Pharmaceutics Flashcards
(66 cards)
1
Q
What are the 3 advantages of the recombinant DNA industry?
A
- More efficient, cheaper, and safer
- Make proteins with therapeutic potential in sufficient quantity to render them of pharmaceutical value
- Production of vaccines
2
Q
What are the 3 purposes of recombinant DNA technology?
A
- Analyse function of genes and their products
- Expression/regulation studies
- Production of industrial and pharmaceutical products
3
Q
What are the 2 ways that DNA recombination occurs in nature?
A
- DNA repair
- Acquire new functions such as multi-drug resistance
4
Q
What are the 3 natural transfers of DNA?
A
Transformation:
- uptake of free DNA (competence)
Conjugation:
- transfer of DNA through cell-cell contact
Transduction:
- transfer of DNA mediated by a virus
5
Q
What are plasmids?
A
- Circular, double stranded DNA molecules
- Replicate independently from chromosomal DNA
- Found in prokaryotes and lower eukaryotes (e.g. yeast)
6
Q
What are the 3 functions of plasmids?
A
- resistance to antibiotics or toxic metals
- metabolic functions
- production of virulence factors
7
Q
What is molecular cloning?
A
- Obtain a defined sequence of DNA and produce multiple copies in vivo
- The DNA sequence can be a gene, but may also contain non-coding elements such as a promoter
8
Q
What are the 3 basic steps of molecular cloning?
A
- Isolation of source DNA
- Inserting source DNA into a cloning vector
- Introduction of cloned DNA into a host organism
9
Q
How do you obtain DNA for cloning when the sequence is known?
A
PCR
10
Q
How do you obtain DNA for cloning when the sequence is not known?
A
May require the creation of a DNA library, followed by “fishing” for the gene of interest
11
Q
What is PCR?
A
Method to amplify section of template DNA
12
Q
What are the 3 steps of PCR?
A
- Denaturation of DNA strands (~30 sec at 94 C)
- Annealing with primers (~30 sec 55-65 C)
- Elongation with thermostable DNA polymerase (~1 min per kb at 72 C)
repeated 25-30x
13
Q
What are restriction enzymes for cloning?
A
- Recognise palindromic sequences: restriction sites
- Restriction sites for cloning usually are 4 or 6 nt
- Cut both DNA strands, creating sticky or blunt ends
- Named after the organism it originates from, plus a number
14
Q
What is DNA ligase?
A
- ATP-dependent enzyme that links DNA strands
- Plays a role in DNA repair and replication
- Can ligate compatible sticky ends, as well as blunt ends
- Ligation of sticky ends is more efficient than ligation of blunt ends
15
Q
What 2 things are required in a plasmid for cloning?
A
- Selection marker (genes for antibiotic-resistance or growth on specific media)
- Region where DNA can be inserted
16
Q
What is insulin?
A
- Hormone produced in pancreas
- Controls blood sugar levels
17
Q
How is recombinant insulin produced?
A
- Short peptides such as insulin are not very stable in cytoplasm of E. coli
- Peptides can be stabilised by fusion to a large protein
- Sequence of insulin can be modified if desired
18
Q
What are the 3 ways to modulate insulin-release profile?
A
- Mix with protein to slow release
- Introduce amino acid changes
- Chemical modification
19
Q
What is Lispro (Humalog)?
A
Rapid release insulin
20
Q
What is Glargine (Lantus)?
A
Slow release insulin
21
Q
What is Detemir (Levemir)
A
Slow release insulin
22
Q
What is Factor VIII?
A
- Essential blood clotting factor
- Used for treatment of haemophilia
- Very large protein of 2332 AA
- Largest recombinant protein that is used commercially
23
Q
How is Factor VIII cloned?
A
- Very large gene with several introns - requires copies to be made from mRNA
- Initial cloning was done in E. coli
- Plasmid integrates in genome; number of copies amplified, and cell line with highest number of copies used for production
24
Q
What are the 3 ways the Factor VIII is produced?
A
Continuous cultures
- Culture fluid with Factor VIII continuously removed and replaced with fresh medium
- Cultures maintained for 6 months
Batch cultures
- Grown for up to 55 days
- All culture medium then harvested
Purification of factor VIII from culture medium
- Combination of gel filtration, ion-exchange, and affinity chromatography
25
What are the 5 possible consequences of denatured proteins?
