Pharmaceutics Flashcards
Recombinant technology: Adv - 3
Adv:
1. Efficient, cheap, & safe production, e.g. insulin, factor VIII
2. Makes rare proteins with therapeutic potential in quantities for pharmaceutical value, e.g. interferon
3. Production of vaccines, e.g. hepatitis B virus, SARS-CoV2, HPV
Recombinant DNA in natural processes - 2
In natural processes:
1. DNA repair
2. Acquire new functions such as multi-drug resistance
Recombinant DNA technology - 3
Recombinant DNA technology:
1. Analyse function of genes and their products
2. Expression/regulation studies
3 Production of industrial & pharmaceutical products
Transcription - 5
- RNA polymerase moves along the DNA unwinding the strand, starting at 5.
- Hydrogen bonds between base pairs break which allows the unzipping of the double helix.
- As RNA polymerase breaks the bonds, it synthesises aprimarytranscript of mRNA using RNA nucleotides. These form hydrogen bonds with the exposed DNA strand by complementary base pairing.
- The primary transcript of mRNA is processed to produce amaturetranscript of mRNA.
- The mature mRNA transcript is now ready to leave the nucleus and travel to the ribosome.
Translation - 7
- mRNA molecule travels through cytoplasm & attaches to the ribosome.
- tRNA molecules transport specific amino acids to the ribosome.
- Each mRNA codon codes for a specific amino acid.
- The anti-codons & codons match up & form complementary base pairs.
- Peptide bonds form between adjacent amino acids to form the polypeptide.
- Used tRNA molecules exit the ribosome & collect another specific amino acid.
- The last codon of an mRNA molecule is a stop codon which signals the end of translation.
DNA transfer: Transformation, Conjugation & Transduction
Transformation: uptake of free DNA (competence), most used in labs
Conjugation: transfer of DNA through cell-cell contact
Transduction: transfer of DNA mediated by a virus
Define: Mobile genetic elements & give examples
Mobile genetic elements: bits of DNA which are efficient in transferring from one to another e.g. plasmids, transposons, prophages
Plasmids: useful genes - 3
Naturally occurring plasmids are not essential, but often encode genes that are helpful
These plasmids may be involved in e.g.:
1. resistance to antibiotics or toxic metals
2. metabolic functions (e.g. growth on lactose, sucrose)
3. production of virulence factors (e.g. toxins such as haemolysin)
Molecular cloning - 2
- Obtain a defined sequence of DNA & produce multiple copies in vivo
- The DNA sequence can be a gene, but may also contain non-coding elements e.g. promoter
Molecular cloning - 7 steps
- DNA fragment is isolated, then using enzymes is inserted into plasmid vector
- Mix recombinant cell with E.coli (easy to transform) in the presence of CaCl2, providing a heat pulse
- CaCl2 is used as both membranes are negatively charged
- Heat pulse generates holes, pushing E.coli to be competent for DNA uptake
- E.coli takes up plasmid (which contains a ampicillin resistance gene)
- The cells are placed in presence of ampicillin, only transformed cells can grow due to resistance
- The plasmid replicates in the plate, ensuring each cell which grows is transformed
Obtaining DNA for cloning (known vs unknown)
Sequence of DNA known:
Polymerase Chain Reaction (PCR) is the most common technique
Sequence of DNA unknown:
May require the creation of a DNA library, followed by “fishing” for the gene of interest
PCR - 6 steps
- Identify target region (e.g. a specific gene)
- Design primers that are complementary to the red and green regions
- Heat up to 94 degrees for 30s, DNA is denatured
- Heat up based on primers so the primers can interact with complimentary sequences
- Thermostable Taq polymerase starts at 3 end and moves to 5, binding nucleotides to make new DNA
- The process is repeated roughly 30 times
Restriction enzymes for cloning - 7
- Recognise palindromic sequences: restriction sites
- Restriction sites for cloning usually are 4 or 6 nucleotides
- Cut both DNA strands, creating sticky or blunt ends
- Named after the organism it originates from, plus a number e.g. EcoR1
