Pharmaceutics (C&T) Flashcards
What are the purposes of each stage of a clinical trial?
Phase 1 = Safety of the medicine
Phase 2 = Focused group of the population
Phase 3 = Open to whole population, this shows a sufficient requirement for the drug – dosing is selected from the phase 2 trial for safety and efficacy
Phase 4 = Post approval, screening through genetic variation to see who will benefit the most
What are some factors that affect a drug’s PK-PD?
What is a potential problem with the usage of drugs?
Drug targets (receptors)
Metabolising enzymes
Transporters (Absorption, efflux, access to action and elimination sites)
They may have off target binding sites — disrupts the homeostatic/physiological function of transporters/receptors.
Drugs rarely only bind to one receptor (allosteric binding?)
Usually intended to protect the individual from the environment
What are some drug targets?
Transporters
Receptor gated channels
GPCR
Nuclear receptors
Enzymes
How might genetic variation affect drug responses?
Drug receptors: altered availability of receptors (Less transcription and lower stability)
Altered affinity of receptor to the drug
Why can’t the use of corticosteroids be stopped suddenly?
The corticosteroid given to the patient takes over the steroidal secretion role of the adrenal glands.
System detects corticosteroids present in the blood, and hence the secretion is down-regulated in the adrenal gland for homeostatic levels to be maintained.
Sudden cessation would therefore have fatal consequences — body can not react adequately to infections and cause inflammation. Thus corticosteroid use has to be slowly reduced, so there is no adrenal gland shutdown.
Give a few ways the body can react to drugs.
Therapeutic response
–Non response
–Hyper response
Adverse response
– Concentration-dependent adverse response
– Idiosyncratic adverse response (Side effect without a known cause)
– Immunological responses (Time dependent development, or genetic)
What is a benefit of an oral lipophilic drugs?
Does not directly go through the liver – goes through the lymphatic system (No hepatic first pass effect).
Thus potential for higher bioavailability.
Give some processes involved in absorption across the GI membrane
Passive diffusion
Transporter mediated
– Influx transporters (Pept1 – nutrient absorption, oligopeptide transporters)
– Efflux transporters (P-gp)
Pre-systemic metabolism in intestine &/or liver
What are the main ways of elimination.
Phase 1 metabolism
– oxidative CYP450 enzymatic reactions
– hydrolysis (esterases, epoxide hydrolases)
Phase 2 metabolism
– conjugation reactions with UDPGT
Filtration
– Transport
– Secretion (transport -> urine)
– Reabsorption via passive diffusion or transport urine to the blood
Describe the transporters present in the blood-brain barrier.
Explain the purpose of this structure
Similar to the GI, contains P-gp to rapidly export molecules that cross the barrier.
Limits access to the target sites in the brain.
BBB is a potent barrier – brain susceptible to any change, (e.g. change in pH could cause death)
Explain what a genotype and phenotype is.
Genotype = an individuals full hereditary information (born with it)
Phenotype = actual expressed properties In debrisoquine,
What is the effect of genetic variability in:
A: Drug metabolising enzymes/drug transport proteins.
B: Drug target/target pathway
A.)
Under exposure = No efficacy, no adverse event
Appropriate exposure = Efficacy, no adverse event
Over exposure = Efficacy, dose related adverse event
B.)
Responders
Response and target related/off target adverse event.
Or response and no adverse event
Non responders
No response and target related/off target adverse event
Or no response and no adverse event
What are the risks of warfarin.
How does it elicit its effects?
Large variation in the dose response, as well as a narrow therapeutic range.
Monitored via INR – target is about 2.5 in patients
Greater than 4 risks haemorrhages
Warfarin is a coumarin anticoagulant.
It inhibits VKORC1 (Vitamin K epoxide reductase complex subunit 1)
–> Antagonises hepatic recycling of Vitamin K
–> Reduction in activation of some clotting factors (Other pathways for clotting are still present)
Describe the use of codeine as an analgesic and the genetic variations in the metabolism.
