End of semester test Flashcards
Target concentration
Target concentration approach links PK with PD to predict the right dose for a patient. A target effect might be pain relief. Ideal dose prediction requires individual estimates of Emax, C50, V and CL. To find the target you randomises concentration controlled trials. PK/PD varies because of systematic (body size, disease state and genotype) and Random (between/within-subject variability) variables.
= (Target effect x C50)/(Emax - target effect)
Theophylline is metabolised by
CYP1A2, which is induced by polycyclic hydrocarbons in cigarette smoke. Enzyme induction reduces variability because there is a max biological limit in the extent of enzyme induction.
Three ways to dose: population
Is commonly used, but patients all receive the same dose, meaning that some patients are over/under dosed.
Three ways to dose: Group
Is when the same dose for similar groups is given, e.g. the same weight, CLcr (CL creatine) and genotype (more so in kids)
Three ways to dose: Individual
Dose is determined by individual response e.g. by BP, international normalised ratio, blood concentration. Used when the within-subject variability is large and predictable individualisation is not really possible.
INR
measures the degree of change in coagulation properties of blood
Target clearance
- Uses responses such as BP as a substitute for being able to measure the clinical disease state that is being treated.
- When medicine is working well or not working at all the clinical disease state may appear to be the same.
- Is used when group based dosing is not enough to reduce the between-subject variability so that the drug can be used safely and effectively.
- Can only work however is the within-subject variability is small enough so that dose individualisation is really predictive for future use of the medicine in the same patient.
Measuring Concentrations
The least informative time to do this is just before the next dose (trough) unless this is paired with another peak. This is because CL determines the average concentration, so measuring concentration in the middle of the dosing interval will be closer to the average and therefore better at predicting CL. A concentration in the middle of the dosing interval (Ctmid) will be closer to the average steady state concentration (Css) then either a peak or a trough. CL= does rate/Css which is approximated by CL= Dose rate/ CTmid.
Gentamicin and dosing intervals
Concentrations vary widely in a dosing interval so two concentrations are needed to reliable estimate CL. The trough concentration at 24h is often unmeasurable because it is below the limit of quantitation. Concentrations are best measures 1h and 8h after the dose.
Therapeutic drug monitoring
Traditional concept, that if the dose is within the therapeutic range then it is not adjusted. A concentration at the bottom of the range (ineffective) is very different from one at the top (possibly toxic), but TDM usually ignores this and are happy to do nothing as long as it is within range.
Target concentration intervention:
Is a science-based method that uses PK and PC principals to identify who patients are different and uses PK-guided does individualisation to achieve a precise target. It has been shown to improve clinical outcome as well as being cost effective.
Three was to think about time course effects
- Drug effects are immediately related to observed drug concentration
- Drug effects are delayed in relation to observed drug concentration
- Drug effects are determined by the cumulative action of drug
Law of mass action
the binding of a drug to a receptor should follow a hyperbolic curve, it is assumed that effect is directly proportional to the binding then the C50 will be the same as Kd
Kd
is the equilibrium binding constant, and the concentration at which 50% of the binding sites are occupied
Emax model
E = (Emax x conc)/(C50 + conc).
- The model is the description of a concentration-effect relationship.
- An important prediction of the model is that all biological systems will reach a maximum.
- Many drugs will have a steeper concentration and effect relationship so that a smaller change is required to see the same change.
The sigmoid Emax model
E = (Emax x conc^hill)/(C50 + conc^hill).
This model is showing 4 different values for the hill coefficient.
When hill >10
The concentration effect relationship is like a switch, the effect turns on at a threshold concentration close to the C50.
When hill = 2
It only takes a 4 fold change in concentration to go from C20 to C80.
When hill <1
the curve is shallower than Emax
When hill >1
the curve is steeper than Emax
When hill = 1
the curve is the same as the Emax model
Conc peak = 10 x C50
This shows how the time course of immediate drug effect depends upon the initial concentration as well as the PK of the drug. Shows the time course concentration after a bolus dose at time zero. Initial conc is 10 x the C50 and produces and effect that is 90% of Emax. After one-half life, the concentration is halved but the effect has changed by less than 10%. As conc falls the effect disappears more quickly
Conc Peak = C50
Initial concentration is the same as the C50 then the initial effect will only be 50% of Emax. At these lower concentrations, the time course of effect is almost parallel to the time course of concentration.
Conc Peak = 100 x C50
When a very big dose is given, the initial effect is close to 100% of Emax. The effect changes very little despite the big changes in drug concentration. After more than 5 half lives when nearly all the initial does will have been eliminated the effect will still be 70% of Emax. This is common for receptor agonists.
Time Course
Time course of effect can be described the three regions by considering if the concentration is about C80 or below C20.
- When the concentration is above C80 the curve is almost flat.
- When the concentration is low, below C20 the curve is almost exponential. The time course of concentration and effect are almost parallel to one another. This is the only time where you can describe the effect as having a “half life.”
- In between C20 and C80 the time course of loos of drug effect is almost a straight line.
Doubling the dose
Prolongs the duration of effect to nearly one half-life, at this time the concentration is equal to 10 mg/L- with the same level of effect (50) that was used to mark the end of response for the lower dose. Doubling the dose does not lead to doubling of effect. Doubling the dose will increase the duration of effect by one-half-life. Concentration-effect curves are non-linear (Emax model) effect do not increase in direct proportion to the dose.
1942 - Goodman/Gilman
Chemo is shown to reduce lymph gland cancer (nitrogen mustard)
1958 - Hertz/Li
Methotrexate cures choriocarcinoma
1978 - Rosenberg
Anticancer platinum complex discovered and approved for medical use to cure testicular cancer. Has a broad range of solid tumour activity (testicular, ovarian, cervical, bladder, head and neck, lung, colorectal and other cancers)
Cancer 1998 (Trastuzumab)
targeted therapies involving specific molecules involved in cancer development and progression. Potential for more effective, less toxic and individualised cancer therapy. Trastuzumab (Herceptin) approved for Her-2 positive breast cancer.