- When cleaved produces sticky ends due to paired bases facing each other
- Blunt ends are more difficult to stick together
- e.g. CGGCCG reverse is CGGCCG
Agarose Gel Electrophoresis - Purpose & 4 steps
When cut DNA fragment, to determine it’s the right size Agarose Gel Electrophoresis
1. Load on agarose gel
2. Run for an hour at negative charge
3. Stain with fluorescent dye to visualize under UV light
4. Size can be calculated from position of MW markers
DNA Ligase - 4
- ATP-dependent enzyme that links DNA strands
- Plays a role in DNA repair & replication
- Can ligate compatible sticky ends, as well as blunt ends
- Ligation of sticky ends is more efficient than ligation of blunt ends
Important regions for cloning - 2
Important regions for cloning:
1. Selection marker (genes for antibiotic-resistance or growth on specific media)
2. Region where DNA can be inserted
Cloning -5 steps
- Vector prepared using restriction digest, then purified
- PCR of insert, followed by restriction digest, then purification
- Vector & insert combined using ligase
- Prepare competent cells (heat pulse on E.coli)
- Plate cells on colonies, these are screened using Blue white-screening
Blue-white screening
- Plasmid contains lacZ gene, which encodes the enzyme β-galactosidase (degrades lactose)
- If lacZ gene is intact: β-gal active and converts artificial substrate X-Gal into blue dye
- If DNA insert disrupts lacZ gene: β-gal inactive no conversion of X-Gal into blue color
- Colonies with intact lacZ (no insert) are blue
- Colonies with inactive lacZ (with insert) are white
5 Traits for Hosts for cloning and expression
- Grows rapidly in inexpensive medium
- Non-pathogenic
- Is genetically stable
- Has many tools for genetic manipulation
- Allows high level of expression of genes
Recombinant insulin - 4
- short peptides such as insulin are not very stable (due to degradation) in cytoplasm of E. coli
- peptides can be stabilised by fusion to a large protein
- sequence of insulin can be modified if desired
- e.g. amino acid changes in insulin can be used to make insulin fast- or slow acting
Production of recombinant insulin - 4
- Clone insulin A & B chains separately in E. coli, as fusions with gene encoding β-galactosidase
- Purify fusion proteins & cleave off β-gal
- Combine A & B chains & refold in oxidising conditions in vitro
- oxidising conditions are required to form disulphide bonds for stability & activity of the protein
Modulating insulin-release profile - 3
- Mix with protein to slow release (eg NPH)
- Introduce amino acid changes
- Chemical modification
Glargine (Lantus) - 3
Glargine:
1. One deletion and 3 additions: pI 5.4 to 6.7
2. pI close to neutral pH: soluble at acidic pH but precipitates when injected subcutaneously – released from precipitate
3. Slow release
Detmir (Levemir) - 3
Detemir:
1. Modified – Thr30 deleted & fatty acid on Lys29
2. 98% binds to albumin in plasma, from which it slowly dissociates
3. Slow release
Factor VIII
- Essential blood clotting factor
- Used for treatment of haemophilia
- Very large protein of 2332 AA
- Largest recombinant protein that is used commercially
- Glycosylated
Cloning of Factor VIII
- 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 genome; number of copies amplified, & cell line with highest number of copies used for production
Protein structure
Primary – order of amino acids
secondary – alpha helices &beta sheets
Tertiary – 3d arrangement
Quaternary – Different proteins binding together
Protein stabilization - 7
- Hydrophobic interactions (80% internal), hydrophobic on inside, cause of folding
- Electrostatic (repulsions, ion pairing)
- H-bonding: Inter- & intramolecular
- VDW forces
- Steric effects
- Hydration
- Disulphide bridges, links distant area into specific conformation
Protein aggregation - 2
- Folded protein may become partially unfolded into aggregates
- When proteins unfold, hydrophobic residues are exposed, & interactions between multiple can produce fibular molecules or aggregates
Possible causes of protein denaturation or aggregation - 9
Summary: Energy change, hydrophobic interfaces or disruption of interactions.