Codeine is partly metabolised by CYP2D6 into morphine
–> Morphine elicits the analgesic effects
Genetic polymorphism can reduce the synthesis of CYP2D6. This results in a poorer analgesic effect in the patient (slow metabolisers)
What are the different types of metabolizer?
Extensive = carries 2 functional genes
Poor/slow = lack functional enzymes or deleted genes
Intermediate = carry 1 functional and one defective, or both partially defective alleles
Ultra-rapid = more than 2 active genes
Where is P-gp found?
What does it result in.
What are the effects of polymorphism here
Important for drug absorption, distribution and elimination.
Mainly affects hydrophobic, and/or organic cations at physiological PH
1< aromatic rings, MW > 400
Anticancers, antibacterial, immunosuppressants, ß-blockers, calcium channel antagonists and HIV protease inhibitors
Not only drugs…
Liver
Kidney
Intestine
Pancreas
Placenta
Adrenal gland
Endothelial cells of the blood brain barrier
Prostate
Lung
ATP-dependent efflux pump – prevents xenobiotic entry
Apical expression of membrane results in…
- reduced drug absorption from GI
- enhanced drug excretion into bile and urine
- impeded entry into the CNS
Modifed BA
Altered disposition to some drugs
Non response to drugs
What is TPMT?
What occurs in heterozygous patients? Homozygous?
Thiopurine methyltransferase
Catalyses S-methylation of thiopurine agents – azathioprine, mercaptopurine, thioguanine (Leukaemia, rheumatic diseases, IBD, solid organ transplants)
Thiopurine agents are activated by HPRT (hypoxanthine phosphoribosyltransferase) to provide thioguanine nucleotides (active entities produce cytotoxicity)
TPMT inactivates these cytotoxic agents via methylation or oxidation
Haemopoietic tissues only have TPMT as the inactivation pathway for thioguanine nucleotides. Low activity of TPMT therefore results in a high risk for severe and fatal haemotological toxicity
Heterozygous = intermediate activity
Homozygous = very low / no activity
– patients must be identified in order for the correct dose to be given to prevent toxicity
What are some key future developments with respect to pharmacogenomics?
Genetic drug response profiles
–finds individual genetic fingerprint in order for a probabe drug response to be found
–personalised medication
Comparisons of old and new drugs
Cost effectiveness?
What are some advances to be made in recombinant DNA
Greater efficiency, cost effective, safer production of therapeutic proteins (insulin and factor VIII)
Produce rare proteins that have therapeutic potential in sufficient quantity for pharmaceutical value (inteferon)
Production of vaccines (hep B)
GM food
What are the hosts required and applications for the production of:
Insulin
Factor VIII
Antithrombin (Atryn)
E. coli, yeast – diabetes
Mammalian cells – haemophilia (poor blood clotting)
Goat – antithrombin deficiency
What is the purpose of recombination of DNA?
Natural processes
– DNA repair
– Multi drug resistance
Recombinant DNA technology
–Analysis of the function of genes and the products
–Expression/regulation studies
–Production of industrial and pharmaceutical products
What direction do polymerases synthesise DNA
5’ -> 3’ direction
Genes always run 5’ - 3’
Properties of bacterial genes
Monocistronic
One mRNA forms one gene
Polycistronic
One mRNA forms 2< genes (organised in an operon)
Other elements present?
– binding sites for regulatory proteins
– transcription terminators
Properties of eukaryotic genes
Monocistronic only (each gene has its own transcriptional control) – no operons
mRNA processed prior to translation
– splicing of introns
– addn of 5’ 7-methylguanylate cap (m7Gppp)
– polyadenylation adds polyA tail after stop codon
n.b. bacterial genes do not contain introns, so cannot clone DNA that have introns in bacteria
How is DNA transferred naturally in the body
Transformation (uptake of free DNA)
Transfection = human
Competence = bacteria
Conjugation – transfer of DNA through cell-cell contact
Transduction – transfer of DNA mediated by virus
Mobile genetic elements
– Plasmids
– Transposons and insertion seuqnce elements
What is the purpose of plasmids?
Naturally occurring plasmids are not essential, but they encode for helpful genes
– antibiotic resistance/toxic metals
– metabolic functions (allowing growth on lactose or sucrose)
– production of virulence factors (haemolysin)
What is molecular cloning
Produces multiple copies of DNA from a defined template in vivo.
