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MPharm2 SL22106 > Pharmaceutics > Flashcards

Pharmaceutics Flashcards

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
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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
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3
Q

What are the 2 ways that DNA recombination occurs in nature?

A
  • DNA repair
  • Acquire new functions such as multi-drug resistance
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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

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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)
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6
Q

What are the 3 functions of plasmids?

A
  • resistance to antibiotics or toxic metals
  • metabolic functions
  • production of virulence factors
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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
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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
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9
Q

How do you obtain DNA for cloning when the sequence is known?

A

PCR

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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

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11
Q

What is PCR?

A

Method to amplify section of template DNA

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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
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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
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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
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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
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16
Q

What is insulin?

A
  • Hormone produced in pancreas
  • Controls blood sugar levels
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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
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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
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19
Q

What is Lispro (Humalog)?

A

Rapid release insulin

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20
Q

What is Glargine (Lantus)?

A

Slow release insulin

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21
Q

What is Detemir (Levemir)

A

Slow release insulin

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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
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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
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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

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25
Q

What are the 5 possible consequences of denatured proteins?

A
  • Altered solubility
  • Hypo-potency
  • Hyper-potency
  • Off target binding
  • Patient may generate neutralizing antibodies (ATAs)
    • Makes drug ineffective
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26
Q

How does amidation affect proteins?

A
  • 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
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27
Q

How does oxidation affect proteins?

A
  • 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
Q

How does hydrolysis affect proteins?

A
  • 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
Q

What are the 5 physical considerations of peptide delivery?

A

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
Q

What 6 things are added to protein therapies in formulation?

A
  • 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
Q

How do Sugars, polyols, polymers aid peptide formulations?

A

Sugars, some amino acids - increased surface tension of water
Glycerol, polyols - repulsion
PEG - steric effects

32
Q

How do Cyclodextrins aid peptide formulations?

A

Suppress aggregation of proteins e.g. insulin and GH

HP-β-CD approved for parental admin of leucine enkephalin

32
Q

How do amino acids and proteins aid peptide formulations?

A
  • Interact with residues of opposite charge which may cause association of proteins
  • Aliphatic regions can cover exposed hydrophobic areas of proteins
32
Q

What are polysorbates?

A
  • Emulsifiers
  • Formation of detergent film in aqueous systems to limit protein exposure to air/water interface
  • Compete with proteins in adsorbing to surfaces
33
Q

What is lyophilisation?

A
  • Freeze drying
    Low temperature liquid phase – prolonged storage
    Freezing – better, but there are problems
34
Q

What are the 4 advantages of IV administration?

A
  • Large doses can be administered with 100% bioavailability
  • Administration can be controlled/discontinued
  • Immediate access to the central compartment
  • Easy weight-based dosing
35
Q

What are the 4 disadvantages of IV administration?

A
  • Additional manipulation
  • Patient inconvenience
  • Multiple materials of construction
  • Risk of microbial exposure before use
36
Q

What are the 4 advantages of SC administration?

A
  • Patient convenience/compliance
  • May require no compounding
  • Can incorporate an autoinjector
  • Best if flat dosing but can accommodate weight-based
37
Q

What are the 3 disadvantages of SC administration?

A
  • Max volume is lower than i.v.
  • Cannot stop dosing once administered
  • Bioavailability is < 100%
38
Q

What is the main advantage of Intravitreal administration?

A

Direct site of action – 100% bioavailability

39
Q

What are the 2 disadvantages of Intravitreal administration?

A
  • Patient convenience/compliance
  • Some risk of infection
40
Q

What is the main advantage of buccal administration?

A

Patient convenience

41
Q

What are the 2 disadvantages of buccal administration?

A
  • < 100% bioavailability
  • Variability
42
Q

What is the main advantage of pulmonary administration?

A
  • Local delivery, local high concentration
43
Q

What are the 3 disadvantages of pulmonary administration?

A
  • Nebulizers are typically large and bulky
  • Proteins not stable in organic solvents
  • Testing required for each nebulizer
44
Q

What are controlled drug delivery systems?

A

Preparations designed in such a way that the release rate or location of active drug is controlled
Referred to as modified-release preparations

45
Q

What are drug-eluting stents?

A
  • 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
Q

Why are induced pleuripotent stem cells useful?

A

Patient-derived cells
Can potentially be grown in large numbers
No issues with rejection

47
Q

What is personalised medicine?

A
  • 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
Q

What is genetic polymorphism?

A

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
Q

What are phase 1 reactions?

A

Oxidative and hydrolysis reaactions

50
Q

What are phase 2 reactions?

A

Conjugation reactions using transferases

51
Q

What are the 3 types of metabolisers?

A
  • 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
Q

How can the two extremes of metabolisers cause varying effects of codeine?

A

Poor metabolisers: cannot convert codeine to morphine (no pain relief)

Ultrarapid metabolisers: too efficient (morphine intoxication, potentially fatal)

53
Q

What is pharmacogenetic testing?

A
  • Screen for gene testing and drug metabolism reducing time and money
  • Currently for research use only
54
Q

How is insulin modified for delivery?

A
  • 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
Q

What is PEGylation of therapeutic proteins?

A

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
Q

What is a PEGylated insulin analogue?

A

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
Q

What is spray drying?

A
  • 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
Q

What are nanoparticles?

A
  • 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
Q

What are polymeric controlled drug delivery systems?

A
  • 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
Q

What is microscale delivery?

A
  • 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
Q

What is cell-based protein delivery?

A
  • Allows combination of immunologically incompatible cells
  • Long-term delivery
  • High cell density
  • Less likely to be rejected by immune system
62
Q
A
63
Q
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64
Q
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MPharm2 SL22106 (4 decks)

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