1. Freezing/thawing
2. Agitation (interfaces)
3. Sonication
4. Contact with silicone oil
5. Low or high pH
6. Low or high salt
7. Specific salts
8. Chemical changes
9. Heat
Consequences of denaturation or aggregation - 4
- Altered solubility: Causes Hypo-potency or Hyper-potency
- Off target binding due to protein structure change
- Patient may generate neutralizing antibodies (anti-therapeutic antibodies)
- Makes drug ineffective, May break tolerance, Cross react with endogenous protein
Deamination - 4
- Low Ph amide can become Aspartic acid
- At 5-12Ph, N attacks amide group, forming either Aspartic acid or Iso-aspartic acid
- Dependant on aspargine residue location, if covered by alpha helices & beta sheets less prone to deamination
- If neighbouring residue large, can inhibit, if no side chain then there is no inhibition
Oxidation - 5
- Can occur from atmosphere or free radicals
- Sulphur residues more susceptible
- Changes shape & activity of proteins
- e.g. Cysteine [O] into Sulphenic acid
- Position & neighbours determine susceptibility
Hydrloysis - 3
- Most bonds stable except aspartic acid, much more easily altered
- Aspartic acid is acid catalysed, protonated then hydrolysed to break amino acid sequence
- Position & neighbours determine susceptibility
Disulphide formation & exchange - 4
- [O] of cysteine to Cystine under alkali conditions.
- Cystine can be cleaved into dehydroalanine which can react with free amines
- Causes cross linking, meaning shape change
- Alterations of Ph changes disulphide bridge melting point, meaning temperature may cause structure to unwind
Excipients used to protect proteins - 6
- Solubility enhancers – e.g. surfactants, amino acids, sugars, polymers
- Anti-absorption & anti-aggregation agents – e.g. surfactants, albumin
- Buffering agents so right pH– usually citrate, phosphate or acetate
- Preservatives & anti-oxidants – e.g. ascorbic acid, antimicrobials (repeated dosing)
- Lyoprotectants/cake formers (powder to protect when frozen)
- Osmotic agents
Amino acids as sacrificial proteins. - 2
- Amino acids can be used to stabilise proteins e.g. Human serum albumin
- Acts as sacrificial molecule if there is an enzymatic reaction which would degrade the therapeutic protein.
Cyclodextrins use with Proteins - 2
- Cyclodextrins can interact with unfolding proteins’ exposed residues
- Prevents further unfolding & aggregate formation
Polysorbate process: (Sorbitan example) - 4
Polysorbate process: (Sorbitan example)
1. Ampicillin molecules with hydrophilic & hydrophobic regions
2. Sorbitan is modified to be amphiphilic
3. Can stabilise proteins by maintaining protein surface or use bond with each region of the protein to prevent further unfolding.
4. Can bind to hydrophobic regions on surface to prevent neighbours aggregating.
Polysorbates use - 4
- Adsorption of proteins to vessels protein is in
- Vessels typically hydrophobic & bind to residues.
- Polysorbate blocks this by binding to hydrophobic material.
- Effects both containers & air
Polysorbates caution - 4
- Can undergo autooxidation or hydrolytic degradation
2.Can form peroxides - Antioxidants used
- Peroxides can form carcinogens (e.g. formaldehyde)
