make liposome without drug but include one or both in aq compartment something that gives a gradient->
perforin liposome, with pH, ionic strength, gel within the aq compartment (ammonium sulfate, citrate salts)
Drug travels up gradient, and/or forms complex with components of compartments
Capture drug and retains in liposome
What are the reasons for encapsulation of drugs within liposomes:
ADMET- more than 50% of drug failures
For altering the pharmacokinetics and bio-distribution of the drug
To function as a drug reservoir (sustained release)
For protecting new drugs from attack e.g peptide, nucleotide drugs
What are the problems with encapsulation?
Poor solubility- in lipid- comes out near target size
Tissue damage on extravasion
Rapid breakdown of drug in vivo
Unfavourable pharmacokinetics (rapidly cleared)- increase circulation time
Poor distribution-only have to deal with pharmacokinetic properties of liposome
Lack of selectivity of target tissue
How are liposomes used in cancer treatment?
IV- injected liposomes interact with blood opsonins
Opsonised liposomes enter the mononuclear phagocytic system (MPS)
This results in a build up of drug containing liposomes at these sites, creates an MPS depot, for slow release of drug into circulation, mimicking slow transfusion
What is the alternative option when MPS deposition is not beneficial:
Surface modification (e.g PEGylation) creates hydrophilic surfaces that repel opsonins and maintain liposomes in circulation (stealth coating)
This enhances opportunities for liposomes to accumulate at pathogenic sites by Enhanced Permeability and Retention (EPR)
Remains longer in the blood stream
What does the opsonin mediated removal of liposomes depend on?
Opsonin-mediated removal of liposomes depends on liposome size (larger more cell like), lipid composition and surface charge
Why would MPS deposition not be beneficial?
Stability, clearance rates and tissue distribution of liposome is now important
Describe Myocet as a treatment for cancer:
Doxorubicin (antrhacycline antibiotic, intercalates into DNA strands and block) in egg phosphatidylcholine/ cholesterol liposomes
1st line treatment for metastatic breast cancer
Describe the structure and route into the body of Myocet:
LUV liposomes, around 180nm diameter
They will be recognised by opsonins- large liposomes without PEG to disguise them, will form MPS depots and slowly release drug into circulation
Describe Caelyx (aka Doxil) as a treatment for cancer:
Doxorubicin in liposomes containing PEG2000- distearoyphosphatidy ethanolamine (DSPE)
Treatment for advanced ovarian and breast cancer, Kaposi’s sarcoma
Describe the structure and route into the body of Caelyx:
SUV liposomes, less than 100nm diameter
Small PEGylated liposomes can avoid opsonisation and so persist in blood circulation
Accumulate in tumours via EPR effect
Describe Abraxane as a treatment for cancer:
Albumin bound paclitaxel
Used in the treatment of breast cancer and non-small cell lung cancer
Describe the structure and route into the body of Abraxane:
Human serum albumin is an endogenous component of the blood (35-50g/L in blood)-non antigenic
So using it as a carrier for drug delivery circumvents many issues associated with liposomes
No need for PEGylation
Describe albumin as a drug carrier:
Capsulates hydrophobic drug- hydrophilic outside, like micelles
Can be taken up by GP60 glycoprotein- using body’s natural response
Describe solid lipid nanoparticles (SLN):
Lipid core matrix comprises exipients such as mono-di and triglycerides, fatty acids and waxes
SLN can be produced by different formulation techniques providing reasonably high drug encapsulation
Self (nano) emulsifying drug delivery systems
Describe the Pfizer/biotech covid vaccination:
Lipid nanoparticle mRNA
70-100nm
Ultralow temp as unstable
PEGylated lipid, cholesterol
Describe the AstraZenca covid vaccination:
Adenoviral vector
80-120nm- exploiting naturally occurring adenovirus
Easier to store- longer at a lower temp
What are the safety precautions with nanoparticles?
Nanometer dimensions- how it can accumulate
Toxicity- composition, surface coating, conc, exposure time
Biodegradability
Metabolism and excretion
Where are renal and hepatic products excreted in?
Renal-> urine
Hepatic-> bile (faeces) clearance
What is clearance?
Volume of blood plasma which is completely cleared of substance (drug) per unit time
What determines whether a substance is cleared renally or hepaticly?
Glomerular filtration-molecules/particles below 6-8nm are efficiently filtered by glomerulus, larger molecules remain in blood
MPS takes up larger particles- can retain months/years
Surface coatings (PEG/Dextrans) can help avoid opsonisation and MPS uptake
If too large for renal clearance and avoiding MPS, hepatic clearance will follow
What is passive targeting in a simple liposome?