DNA sequence can be a gene, but can have non-coding elements (promotors)
What are the steps to molecular cloning?
Isolate the source DNA – restriction enzyme used to cut out the required sequence and also used in the plasmid so that the DNA can be inserted to that area
Insert the source DNA into the cloning vector
– DNA fragment enzymatically inserted into the DNA of a plasmid vector using DNA ligase
– Marker (ampicillin resistance gene and lacZ gene) also inserted so to identify which have taken up the DNA fragment (white colonies) and kill the hosts that have not taken it up (blue colonies).
–> Recombinant plasmid formed
Introduce the recombinant plasmid into a host organism
– E. coli is mixed with the plasmid with CaCl2 – heat pulse
– Culture the bacteria on nutrient agar plates impregnated with ampicillin
–> Cells that have been transformed survive, those that did not take up the plasmid die
Plasmid replication then occurs to produce more of the inserted DNA
—> Cell multiplication occurs producing a large quantity of cells containing the desired DNA
What is PCR?
What is it used for?
Polymerase chain reaction
Method to produce a large quantity of the template DNA
Reaction requires water, DNA template, nucleotides, primers (oligonucleotides, polymerase, and a buffer.
There are 3 steps, which are repeated multiple time (25-35)
Locate the target sequence to be cloned
Primers are designed so that they are complementary just up and downstream of the target gene
- ) Denature the DNA strands – 30s at 94C
- ) Anneal with primers – 30s at 55-65C
- ) Elongation with thermostable DNA polymerase (Taq polymerase - derived from thermophilic bacteria or archae) – 1 min per kb at 72C
How is recombinant DNA produced?
Palindromic sites are recognised by the enzyme (restriction sites)
Typically 4-6 nt long
Resstriction enzyme then cleaves these areas – produces sticky or blunt ends.
The enzyme then binds to the sticky ends of the sequence and is bound using DNA ligase.
What is agarose gel electrophoresis?
A method using agarose in order to find the number of base pairs in a fragment.
Requires…
DNA
Water
Concentrated buffer
Restriction enzyme
An electric current
A dye
UV Light
The size of the fragments can be determined by using a known set of reference bands.
What is DNA ligase?
How can it be used in an experimental environment?
An enzyme that ligates (binds) compatible DNA strands
– Sticky or blunt (though more efficient as linking compatible sticky ends)
Usually plays a role in repairing DNA and replication
____
Requires…
Vector for the DNA to be inserted into
The insert DNA
Concentrated buffer
Water
Buffer
What are the benefits/properties of cloning vectors?
Clones independent of DNA
Available commercially or distributed freely
Accepts inserts of 10kb> (greater than this causes instability)
Easy to use
Narrow range of compatible hosts
What is blue-white screening in plasmids?
The plasmid contains the lacZ gene – encodes ß-galactosidase.
This degrades lactose…
MCS is part of the lacZ gene
–> If DNA is inserted. it inserts into the lacZ gene, causing inactivation of it.
So, hosts that have taken up the DNA have ß-galactosidase inactivated, so that the artificial substrate x-gal is not degraded into the blue dye.
—> Observe a white colony (desired)
Hosts that have not taken up the DNA have the enzyme remaining active. This degrades the x-gal substrate, resulting in a blue dye being produced.
—> Observe a blue colony (undesired)
What are some types of plasmids?
Shuttle plasmids
–> Plasmid can replicate in a minimum of 2 different hosts
Integration vectors
–> Cannot replicate, but integrates itself into the chromosome
–> Useful for knockouts
ƛ Cloning vectors (spp to g(-) bacteria)
–> Based on bacteriophage lambda (ƛ)
–> Accomodates to larger inserts (up to 22kb)
Artificial chromosomes
–> Construct is based off of bacterial (BAC) or yeasts (YAC) DNA
–> Can contain very large inserts (75-800kb)
–> Useful for cloning very large genes and genome mapping/sequencing
What are some characteristic desired in hosts for cloning/expression.
