(chan) 1. Pharmaceutics Flashcards
Define ‘Pharmacogenetics’ and its objective
- Study of the effect of inherited variations on drug response
- Objective = to link differences in gene structure with differences in
: drug absorption, transport, metabolism
: pharmacological (therapeutic/toxicity) effect
Define ‘Genotype’ and ‘Phenotype’
Genotype
: Differences observed at the genetic level
Phenotype
: Differences observed at the enzyme/transporter activity level
Which gene products influence drug’s PK-PD effect?
- Receptors (drug targets)
- Metabolizing enzymes
- Transporters
How does genetic variation effect drug responses?
- Drug receptors
: altered availability of receptors
: altered affinity of receptor to drug - Drug transporters and enzymes
: rate of metabolism
: rate of transport
What is the aim of pharmacogenetic studies?
- To understand genetic basis for variation in the therapeutic and adverse drug response
What occurs in Phase I and Phase II of liver metabolism?
Phase I
- oxidative reactions
- hydrolysis reactions
Phase II
- conjugation reactions
What is the advantage of DNA industry?
- More efficient, cheaper, safer production of therapeutic proteins e.g insulin
- To make rare proteins with therapeutic potential in sufficient quantitiy
- Production of vaccines
- GM food
What is the aim of ‘Recombinant DNA technology’?
- Analyse function of genes and their products
- Expression/regulation studies
- Production of industrial and pharmaceutical products
In which direction do polymerases synthesise in?
5’ -> 3’ direction
What is monocistronic & polycistronic?
monocistronic
: One mRNA, one gene
polycistronic
: One mRNA, 2 or more genes (genes organised in operon)
What do the following terms mean regarding transfer of DNA?
- Transformation
- Conjugation
- Transduction
- Transformation
- uptake of free DNA - Conjugation
- transfer of DNA through cell-cell contact - Transduction
- transfer of DNA mediated by a virus
Characteristics of plasmids
- Most are circular, double stranded DNA molecules
- Replicate independently from chromosomal DNA
- Found in prokaryotes and lower eukaryotes
What are plasmids involved in?
- Resistance to antibiotics or toxic metals
- Metabolic functions
- Production of virulence factors
What is molecular cloning?
- Obtaining a defined sequence of DNA and produce multiple copies in vivo
What are the 3 basic steps in molecular cloning?
- Isolation of source DNA
- Inserting source DNA into a cloning vector
- Introduction of cloned DNA into a host organism
What is Polymerase Chain Reaction (PCR)?
- most common technique for obtaining DNA fragments for cloning
- method to amplify section of template DNA
What does Polymerase Chain Reaction require and how is it done?
- Water, DNA template, Nucleotide, primers, polymerase, buffer
Three steps (repeated 25-35 times) 1. Denaturation of DNA strands (~30s at 94degrees)
- Annealing with primers (~30s at 55-65degrees)
- Elongation with thermostable DNA polymerase (~1min per kb at 72degrees)
Recombinant DNA is generated by combining DNA from a source with a vector.
Which specific enzymes are used for ‘cutting’ and ‘pasting’?
Cutting
- Restriction enzymes
Pasting
- Ligase
What do restriction enzymes do in cloning?
- recognise palindromic sequence: restriction sites
- Cut both DNA strands, creating sticky and blunt ends
What are the 3 important regions of plasmids regarding cloning?
- Replication origin
- Selection marker
- Region where DNA can be inserted
What is the procedure of ‘Cloning’?
- Cut source DNA and plasmid with restriction enzymes
- Mix source DNA and plasmid, and add ligase
- Use ligation mixture to transform E.coli
- Grow on agar plates
- Identify colonies containing recombinant DNA
What is ‘blue-white’ screening?
- plasmid contains lacZ gene, which encodes the enzyme β-galactosidase
- MCS is part of lacZ gene
- if no DNA in MCS, β-gal is active and converts artificial substrate into blue dye
- if foreign DNA in MCS, β-gal is inactive and no blue colour (white)
What are the requirements of hosts for cloning and 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
What is insulin?
