Pharmaceutics (C&T) Flashcards
What are the purposes of each stage of a clinical trial?
Phase 1 = Safety of the medicine
Phase 2 = Focused group of the population
Phase 3 = Open to whole population, this shows a sufficient requirement for the drug – dosing is selected from the phase 2 trial for safety and efficacy
Phase 4 = Post approval, screening through genetic variation to see who will benefit the most
What are some factors that affect a drug’s PK-PD?
What is a potential problem with the usage of drugs?
Drug targets (receptors)
Metabolising enzymes
Transporters (Absorption, efflux, access to action and elimination sites)
They may have off target binding sites — disrupts the homeostatic/physiological function of transporters/receptors.
Drugs rarely only bind to one receptor (allosteric binding?)
Usually intended to protect the individual from the environment
What are some drug targets?
Transporters
Receptor gated channels
GPCR
Nuclear receptors
Enzymes
How might genetic variation affect drug responses?
Drug receptors: altered availability of receptors (Less transcription and lower stability)
Altered affinity of receptor to the drug
Why can’t the use of corticosteroids be stopped suddenly?
The corticosteroid given to the patient takes over the steroidal secretion role of the adrenal glands.
System detects corticosteroids present in the blood, and hence the secretion is down-regulated in the adrenal gland for homeostatic levels to be maintained.
Sudden cessation would therefore have fatal consequences — body can not react adequately to infections and cause inflammation. Thus corticosteroid use has to be slowly reduced, so there is no adrenal gland shutdown.
Give a few ways the body can react to drugs.
Therapeutic response
–Non response
–Hyper response
Adverse response
– Concentration-dependent adverse response
– Idiosyncratic adverse response (Side effect without a known cause)
– Immunological responses (Time dependent development, or genetic)
What is a benefit of an oral lipophilic drugs?
Does not directly go through the liver – goes through the lymphatic system (No hepatic first pass effect).
Thus potential for higher bioavailability.
Give some processes involved in absorption across the GI membrane
Passive diffusion
Transporter mediated
– Influx transporters (Pept1 – nutrient absorption, oligopeptide transporters)
– Efflux transporters (P-gp)
Pre-systemic metabolism in intestine &/or liver
What are the main ways of elimination.
Phase 1 metabolism
– oxidative CYP450 enzymatic reactions
– hydrolysis (esterases, epoxide hydrolases)
Phase 2 metabolism
– conjugation reactions with UDPGT
Filtration
– Transport
– Secretion (transport -> urine)
– Reabsorption via passive diffusion or transport urine to the blood
Describe the transporters present in the blood-brain barrier.
Explain the purpose of this structure
Similar to the GI, contains P-gp to rapidly export molecules that cross the barrier.
Limits access to the target sites in the brain.
BBB is a potent barrier – brain susceptible to any change, (e.g. change in pH could cause death)
Explain what a genotype and phenotype is.
Genotype = an individuals full hereditary information (born with it)
Phenotype = actual expressed properties In debrisoquine,
What is the effect of genetic variability in:
A: Drug metabolising enzymes/drug transport proteins.
B: Drug target/target pathway
A.)
Under exposure = No efficacy, no adverse event
Appropriate exposure = Efficacy, no adverse event
Over exposure = Efficacy, dose related adverse event
B.)
Responders
Response and target related/off target adverse event.
Or response and no adverse event
Non responders
No response and target related/off target adverse event
Or no response and no adverse event
What are the risks of warfarin.
How does it elicit its effects?
Large variation in the dose response, as well as a narrow therapeutic range.
Monitored via INR – target is about 2.5 in patients
Greater than 4 risks haemorrhages
Warfarin is a coumarin anticoagulant.
It inhibits VKORC1 (Vitamin K epoxide reductase complex subunit 1)
–> Antagonises hepatic recycling of Vitamin K
–> Reduction in activation of some clotting factors (Other pathways for clotting are still present)
Describe the use of codeine as an analgesic and the genetic variations in the metabolism.
Codeine is partly metabolised by CYP2D6 into morphine
–> Morphine elicits the analgesic effects
Genetic polymorphism can reduce the synthesis of CYP2D6. This results in a poorer analgesic effect in the patient (slow metabolisers)
What are the different types of metabolizer?
Extensive = carries 2 functional genes
Poor/slow = lack functional enzymes or deleted genes
Intermediate = carry 1 functional and one defective, or both partially defective alleles
Ultra-rapid = more than 2 active genes
Where is P-gp found?
What does it result in.
What are the effects of polymorphism here
Important for drug absorption, distribution and elimination.