Not manipulating the body, just allowing it to react
A process in which deposition of nano sized systems (1-100nm) within the tumour microenvironment is enhanced due to distinctive characteristics inherent to the tumour mildew, not normally present in healthy tissues
What is the goal (benefits) of nano medicine?
Small molecule drugs are poorly targeted and rapidly cleared in urine
Few of them reach their target and the rest can accumulate in other tissue, causing SEs (like chemo as doesn’t target specific cells)
Nanomedicines are larger (not excreted in urine), so are cleared from the body more slowly
They can also accumulate at the therapeutic site through targeting methods
Less drug is released in other tissues so fewer SEs
What is the EPR effect in nanomedicine?
Enhanced Permeation and Retention
Taking advantage of blood vessel damage for the drug to get into the cells
Describe the EPR effect in normal BVs:
Healthy endothelial cells, barrier to BV so stops blood into tissue
Lymphatic system drains waste material from cells
Nanoparticles just flow through in blood stream as a strong barrier
Describe the EPR effect in tumour BVs:
BV distorted/ degraded- angiogenic endothelial cells is erratic, leaky vasculature
Bvs break down and blood goes into tumour tissue
So nanoparticles can also enter tumour tissue
Enhanced retention in tumour, lymphatic system breaks down so material isn’t being drained away and will accumulate their (nanoparticles)
Describe the lymphatic system:
One way stream from peripheral tissues to blood
Doesn’t carry O2 or essential nutrients
It absorbs extraverted protein rich fluids, lipids, macromolecules and immunocompetent cells from the interstitial spaces within tissues
After resorption from lymphatic capillaries, lymph is transported to larger vessels and flows back into blood stream via the left lymphatic duct (thoracic duct)
What is the normal function of the lymphatic vessels?
Maintain plasma volume
Prevent increase tissue pressure
Permit passage of leukocytes, so proper functioning of IS
How specific is EPR?
Provide relatively modest specificity offering 20-30% increase in delivery compared with critical normal organs e.g estimated only 0.7% of injected dose accumulated
EPR is highly heterogeneous, changing over time during tumour development and possibly being transient
What are clinical barriers of oral drug delivery?
Fed and fasted state variability in drug absorption
Inter and intra individual differences in oral bioavailability
Differences in gastric emptying time and GI transit time
Disease state
What are physiochemical (drug related) barriers of oral DD?
Poor aq solubility
pH dependent solubility
Extensive ionisation at GI pH range
Extreme lipophilicity
High MW
Susceptibility to pH mediated degradation
What are the physiological barriers of oral DD?
Mucus barrier
Diverse pH range of GIT
Rapid gastric emptying
Gastric and intestinal motility
First pass metabolism
Presence of digestive enzymes
GI microflora and their secretion
What are the biopharmaceutical barriers of oral DD?
Poor drug permeability
Pre systemic metabolism in GIT
Presence of drug efflux transporters
pH and mucosal layer thickness variations in GIT depending on location
Describe the role of mucus:
A ubiquitous protective barrier
Any wet surface on body has mucus layer
Role is to:
Protect epithelium against particulate damage and pathogenic attack
Lubricates to allow passage of food through GIT etc
Hosts commensal bacteria
Selectively allows nutrients passage to the epithelium during digestion
Poses a barrier to the oral and nasal delivery of drugs
What occurs in the absence or excess of mucus?
Absence leads to colitis in GIT, dry eye syndrome in tear film
Modification of respiratory mucus in CF leads to inflammation and infection- thickened and over production of mucus
What is mucus made from?
Mucins from the mucus layer
Water, salts, bile acids, lipids and proteins
Describe the structure of mucins:
Are glycoproteins
Protein backbone, glycosylated with sugar side chains, O-glycosylation, connected with serine/ threonine
Stiff extended polymer structure
Where can mucins be excreted?
Can be excreted into mucous (individual long chain polymers) or membrane bound at epithelial surface
Where are mucins made?
Made in the ER to Golgi where glycosylated to vesicles to mucus
What proteins are in the mucus of the GI?
SI=MUC2
LI=MUC2
+small amounts of 5AC and 6
Describe the layer of mucus in the SI:
1 layer of mucus in lumen
Bacteria can pass fairly freely
Small molecules can pass through mucus layer to the epithelium
Describe the layer of mucus in the LI:
Thick mucus layers in lumen, stops bacteria passing through
In the LI, no bacteria in inner mucus layer as so dense, protects against diseases
Can nanoparticles move through mucus and why/ why not?
