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Flashcards in (chan) 1. Pharmaceutics Deck (55):

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'

: Differences observed at the genetic level

: 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?

1. Drug receptors
: altered availability of receptors
: altered affinity of receptor to drug

2. 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?

: One mRNA, one gene

: One mRNA, 2 or more genes (genes organised in operon)


What do the following terms mean regarding transfer of DNA?

1. Transformation

2. Conjugation

3. Transduction

1. Transformation
- uptake of free DNA

2. Conjugation
- transfer of DNA through cell-cell contact

3. 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)

2. Annealing with primers (~30s at 55-65degrees)

3. 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'?

- Restriction enzymes

- 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'?

1. Cut source DNA and plasmid with restriction enzymes

2. Mix source DNA and plasmid, and add ligase

3. Use ligation mixture to transform E.coli

4. Grow on agar plates

5. 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?

1. Conformational changes
: Formation of incorrect structures
: Aggregation

2. 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?

1. Temperature

2. pH

3. Adsorption & Interfaces

4. Salts and metal ions

5. 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?

1. Sugars

2. Amino acids

3. Cyclodextrins

4. Polysorbates

1. Sugars
- increase surface tension of water

2. Amino acids
- interact with residues of opposite charge which may cause association of proteins

3. Cyclodextrins
- suppress aggregation of proteins

4. 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?

- Large doses can be administered with 100& Bioavailability
- Administration can be controlled/discontinued
- Immediate access to the central compartment
- Easy weight-based dosing

- 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?

1. Air-water
- Vials, IV bags

2. Oil-water
- Silicone-coated syringes

3. Hydrophobic surfaces
- IV set & bag - PVC vs polyolefin


Advantages and Disadvantages of subcutaneous delivery of proteins?

- Patient convenience/compliance
- Best in flat dosing but can accomodate weight-based

- Maximum dose is lower (than iv)
- Cannot stop dosing once administered


Advantages and Disadvantages of Intravitreal(eye injection) delivery of proteins?

- Direct site of action (100% bioavailability)

- Patient inconvenience/compliance
- Risk of infection


Advantages and Disadvantages of Buccal delivery of proteins?

- Patient convenience/compliance

- Drug loss, <100% bioavailability
- Variability


Advantages and Disadvantages of Pulmonary delivery of proteins?

- Local delivery, local high concentration

- 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?

1. Hydrophilicity of PEG
- Improved solubility
- Reduced protein binding
- Improved bioavailability
- Avoidance of phagocytosis

2. Flexibility of PEG
- Shielding antigenic sites
- Reduced toxicity
- Proteolytic resistance
- Reduced clearance
- Improved thermal and mechanical stability

3. 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


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?

1. Poke and patch

2. Coat and poke

3. Poke and release

4. Poke and flow


In Responsive Drug Release under CDDS (Controlled Drug Delivery System), what are the potential controls?

1. pH

2. Chemicals

3. Enzymes

4. Ultrasound

5. Magnetism

6. Light

7. Electronics