Protein admin IV: Adv 4 vs Dis 5
Adv
1. 100% BA
2. Administration controlled/discontinued
3. Immediate access to central compartment
4. Easy weight-based dosing
Dis
1. Additional manipulation
2. Patient inconvenience/compliance
3. Dose usually diluted into prefilled i.v. bag
4. Adsorption leading to [lower]
5. Risk of microbial exposure
Protein admin SC: Adv 3 vs Dis 3
Adv
1. Patient convenience/compliance
2. May require no compounding
3. Can incorporate an autoinjector
Dis
1. Max volume is lower than i.v.
2. Can’t stop dosing once administered
3. BA is < 100%
Protein admin Intravitreal: Adv 1 vs Dis 3
Adv
1. 100% BA
Dis
1. Patient convenience/compliance
2. Some risk of infection3. immunologically privileged
Protein admin Buccal: Adv 1 vs Dis 2
Adv
1. Patient convenience/compliance
Dis
1. Drug loss, < 100% BA
2. Variability
Protein admin Pulmonary: Adv 2 vs Dis 6
Adv
1. Local delivery
2. Local [high]
Dis
1. Nebulizers typically large & bulky
2. Proteins not stable in organic solvents (e.g. HCFCs)
3. Not all nebulizers are the same
4. Particle size distribution
5. Shear or air-water interface denaturation
6. Testing required for each nebulizer
Protein modification - 3
- Modified to improve PK
- Formed with recombinant DNA technology (i.e. altering gene’s sequence)
- or chemical modification of the protein
e.g.
- Humanization of mAbs
- Modified insulins
Protein mod: Humanization of mouse antibodies - 2
- Mouse antibodies may be altered to chimeric
- Modified so only antigen complimentary region is mouse, rest human
Protein mod: Insulin - 6
- Monomer only at [low]
- Forms Dimer at [higher]
- B24-B26 & B28-B29 crucial for dimerization & binding to IR
- Insulin forms hexamers in the presence of zinc ions
- Excipients in insulin formulations can affect conformation
- e.g. Chloride groups form tense (t6) & relaxed (r6) states
Long acting Insulin - what is promoted?
Modifications to insulins may change quaternary structure, e.g.:
Long-acting use hexamer - promote hexamer formation, reduce solubility, promote binding to plasma proteins
What is promoted in fast acting Insulin?
Modifications to insulins may change quaternary structure, e.g.:
Fast-acting use monomers - prevent formation of dimers and hexamers, increase solubility
Production of Fast-acting Insulin - 5
Fast-acting:
1. e.g. Lysin & proline swapped to destabilize dimerization
2. Promotes maintenance of insulin monomer
3. Proline modified to aspartic acid
4. Inhibits formation of dimers
5. Means monomer promoted: fast absorption
Production of Long-acting Insulin - 2
Long-acting:
1. Arginine added to protein chain, reducing solubility of insulin
2. Less solubility means forms dimers & monomers slower, ergo longer acting
PEG’s (polyethylene glycol) use on proteins - 3
- PEG ligated to proteins, reducing kidney Cl
- Has larger hydrodynamic radius
- Appears larger to filter in kidney, circulates for longer.
PEGylation benefits - 7
- Improves solubility (hydrophilic)
- Reduces protein binding
- Improved BA (reduces serum protein binding)
- Avoids phagocytosis
- Shields antigenic sites
- Reduces toxicity
- Reduces Cl
PEGylation - Specificity - 2
- PEGylation can negatively affect protein activity
- Site-specific PEGylation can be performed so functional regions aren’t blocked:
- PEG-maleimide to not alter activity
- Enzyme-catalysed PEGylation using suitably modified PEGs
Define: Pharmacogenetics
Pharmacogenetics: how genetic variability influences drug treatment outcomes. Often focused on specific genes
Define: Pharmacokinetics (PK)
Pharmacokinetics (PK): the movement of drugs through the body
ADME: absorption, distribution, metabolism, excretion
Define: Pharmacodynamics (PD)
Pharmacodynamics (PD): relationship between [drug]/ dose & response (pharmacologic/ toxicologic)
Define: Pharmacogenomics
Pharmacogenomics: broader than pharmacogenetics (collective influence of entire genome rather than specific genes)
Personalized medicine - 3
- Close relatives of pharmacogenetics
- Covers also non-genetic factors (weight, other diseases, diet…)
- Potential to tailor drugs to individuals
Define: Genotype
Genotype: an individual’s collection of genes (differences at the genetic level)
Define: Phenotype
Phenotype: observable traits (includes observed differences in enzyme/ receptor response)
Define: Polymorphism - 3
Polymorphism:
1. genetic morphs that affects PK & PD.