- Altered solubility
- Hypo-potency
- Hyper-potency
- Off target binding
- Patient may generate neutralizing antibodies (ATAs)
- Makes drug ineffective
26
How does amidation affect proteins?
- Rate of asparagine (Asn) deamidation can be faster than hydrolysis of amide bond
- Favoured at pH 5 and above
- Position of residue affects relative rate – α-helices & β-sheets stabilize
- Neighbouring residues have an influence too
27
How does oxidation affect proteins?
- Trace amounts of transition metals or chemical oxidants
- His, Met, Cys, Trp and Tyr are potential sites
- Again, location of the residue in the protein is important
28
How does hydrolysis affect proteins?
- Most peptide bonds are stable, except X-Asp-Y
- Asp-Y may be >100 times more labile than any other in dilute acid
- Asp-Gly and Asp-Pro particularly labile e.g., rhM-CSF at Asp169-Pro170 and Asp213-Pro214
29
What are the 5 physical considerations of peptide delivery?
1) Temperature
Tm = temperature at which 50 % of molecules are unfolded, usually 40-80 ºC
Thermophilic vs mesophilic proteins – hydrophobics, H-bonds, salt bridges
Denaturation can lead to aggregation
Chemical reactions more rapid at elevated temperatures - proteins are stored in the fridge or freezer
2) pH
3) Adsorption & interfaces
Air/water, organic solvents, vessels
4) Salts and metal ions
Formulation and equipment, e.g., rHuMAb HER2 stainless steel filler = 52 % oxidation vs 18 %
5) Concentration
30
What 6 things are added to protein therapies in formulation?
- 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
31
How do Sugars, polyols, polymers aid peptide formulations?
Sugars, some amino acids - increased surface tension of water
Glycerol, polyols - repulsion
PEG - steric effects
32
How do Cyclodextrins aid peptide formulations?
Suppress aggregation of proteins e.g. insulin and GH
HP-β-CD approved for parental admin of leucine enkephalin
32
How do amino acids and proteins aid peptide formulations?
- Interact with residues of opposite charge which may cause association of proteins
- Aliphatic regions can cover exposed hydrophobic areas of proteins
32
What are polysorbates?
- Emulsifiers
- Formation of detergent film in aqueous systems to limit protein exposure to air/water interface
- Compete with proteins in adsorbing to surfaces
33
What is lyophilisation?
- Freeze drying
Low temperature liquid phase – prolonged storage
Freezing – better, but there are problems
34
What are the 4 advantages of IV administration?
- Large doses can be administered with 100% bioavailability
- Administration can be controlled/discontinued
- Immediate access to the central compartment
- Easy weight-based dosing
35
What are the 4 disadvantages of IV administration?
- Additional manipulation
- Patient inconvenience
- Multiple materials of construction
- Risk of microbial exposure before use
36
What are the 4 advantages of SC administration?
- Patient convenience/compliance
- May require no compounding
- Can incorporate an autoinjector
- Best if flat dosing but can accommodate weight-based
37
What are the 3 disadvantages of SC administration?
- Max volume is lower than i.v.
- Cannot stop dosing once administered
- Bioavailability is < 100%
38
What is the main advantage of Intravitreal administration?
Direct site of action – 100% bioavailability
39
What are the 2 disadvantages of Intravitreal administration?
- Patient convenience/compliance
- Some risk of infection
40
What is the main advantage of buccal administration?
Patient convenience
41
What are the 2 disadvantages of buccal administration?
- < 100% bioavailability
- Variability
42
What is the main advantage of pulmonary administration?
- Local delivery, local high concentration
43
What are the 3 disadvantages of pulmonary administration?
- Nebulizers are typically large and bulky
- Proteins not stable in organic solvents
- Testing required for each nebulizer
44
What are controlled drug delivery systems?
Preparations designed in such a way that the release rate or location of active drug is controlled
Referred to as modified-release preparations
45
What are drug-eluting stents?