Yes, they can move quite freely through mucus
Particle diameter below 200nm so can move quite freely
Describe the different types of drug permeability across the intestinal mucosa and explain the outcomes:
Need to generate a steep conc (of drug) gradient just outside the point of absorption
Need very careful control of release of drug
-too fast=drug doesn’t reach epithelium
-too slow=conc gradient too shallow so drug doesn’t cross epithelium
What is the estimated ideal time of drug release time across the SI?
Around 2-3 hours
Drug needs to cross the mucus barrier
Name 2 approaches of drug delivery in terms of moving a drug with mucus and why?
Mucus moves down the GIT so drug has to go with flow
-mucoadhesive
-mucopenetrant
Describe the mucoadhesive drug delivery system:
Drug/carrier adheres to mucus, is carried along GIT, drug released along the way, drug retained for lifetime of mucus transit, but still has to pass through mucus going ‘against the flow’
Describe the mucopenetrant drug delivery system:
Drug/carrier penetrates through mucous to get to epithelium (against flow), difficult to avoid mucoadhesion
Need nano-sized vehicles with ‘stealth’ coating e.g PEG
Name the 5 routes of drug absorption across the mucosa:
- Transcellular route: passive diffusion
- Paracellular route: passive diffusion
- Transcellular route: active transporter utilisation
- Lipid absorption via micelles/bile salts
- Particulate absorption via GALT (gut associated lymphatic tissue)
Describe the mechanism for the passive trans cellular route:
Entering and leaving cell on basolateral side
Passive diffusion (Fick’s Law):
-high conc on apical side of cell (on epithelium)
-low conc inside the cell
-molecule diffused into cell and out the other side into blood
Describe the type of drugs used in passive the trans cellular route:
Most drug molecules
Neutral molecules- unionised
LogP-needs to partition into membrane so has to have some degree of lipophilicity
What does a parabolic relationship mean?
At some point, LogP (0), the absorption (and biological activity) reaches a max
What occurs with a drug if you increase its Log P?
Decrease aq solubility
Decreases plasma protein binding
Increases binding to non-target sites
Describe the relationship between drug size and absorption:
Larger molecules cross cell membranes more slowly than smaller
Tight junctions restrict the diffusion of polar molecules
Diffusion Coefficients decrease (Stokes-Einstein)
Active transporters responsible for fast transport of some polar molecules
What are tight junctions?
Two cells next to each other, interacting plasma membranes
Essential for structural integrity of GIT epithelium
Used for passive paracellular route
Describe the mechanism for the passive paracellular route:
Absorption through the tight junctions
What pore size are in the different parts of the GIT and what size drugs can be transported through the passive paracellular route?
Calculated as 0.8nm in the jejunum and 0.3nm in the ileum and colon
These are calculated at the average size over time so drugs the diameter of less than 1.15nm, can be larger than pore size due to fluctuations
Name and describe the drugs used in passive paracellular route:
Small hydrophobic molecules with a diameter of less than 1.15nm
E.g Mannitol (0.67nm)
PEG (0.53nm)
Lactulose (0.95nm)
Describe the mechanism of active transport trans cellular route:
Molecule ‘piggybacks’ into the cell using the cells natural system seasoned for natural substrates such as a.as and vitamins- against the conc gradient
Name and describe the substrates used in active transport trans cellular route:
L-dopa and D-cycloserine utilise the a.a transporter
ACE inhibitors utilise the oligo (2to3) peptide transporter (PEPT)
Where are active transporters located?
Present on apical brush border membrane of intestinal epithelium
Generally restricted to specific segments of intestinal mucosa e.g folate transporter, PEPT transporter etc
Name drugs that go via the passive transcellular route and their Log P:
Propranolol- 2.6
Testosterone- 3.0
Naproxen-3.3
Name drugs that go through the paracellular route and their log P:
Cimetidine- 0.4
Atenolol- 0.2
Both long and thin
Name drugs that go through the carrier mediated route and their log P:
Cefalexin- 0.7
Levodopa- -2.4
Captopril- 0.3
What does a low and high log P mean?