- Grows rapidly in inexpensive medium
- Non-pathogenic
- Easily takes up DNA
- Is genetically stable
- Allows replication of vector
- Has many tools for genetic manipulation
- Allows high level of expression of genes
–> High expression -> more mRNA -> more protein produced
•N.B. Hosts for cloning and expression are not necessarily the same
Describe the mechanism of physiological insulin production.
Stimulus causes ER to produce preproinsulin via ribosomes.
The protein produced from mRNA translation contains an A chain, B chain and C chain.
A and B chain are linked via the C chain.
As the protein is being made, it is transported into the lumen of the ER
The signal peptide is then cleaved from the preprohormone, to produce proinsulin. A disulfide link is also produced between the A chain and B chain
Proinsulin is transported to the golgi complex using vesicles
As proinsulin is packaged into vesicles by the golgi complex, the C chain is cleaved, leaving the disulfide bond linking the A and B chain, as well as the internal disulfide bond in the A chain.
How is recombinant insulin produced? Describe 2 ways it can be done.
The peptide is short, therefore it is degraded in the cytoplasm of E. coli – unstable.
Thus it can be stabilized by fusion to a larger protein.
Method 1
- A chain and B chain are produced separately in E. coli, as fusions with the gene encoding for ß-galactosidase.
- ß-galactosidase is then cleaved from the fusion proteins, and purified.
- A chain and B are combined and refolded in oxidising conditions to form the disulfide bonds
Method 2
- Clone the gene for proinsulin
- Attach ß-galactosidase encoding gene to proinsulin of E. coli
- Extract the protein, purify and cleave off ß-galactosidase
- Refold proinsulin
- Enzymatic removal of the C chain to produce insulin
How is factor VIII produced commercially?
Initially produced using mRNA, and cloned using E. coli.
However, factor VIII is glycosylated, so transfection into mammalian cell line is required.
The plasmid is transfected into the genome. The cell line that produces the greatest number of Factor VIII genes is used for production.
How does antithrombin work?
Inactivates thrombin, Factor Xa and Factor IXa
->regulates normal blood coagulation
How are biotherapeutics produced in goats?
•Gene linked to mammary gland-specific regulatory elements
–>gene is expressed only in milk
- transgene is introduced into embryo by injection
- embryos transferred to oviduct/uterus
- transgenic offspring identified
- lactation induced and production in milk is tested
What is the general rule for production of biotherapeutics, with respect to the choice of organism.
Bacteria / yeast preferred when the protein does not need to be modified post-translation.
Choice is made dependent on the biotherapeutic being produced.
Proteins of mammalian origin (require glycosylation or are large) are produced in insects, animals, or animal cells.
What is the stability of proteins/peptides dependent on?
Environmental conditions
Excipients present
Delivery route/vehicle
What are some of the factors that contribute to the protein’s folding and stabilization.
- Hydrophobic interactions (80% internal)
- Electrostatic (repulsions, ion pairing)
- H-bonding
–>Inter- and intramolecular
- VDW forces
- Steric effects
- Hydration
- Disulphide bridges
What are some ways in which irreversible protein inactivation occurs.
- Conformational changes:
- Formation of incorrect structures
- Aggregation (partially hydrophobic residues interacting with another partialsly hydrophobic residue)
- Chemical changes:
e. g. Hydrolysis, oxidation, deamidation,
glycation, disulphide bond rearrangment
- Break peptide backbone
- Modification of important residues
- Change protein shape (i.e. conformational changes & aggregation!)
What are some actions that can denature or aggregate proteins?
- Freezing/thawing
- Agitation (interfaces)
- Sonication (use of ultrasound to unfold proteins)
- Contact with silicone oil
- Low or high pH
- Low or high salt
- Specific salts
- Chemical changes
- Heat
What are the effects of aggregation/denaturing of proteins?
- Altered solubility
- Hypo-potency (most likely)
- Hyper-potency
- Off target binding – Adverse events, faster clearance
- Patient may generate neutralizing antibodies (ATAs)
–Makes drug ineffective
–May break tolerance
–Cross react with endogenous protein
Give some chemical changes that can result in aggregation/altered folding
Deamidation
Hydroylsis of peptide bonds
Elimination
Shuffling of disulfide bonds
Oxidation
Cross linking
Thiol-disulphide exchange
What exicipients are added for parenteral delivery of proteins.