- Limit restenosis
- Common intervention
- Drug release controlled by diffusion or erosion - drugs prevent cell growth
- Stent bioabsorbed in 2 years - bulk erosion
- Restenosis prevented, clinically safe
46
Why are induced pleuripotent stem cells useful?
Patient-derived cells
Can potentially be grown in large numbers
No issues with rejection
47
What is personalised medicine?
- Close relatives of pharmacogenetics
- Broader still covering also non-genetic factors
- By understanding how individuals respond to drugs there is potential to design/tailor drug therapies according to
individual genotypes
48
What is genetic polymorphism?
Can affect: specific receptors, enzymes, ion channels, transporters for physiological mediators
- Alters the amino acid sequence of the protein
- Affects how cells interact with that protein
- Affects how drugs interact with that protein
- Affects enzyme activity
- Affects binding affinities
49
What are phase 1 reactions?
Oxidative and hydrolysis reaactions
50
What are phase 2 reactions?
Conjugation reactions using transferases
51
What are the 3 types of metabolisers?
- Poor metabolisers: 2 loss-of-function alleles (homozygous). Half-life of prodrug longer
- Intermediate metabolisers: 1 loss-of-function allele (heterozygous)
- Ultrarapid metabolisers: gene duplication or gain-of-function alleles (homozygous/heterozygous). Half-life of prodrug shorter
52
How can the two extremes of metabolisers cause varying effects of codeine?
Poor metabolisers: cannot convert codeine to morphine (no pain relief)
Ultrarapid metabolisers: too efficient (morphine intoxication, potentially fatal)
53
What is pharmacogenetic testing?
- Screen for gene testing and drug metabolism reducing time and money
- Currently for research use only
54
How is insulin modified for delivery?
- Monomer only at low concentrations
- Dimer at higher concentrations
- B24-B26 and B28-B29 crucial for dimerization and binding to IR
- Insulin forms hexamers in the presence of zinc ions
- Excipients in insulin formulations can affect conformation
- Regular insulin will exist in the hexameric form following s.c. injection, must dissociate for absorption
- Modifications to insulins may change quaternary structure, e.g.:
* Long acting - promote hexamer formation, reduce solubility, promote binding to plasma proteins
* Fast-acting - prevent formation of dimers and hexamers, increase solubility
55
What is PEGylation of therapeutic proteins?
Hydrophilicity of PEG
- improved solubility
- reduced protein binding
- improved bioavailability
- avoiding phagocytosis
Flexibility of PEG
- shielding antigenic sites
- reduced toxicity
- proteolytic resistance
- reduced clearance
56
What is a PEGylated insulin analogue?
Long-acting, once-daily basal insulin
PEGylation:
- Reduces diffusion rate
- Reduces renal clearance
- Improves stability vs proteases
Phase I trials:
- Flat PK and PD profiles
- Glucose normalization
- Prandial insulin dose reduction
- Significant reduction in HbA1c vs insulin glargine
- Liver-specific action
57
What is spray drying?
- Continuous process vs batch process for lyophilization
- Gentler than freeze drying
- Forms a powder - doesn’t require milling or sieving
- Particle size/area can be adjusted to change dissolution rate
- Can be done aseptically
58
What are nanoparticles?
- Polymer and lipid-based nanoparticles (e.g. liposomes) are able to encapsulate proteins
- Interest in many routes, including oral
- Can be modified with shielding molecules and targeting ligands, may facilitate intracellular delivery
- Phagocytosis needs to be avoided - alter particle size
59
What are polymeric controlled drug delivery systems?
- Polymeric systems can be used for controlled protein delivery over long time periods - protein released as polymer degrades
- The most widely used polymers are hydrophobic and only soluble in organic solvents - fine for many traditional drugs, but not for biologicals
- Often need to generate emulsions to get the hydrophilic biological drug into the hydrophobic polymer
60
What is microscale delivery?
- Study designed to deliver hPTH(1–34) and compare PK profile with that of approved drug FORTEO (Eli Lilly)
- Device implanted during out-patient visit
- S.C. space of abdomen, just below waistline
- Each reservoir contained 40 µg drug
61
What is cell-based protein delivery?
- Allows combination of immunologically incompatible cells
- Long-term delivery
- High cell density
- Less likely to be rejected by immune system
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