High log P- above 3= lipophilic
Low log P- below 3= hydrophilic
Describe the mechanism of lipid absorption via micelles/bile salts:
Bile salts secreted into small intestine to emulsify lipid molecules
Lipids then hydrolysed by lipase to give MGs and FAs
Formation of mixed micelles of MG, FA and bile salts
Lipid molecules absorbed either directly ion micelle or by partition from micelle into the cell
Describe the substrates used in lipid absorption via micelles/ bile salts:
Poor water soluble drugs (fat soluble) so lipophilic drugs
Describe the mechanism of particulate absorption via GALT:
Endocytosis via M (membranous) cells (which sample contents of intestinal lumen) in the Peyer’s Patches of GALT in the SI
Subsequent absorption into the lymphatic system
Eventual distribution to liver and spleen
Describe the substrates used for the particulate absorption via GALT:
Macromolecules
Microparticles (<10µm)
What are 2 factors in the body which can mitigate against the absorption from the GIT lumen?
P glycoprotein
Cyp3A4 (CYP450)
How does Cyp3A4 act to stop absorption?
Acts to decrease the amount of drug absorbed through the gut (metabolise to non active form)
Stop from passing to basal layer into systemic circulation
How does P-gp act to stop absorption?
Efflux transporter protein
Acts to remove drug back into the intestinal lumen
May also remove drug metabolites from the cell
Stop from passing to basal layer into systemic circulation
Describe the Cyp450 family:
Family of enzymes
Responsible for much of the metabolism of administered drugs
There are several Cyp sub families
The major one is the Cyp3A subfamily- predominant forms for human drug metabolism is the Cyp3A4
Where are the CYP enzymes located?
Liver, SI (brush border)
Levels decrease from stomach to colon
Levels in SI are approx 10-50% of those in the liver
Liver and SI expression is not co-ordinated
Where are the P-gps located?
Apical membrane (bound)
Levels increase from stomach to colon, also found in tumour cells, BBB, kidney etc
Encoded by ABCB1 gene
Describe the P glycoproteins:
ATP-dependent
Saturable kinetics
How can P-gps undergo resistance?
Restance on some tumult cells to several anti-cancer drugs after initial treatment- the drug is pumped back out so can’t exert its therapeutic effect
Maintains its role in normal physiological functions-recognises drug as toxic
What is the function of P-gps?
Efflux transporter protein (one of several)
Removal or toxic materials from the cell
What are the substrates like in CYPs?
Many current therapeutic drugs
Structurally diverse e.g midazolam, saqunavir, simvastatin, ciclosporin
What are the substrates like in P-gps?
Structurally diverse e.g doxorubicin, ciclosporin, tacrolimus, saqunavir
Large and amphiphatic (has charge)
Name inhibitors of CYPs and what is the consequence of this?
Enhance absorption of drugs
Ketoconazole
Grapefruit juice (intestine only)
Name inhibitors of Pgp and why?
Drugs: verapamil, ketoconazole
Competition for Pgp in many drug cocktails- reach saturation limit
Excipients: PEG, TWEEN
Name inducers of CYP and Pgp:
Rifampicin, phenytoin, dexamethasone, phenobarbital
What are key questions to consider when assessing biopharmaceuticals?
Is drug released from the dosage form?
Is the drug stable in physiological fluids?
Is the tissue permeable to the drug?
Is the drug metabolised before it reaches systemic circulation?
What factors can affect bioavailability?
Dissolution and solubility
Dosage form and particle size
Drug form
Absorption processes-dissociation into ionised and unionised forms affecting partition into plasma or lipid solubility
Active processes can change absorption-efflux transports, metabolism
Describe how liphophilicty of a drug is critical to its absorption and bioavailability:
Large poorly lipid soluble drugs (P<0) are poorly absorbed after oral admin so given IV e.g heparin
Small poorly lipid soluble drugs can be absorbed by the paracellular route (tight junctions)
Lipid soluble drugs (P 0-3) are readily absorbed across the mucosal epithelium and so are suited to oral administration
Very lipid soluble drugs are also readily absorbed but are more susceptible to metabolism and biliary clearance (incorporation into bile acid micelles)
How would you calculate if the tissue is permeable for the drug?
Many methods:
-computational (stimulate variants of logP/D)
-In vitro cell culture
^both used early on as cheap, allows for molecule optimisation
-Tissue studies (ex vivo)
-Human studies
^these 2 more expensive
What is LogP?
Drug conc in octane and water phases
How would you study the in vitro epithelial permeability?
Intestinal epithelial cells are grown on membrane filter
Add drug to lower compartment and sample from higher compartment over time
How would you calculate if the drug is metabolised before it reaches systemic circulation?
Add drug to homogenised gut tissue/ brush border homogenate
Measure the extent of metabolite by HPLC/mass spec
Mass spec allows the identification of metabolite structure