- Hormone produced in pancreas; secreted into bloodstream
- Controls blood sugar levels
- Faulty production or ineifficient utilisation leads to diabetes
- Small protein
Recombinant human insulin production method 1
- Clone insulin A and B chains seperately in E.coli as fusions with gene encoding β-galactosidase
- Purity fusion proteins and cleave off β-gal
- Combine A and B chains and refold in oxidising conditions in vitro
Recombinant human insulin production method 2
- Clone gene for proinsulin, fused to gene encoding β-galactosidase in E.coli
- Extract protein, purify, and cleave off β-gal
- Refold proinsulin
- Cleave proinsulin enzymatically
What is Factor VIII?
- Essential Blood Clotting factor
- Used for treatment of haemophilia
- Very large protein
- Largest recombinant protein used commercially
How is Cloning of Factor VIII done?
- Very large gene with several introns (requires copies to be made from mRNA)
- initial cloning was done in E.coli
- Plasmid containing F8 gene used to transfect mammalian cell lines
- Plasmid integrates in genone; cell line with highest number of F8 gene copies used for production
What is antithrombin?
- Glycoprotein, made in liver
- Inactivates thrombin, Factor Xa and Factor IXa
: regulates normal blood coagulation - Used in patients with antithrombin deficiency
Cloning Key points (JUST READ)
- Recombinant DNA can be made using restriction enzymes (cutting) and ligase (pasting)
- Choice of vector/host system depends on the nature of the protein that has to be produced
- For small proteins that are not post-translationally modified, bacteria or yeast are preferred
- Proteins of mammalian origin that are e.g large and glycosylated need to be produced in insect or mammalian cells, or in whole animals
Which forces exist in Protein Folding & Stabilisation
- Hydrophobic interactions (80% internal)
- Electrostatic (repulsions, ion pairing)
- H-bonding (Inter, intra)
- VDW forces
- Steric effects
- Hydration
- Disulphide bridges
How can a protein be irreversibly inactivated?
- Conformational changes
: Formation of incorrect structures
: Aggregation - Chemical changes
: Hydrdolysis, oxidation, deamidation, glycation, disulphide bond rearrangement
Examples of operations that may Denature or Aggregate proteins
- Freezing/thawing
- Agitation (interfaces)
- Sonication
- Contact with silicone oil
- Low or high pH
- Low or high salt
- Specific salts
- Chemical changes
- Heat
What are the possible consequences of Protein Aggregation/Denaturation?
- Altered Solubility
- Hypo-potency
- Hyper-potency
- Off target binding
: adverse events, faster clearance - Patient may generate neutralising antibodies (ATAs)
: makes drug ineffective
: may break tolerance
What are the Physical considerations for stabilised protein?
- Temperature
- pH
- Adsorption & Interfaces
- Salts and metal ions
- Concentration
Most therapeutic proteins are formulated as liquids for parenteral delivery
What extra ingredients are added to the active protein?
- Solubility enhancers
- Anti-absorption & anti-aggregation agents
- Buffering agents
- Preservatives & anti-oxidants
- Lyoprotectants/Cake formers
- Osmotic agents
Formulation of Therapeutic Proteins
What are the roles of the following ingredients?
- Sugars
- Amino acids
- Cyclodextrins
- Polysorbates
- Sugars
- increase surface tension of water - Amino acids
- interact with residues of opposite charge which may cause association of proteins - Cyclodextrins
- suppress aggregation of proteins - Polysorbates
- thought to stabilise proteins, preventing denaturation and aggregation
What is Lyophilization (Freeze Drying) of protein?
- Low temperature liquid phase
: prolonged storage
Advantages and Disadvantages of Intravenous delivery of proteins?