- Solubility enhancers – e.g. surfactants, amino acids, sugars, polymers
- Anti-absorption & anti-aggregation agents – e.g. surfactants, albumin
- Buffering agents – usually citrate, phosphate or acetate
- Preservatives & anti-oxidants – e.g. ascorbic acid, antimicrobials (repeated dosing)
- Lyoprotectants/cake formers
- Osmotic agents – NaCl, mono- or disaccharides
Give some commonly co-formulated chemicals with therapeutic proteins.
- Sugars, some amino acids - increased surface tension of water
- Glycerol, polyols - repulsion between molecules, this maintains a water layer, and hence preserves the protein’s structure
- PEG - steric effects
HSA – human serum albumin (Indirect and direct stabilization of the protein)
Polysorbates – prevents aggregation and denaturing via..
- Preferential exclusion of solutes
- Acting as a “chemical chaperone” aiding protein refolding
- Binding to hydrophobic patches of proteins
– can reduce protein exposure to interfaces (can cause aggregation)
– forming of detergent film, in aqueous systems. This reduces protein exposure to air/water
Methionine – prevents oxidation of the protein
- Interact with residues of opposite charge which may cause association of proteins
- Aliphatic regions can cover exposed hydrophobic areas of proteins
Cyclodextrins (prevents aggregation of proteins)
– HP-ß-CD is now approved for parenteral administration of leukine-enkephalin
What are some caution associated with polysorbates?
What can cause changes in the intensity of the effects.
Auto-oxidation/hydrolytic degradation can result in aggregation and denatuaring of enzymes. They produce…
Reactive peroxides (antioxidants may help here)
Aldehydes – immunogenicity (even without aggregation)
–Formaldehyde & acetaldehyde are potential carcinogens, and cause contact allergies.
Extent is controlled by:
- Identity of the protein
- Protein and surfactant concentration
- Other excipients
- Temperature
- Light exposure
What is lyophilization?
What are the benefits?
What are the negatives?
Freeze drying
–Restricts mobility
–Reduces relative humidity
–Allows storage at room temp.
Requires reconstitution
Takes time for full dissolution
Agitation
Air entrapped
Potential for mistakes – dilution? microbial contamination?
What are the pros and cons of transporting/storage of proteins as liquids.
Volume can be directly withdrawn
Less manipulation compared to lyophilization
Agitation
Shipping and storage between 2-8C
Inadvertent freezing
Compare the pros and cons of using IV formulations
Advantages…
- Large doses can be administered with 100% bioavailability
- Administration can be controlled/discontinued
- Immediate access to the central compartment
- Easy weight-based dosing
Disadvantages
- Additional manipulation
- Patient inconvenience/compliance
- Dose usually diluted into prefilled i.v. bag
—-Adsorption leading to lower concentration
- Multiple materials of construction (polyolefin, PVC, etc)
- Agitation during transport may be significant
- Risk of microbial exposure before use
What types of interfaces can cause aggregation of proteins.
Air-water (Vials, IV bags..)
Oil-water (Silicone coated syringes)
Hydrophobic surfaces (IV set and bag)
Compare normal saline and dextrose as diluents in parenteral administration of medicines.
Normal saline is normally fine.
Some hydrophobic proteins are less soluble/insoluble in high salt conc, it may be better to formulate lower salt content in these cases.
Dextrose is usually NOT fine.
Can react with lysine on protein surfaces to produce Schiff bases relatively quick. This can occur in vivo, esp in patients with uncontrolled diabetes
What is etanercept (enbrel)?
TNF-⍺ antagonist
Consists of Fc region of IgG1 (prolongs the half life) fused with the extracellular domain of the TNF receptor
Soluble protein given SC
50-100x better at binding to TNF-⍺ than the endogenous receptor
Dimeric, so each molecule can bind to a maximum of 2 molecules
What the forms polymeric forms insulin is available in?