Mainly affects hydrophobic, and/or organic cations at physiological PH
1< aromatic rings, MW > 400
Anticancers, antibacterial, immunosuppressants, ß-blockers, calcium channel antagonists and HIV protease inhibitors
Not only drugs…
Liver
Kidney
Intestine
Pancreas
Placenta
Adrenal gland
Endothelial cells of the blood brain barrier
Prostate
Lung
ATP-dependent efflux pump – prevents xenobiotic entry
Apical expression of membrane results in…
- reduced drug absorption from GI
- enhanced drug excretion into bile and urine
- impeded entry into the CNS
Modifed BA
Altered disposition to some drugs
Non response to drugs
What is TPMT?
What occurs in heterozygous patients? Homozygous?
Thiopurine methyltransferase
Catalyses S-methylation of thiopurine agents – azathioprine, mercaptopurine, thioguanine (Leukaemia, rheumatic diseases, IBD, solid organ transplants)
Thiopurine agents are activated by HPRT (hypoxanthine phosphoribosyltransferase) to provide thioguanine nucleotides (active entities produce cytotoxicity)
TPMT inactivates these cytotoxic agents via methylation or oxidation
Haemopoietic tissues only have TPMT as the inactivation pathway for thioguanine nucleotides. Low activity of TPMT therefore results in a high risk for severe and fatal haemotological toxicity
Heterozygous = intermediate activity
Homozygous = very low / no activity
– patients must be identified in order for the correct dose to be given to prevent toxicity
What are some key future developments with respect to pharmacogenomics?
Genetic drug response profiles
–finds individual genetic fingerprint in order for a probabe drug response to be found
–personalised medication
Comparisons of old and new drugs
Cost effectiveness?
What are some advances to be made in recombinant DNA
Greater efficiency, cost effective, safer production of therapeutic proteins (insulin and factor VIII)
Produce rare proteins that have therapeutic potential in sufficient quantity for pharmaceutical value (inteferon)
Production of vaccines (hep B)
GM food
What are the hosts required and applications for the production of:
Insulin
Factor VIII
Antithrombin (Atryn)
E. coli, yeast – diabetes
Mammalian cells – haemophilia (poor blood clotting)
Goat – antithrombin deficiency
What is the purpose of recombination of DNA?
Natural processes
– DNA repair
– Multi drug resistance
Recombinant DNA technology
–Analysis of the function of genes and the products
–Expression/regulation studies
–Production of industrial and pharmaceutical products
What direction do polymerases synthesise DNA
5’ -> 3’ direction
Genes always run 5’ - 3’
Properties of bacterial genes
Monocistronic
One mRNA forms one gene
Polycistronic
One mRNA forms 2< genes (organised in an operon)
Other elements present?
– binding sites for regulatory proteins
– transcription terminators
Properties of eukaryotic genes
Monocistronic only (each gene has its own transcriptional control) – no operons
mRNA processed prior to translation
– splicing of introns
– addn of 5’ 7-methylguanylate cap (m7Gppp)
– polyadenylation adds polyA tail after stop codon
n.b. bacterial genes do not contain introns, so cannot clone DNA that have introns in bacteria
How is DNA transferred naturally in the body
Transformation (uptake of free DNA)
Transfection = human
Competence = bacteria
Conjugation – transfer of DNA through cell-cell contact
Transduction – transfer of DNA mediated by virus
Mobile genetic elements
– Plasmids
– Transposons and insertion seuqnce elements
What is the purpose of plasmids?
Naturally occurring plasmids are not essential, but they encode for helpful genes
– antibiotic resistance/toxic metals
– metabolic functions (allowing growth on lactose or sucrose)
– production of virulence factors (haemolysin)
What is molecular cloning
Produces multiple copies of DNA from a defined template in vivo.
DNA sequence can be a gene, but can have non-coding elements (promotors)
What are the steps to molecular cloning?
Isolate the source DNA – restriction enzyme used to cut out the required sequence and also used in the plasmid so that the DNA can be inserted to that area
Insert the source DNA into the cloning vector
– DNA fragment enzymatically inserted into the DNA of a plasmid vector using DNA ligase
– Marker (ampicillin resistance gene and lacZ gene) also inserted so to identify which have taken up the DNA fragment (white colonies) and kill the hosts that have not taken it up (blue colonies).
–> Recombinant plasmid formed
Introduce the recombinant plasmid into a host organism
– E. coli is mixed with the plasmid with CaCl2 – heat pulse
– Culture the bacteria on nutrient agar plates impregnated with ampicillin
–> Cells that have been transformed survive, those that did not take up the plasmid die
Plasmid replication then occurs to produce more of the inserted DNA
—> Cell multiplication occurs producing a large quantity of cells containing the desired DNA