Advantage
- Large doses can be administered with 100& Bioavailability
- Administration can be controlled/discontinued
- Immediate access to the central compartment
- Easy weight-based dosing
Disadvantage
- Additional manipulation
- Patient inconvenience/compliance
- Multiple materials of construction
- Agitation during transport
- Risk of microbial exposure before use
What are the examples of interfaces that can cause protein aggregation?
- Air-water
- Vials, IV bags - Oil-water
- Silicone-coated syringes - Hydrophobic surfaces
- IV set & bag - PVC vs polyolefin
Advantages and Disadvantages of subcutaneous delivery of proteins?
Advantage
- Patient convenience/compliance
- Best in flat dosing but can accomodate weight-based
Disadvantage
- Maximum dose is lower (than iv)
- Cannot stop dosing once administered
Advantages and Disadvantages of Intravitreal(eye injection) delivery of proteins?
Advantage
- Direct site of action (100% bioavailability)
Disadvantage
- Patient inconvenience/compliance
- Risk of infection
Advantages and Disadvantages of Buccal delivery of proteins?
Advantage
- Patient convenience/compliance
Disadvantage
- Drug loss, <100% bioavailability
- Variability
Advantages and Disadvantages of Pulmonary delivery of proteins?
Advantage
- Local delivery, local high concentration
Disadvantage
- Nebulisers typically large and bulky
- Proteins not stable in organic solvents
Peptide and Protein Therapeutics Summary (JUST READ)
- Many mechanisms can denature, degrade or aggregate proteins
- Excipients in protein formulations can help stabilize the protein
- All proteins are different so follow the Summary of Product Characteristics / package insert / Investigator’s Brochure! \: With what to dilute \: How to mix \: IV bag headspace considerations \: How to reconstitute if necessary \: Dating after reconstitution or dilution \: Storage conditions \: Agitation considerations
What is the aim of protein modifications?
- Improving stability, efficacy and pharmacokinetics
- Therapeutic and commercial drivers
What is PEGylation?
- process of both covalent and non-covalent attachment of PEG polymer chains to therapeutic protein
- PEG ‘mask’ the agent from host’s immune system prolonging its circulatory time by reducing renal clearance
- Can also provide water solubility to hydrophobic drugs
What are the 3 main benefits of PEGylation of Therapeutic proteins?
- Hydrophilicity of PEG
- Improved solubility
- Reduced protein binding
- Improved bioavailability
- Avoidance of phagocytosis - Flexibility of PEG
- Shielding antigenic sites
- Reduced toxicity
- Proteolytic resistance
- Reduced clearance
- Improved thermal and mechanical stability - Attachment of other drugs and targeting ligands
What is Spray Drying?
- Method of producing a dry powder from a liquid or slurry by rapidly drying with a hot gas
- Consistent particle size distribution is a reason for spray drying
- Particle size/area can be adjusted to change dissolution rate
What is the following protein delivery technology ‘Nanoparticles’?
- Polymer and lipid-based nanoparticles are able to encapsulate proteins
What is Polymeric Controlled Drug delivery system?
- Controlled protein delivery over long time periods: proteins released as polymer degrades
(JUST READ)
Peptide and Protein Formulation and Delivery Summary
- Protein activity, stability and PK can be improved by changing structure or PEGylation
- Changes to protein formulation can improve delivery and open up new routes
- Long-term delivery system is possible using microchips or encapsulate cells
What is the limitation of Oral Insulin Delivery and its solution?
- limited by acidic gastric juices & proteases in addition to MW
- Extensive interest in nanoparticlebased protein delivery
What are the 4 types of Microneedles (Transdermal) Delivery methods?
- Poke and patch
- Coat and poke
- Poke and release
- Poke and flow
In Responsive Drug Release under CDDS (Controlled Drug Delivery System), what are the potential controls?
- pH
- Chemicals
- Enzymes
- Ultrasound
- Magnetism
- Light
- Electronics