2. Polymorphisms can occur from insertion, deletion, tandem repetition of segments.
3. Can also occur from SNP (single nucleotide polymorphism): single allele change – nucleotide variation
Genetic polymorphism affects - 6
- 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
- Cause adverse response
Human genome product - 4
Human genome product:
1: take sequence of a known gene & find instances of SNPs
2: perform clinical association studies & cell-animal based studies
3: use SNP map to associate polymorphism to phenotype
4: use pharmacogenetic profile of patient to change drug response
Cytochrome P450 - 5
- Cytochromes P450 (CYP): most important family of genes contributing to Phase I metabolism in a range of drugs
- Phase I (oxidative & hydrolysis reactions): e.g; CYP2D6, CYP3A4,
3.Each encoded by different genes - Sig. population lack CYP2D6 or CYP2C19 enzymes (alleles encoding inactive forms)
- Some have higher activity: ultrarapid metabolizers
TPMT - 3
- Phase II (conjugation reactions using transferases): e.g., thiopurine methyltransferase (TPMT)
- Some people lack TPMT (inactivating genetic polymorphism on both copies of gene)
- TPMT helps body process thiopurine-based drugs (which inhibit the immune system)
Define: Poor metabolisers
Poor metabolizers: 2 loss-of-function alleles (homozygous). Half-life of prodrug longer
Define: Intermediate metabolisers
Intermediate metabolizers: 1 loss-of-function allele (heterozygous)
Define: Ultrarapid metabolisers
Ultrarapid metabolizers: gene duplication or gain-of-function alleles (homozygous/ heterozygous). Half-life of prodrug shorter
Anti-coagulant agent - 2
Anti-coagulant agent
1. Polymorphism in CYP2C9 & vitamin K epoxide reductase required for the oxidation could lead to slow oxidation, & potentially death.
2. Genotypes used to identify polymorphism in metabolism to prevent possible harm.
Dose variation of TPMT - 3
Immunosuppressants
1. High levels of thioguanine nucleotides in population without TPMT enzyme
2. High levels associated with myelosuppression & use of thiopurine drugs could lead to serious toxicity
3. Determine TPMT status before thiopurine drug is prescribed
- measure levels of enzyme in RBCs
- genotype for common variant alleles
- Consider reduced dose
Pharmacogenetic polymorphism - 2
- Pharmacogenetic polymorphism is higher when parent drug is already active.
- Means prodrugs less impacted by polymorphism.
Pain management - 4
Pain management:
1. Some Codeine converted to norcodeine & codeine-6-glucuronide
2. CYP2D6 required to metabolise codeine to morphine (most active metabolite)
3. Variation in metabolism can result in accumulation of drug or none production.
4. e.g. neonate died from morphine poisoning because mother was ultrarapid metaboliser, morphine accumulated in breast milk
Clopidorgel - 4
- Clopidogrel: antiplatelet prodrug
- CYP2C19 contributes to activation steps
- Drug not as effective in poor metabolisers
- Alternative antiplatelet drug recommendation: prasugrel or ticagrelor
MDR1 polymorphism - 3
- MDR1 gene: codes for P-glycoprotein (P-gp)
- Efflux transporter designed as a defense against xenotoxins (pumps substrates from inside of cells to outside)
- Polymorphism in MDR1: altered BA, modified disposition of some drugs, no response to some drugs used to target specific tissue diseases
Idiosyncratic toxicity: Abacavir - 6
Idiosyncratic toxicity – toxic to only some populations
Abacavir
1. Drug used to treat HIV
2. Hypersensitive: skin rash, GI & respiratory symptoms
3. Re-exposure potentially fatal
4. Association between hypersensitivity & HLA haplotype
5. (polymorphism by combo of alleles or set of SNPs on the same chromosome)
6. Genetic testing before administration