How can insulin be modified?
Monomers are formed at low conc
Dimers are formed at high concs.
Zinc ions promote the hexamer form of insulin
Modification of quarternary structure
–Long acting insulin promotes the hexamer form, has a reduced solubility and promoted binding to plasma proteins
–Fast acting insulin prevents hexamer/dimer formation and increases solublity
What is insulin degludec?
Give an example of one.
Basal insulin
Des(B30) human insulin
C16 chain promotes the formation of multi-hexamers following injection
Tresiba
What is PEGylation, and what are the benefits of it.
PEGylation is a modification that can be performed on proteins. The apparent diameter is increased, and so clearance is reduced.
Must not block the functional parts of the protein
Hydrophilicity of PEG
- Improved aqueous solubility
- Reduced protein binding
- Improved bioavailability
Can allow attachment of other ligands or drugs
-Avoids phagocytosis
Flexibility of PEG
- Shields antigenic sites (prevents its destruction by antibodies etc)
- Reduced toxicity
- Increased proteolytic resistance
- Reduced clearance
- Improved mechanical & thermal stability
What is spray drying? What are the benefits of it.
A liquid form of the drug is produced, and an aerosol is formed in a drying chamber (filtered hot air).
The droplets dry very quickly to produce micron sized particles of the protein and excipients.
Particles that are too large are recirculated in the system.
Benefits.
- Continuous process (vs batch in lyophilization)
- Gentler than freezing
- Produces a powder – no sieving or milling required
- Particle size/area can be modified to change the dissolution rate
- Can be performed aseptically
- Can allow for room temp storage
What is Raplixa?
Thrombin/Fibrinogen spray dried to produce a powder.
This is used in surgery for clotting.
What is the use of “Polymeric Controlled Drug Delivery Systems”.
What are the common limitations?
Method of drug delivery that prolongs the release of drug.
As the polymer degrades, the drug is released (commonly used polymer is PLGA).
Commonly used polymers are typically hydrophobic and only soluble in organic solvents. This is fine for most drugs – not biologicals. Emulsions are usually required to get the hydrophilic drug into the polymer.
Issues
- Interfaces with the air, solvents and surfaces
- Temperature
- Degradation products at at release site
What is encapsulated cell technology?
Implanted cells are coated with a semi-permeable membrane. This allows the flow of nutrients into the cell, but prevents damage to the cells from the host immune cells.
–> Prevents the requirement of immunosuppressants being used when external cells are implanted.
–> Potential for long term drug delivery
What are the new types of transdermal delivery of insulin?
- Poke and patch (Solid microneedle)
- Coat and poke (Coated microneedle)
- Poke and release (Dissolving microneedle)
- Poke and flow (Hollow microneedles)
What are some of the types of Controlled Drug Delivery Systems?
Bulk erosion
Surface erosion
Diffusion
-> Dependent on the polymer used.
Give an example of when controlled drug delivery systems would be used.
Treatment of stenosis with stents.
Restenosis is common after using stents.
Drug eluting stents can be used that elicit its effects via diffusion/erosion of cell growth inhibiting drugs.
e.g. Rapamycin (G1 cell cycle inhibition)
Paclixatel (M phase arrest)
What are some ways that drug release can be controlled? (CDDS)
Drug release controlled by:
- pH
- Chemicals (including metabolites)
- Enzymes
- Ultrasound
- Magnetism
- Light
- Electronics
What are some CDDS for insulin
PBA triggered release – glucose competitively binds to PBA resulting in the polymer breaking apart and releasing insulin
Glucose oxidase-triggered release – increase of glucose concentration results in a decrease of pH. Polymer breaks apart.
Glucose + water + oxygen —(Glucose oxidase)—> Gluconic acid + Hydrogen peroxide
ConA triggered release – glucose competitively binds to ConA, resulting in polymer breakdown
PBA = phenylboronic acid
Give the process of tissue regeneration derived from the patient.
- Cell isolation, purification/enrichment
- Expansion of number of cells
- Seeding on a suitable scaffold
- Maturation of the tissue
- Implantation into the patient