Cancer 2001
Imatinib (Glivec) approved for chronic myelogenous Leukaemia
Cancer 2011
immune checkpoint modulation: Monoclonal antibodies binding Cytotoxic T-lymphocyte Antigen 4 (CTLA-4) (eg. ipilimumab) or programmed death 1/programmed Death ligand 1 (PD1/PDL1) e.g. nivolumab. Results in durable responses in subgroups of patients with melanoma, non-small cell lung cancer and others. Immune-related side effects, colitis and dermatitis.
Chronic myelogenous Leukaemia:
Is the acquisition of the Philadelphia chromosome [t(9;22) translocation], abnormal fusion protein (bcr-abl) and self-sufficiency of growth signals. Survival of CML patients treated with Imatinib had a 95% survival rate over the first few years
5 levels of selection for animal tests - species/strain
May not have the relevant target, may be subject to diurnal variation, time of dosing might be important
5 levels of selection for animal tests - end-points
pharmacological, direct toxicity (skin irritancy), genotoxicity( bacterial mutagenicity tests) and immunotoxicity (immune suppression/allergic reactions)
5 levels of selection for animal tests - dose
response curves, toxicity can be represented on a log dose response curve. Populations, due to non-reversibility of many toxic end points it is not possible to look at the response of a tissue to increasing doses, instead, the dose required to produce a desired end point is studied in population. Acute toxicity taste, determine the effects that occurs within a short period after dosing, only a single dose given by different routes.
Testing involves 5 levels of selection for animal tests- what are they?
Species/strain End-points Dose Route Duration of tests.
C x T = K (Harber’s rule)
noted that exposure to a low concentration of a poisonous gas for a long time often had the same effect as exposure to a high concentration for a short period of time.
Sub Acute toxicity tests
Involve exposing animals to the compound for 28-90 days. Exposure is frequent and usually daily. The test provides information in the target organs and major toxic effects, that may have a slow onset can be detected. Clinical measurements may indicate the development of any pathological lesions.
Chronic toxicity tests
involves lifetime exposure of animals to the compound, changes in simple measurements (weight, food) can be recorded. The choice of dose, species, strain, route are influenced by the type of chemical. Often rats and dogs are used, and the drug is often administered in the food, or inhabited.
Limitations of animal testing
limited choice of species, species switching between taste, interspecies variability in metabolism and response, lack of subjective ADR, and lack of suitable human models.
ADR
leading cause of medical injury. Most reactions are definitely or possibly avoidable.
Type 1 ADR
predictable, dose-dependent based in the known pharmacology of the drug.
Type 2 ADR
not predictable, no clear dose dependency and not due to the known pharmacology of the drug.
Type A ADR - agumented.
predicted from known pharmacology of the drug, usually drug-dependent and reduced by dose reduction.
Type B ADR - bizarre
not predicted from basic pharmacology on simple dose-response relationship
Type C ADR - chemical
biological characteristics can be predicted or rationalised in terms of chemical structure of the drug
Type D ADR - delayed
Occur after many years of treatment
Type E ADR - end of treatment
due to withdraw of treatment, especially if done suddenly
NSAID induced gastro-duodenal ulceration
Ulceration/bleeding reported in 15-30% of chronic users. NSAID may induce lesions by interacting with phosphatidylcholine and reduce the ability of gastric mucosa to protect itself from e.g. HCL. Inhibition of COX important because some prostaglandins are cytoprotective and so initial lesion results in overt damage.
Renal toxicity (acute)
PGs are crucial for maintenance of renal perfusion through renal blood flow. PGI2 and PGE2 cause vasodilation ( inc bf, dec proximal reabsorption of na+). Renin release controlled by PIG1 so NSAIDs can influence RAAS (dec aldosterone recreation, so inc k+ - hyperkalemia). HETES inhibit Na+/K+atpase. NSAIDs may reduce the effectiveness of diuretics.
Acute ischemic renal insufficiency
May occur within hours of the initial dose of susceptible patients. Characterised by a marked decrease in urine, weight gain, increase BUN, increased serum creatine. Readily reversed when drugs were withdrawn.
Analgesic-Associated Nephropathy
may be secondary to acute interstitial nephritis, and requires continuous analgesic abuse over many years. Patients present with hypertension, GI ulceration, UTI, headaches, depression and CVD. 5-year survival (50%). Involves necrosis within the loop of hence and medullary capillaries spreading throughout the papilla.
Importance of Metabolism
metabolism may lead to the formation of a chemically-receive species that binds to and inhibits the biological function of a macromolecule. Formation of toxic metabolites may be influenced by dose, inter-individual variability in enzyme expression of PK interactions.
Inter-individual variation in drug metabolism
- Lack of metabolism: enhanced plasma concentrations and exaggerated pharmacological responses.
- Lack of a metabolite pathway in certain individuals: compound is bioactivated in a different enzyme
- Enhanced toxicity: due to the lack of a detoxification pathway
- Lack of bioactivation pathway: poor metabolisers are at less risk
- Increased protein expression or catalytic activity: with subsequent increase in the formation of toxic metabolites.
Paracetamol:
In an overdose, normal pathways of metabolism are saturated, so other routes of metabolism may take place. Metabolites can bind to and destroy key proteins, leading to cell death in the liver, potentially leading to liver failure and eventually death. Loss of intracellular calcium regulation disrupts mitochondrial function and leads to necrotic cell death. The toxic metabolite is a quinonimine that can react with sulfhydryl groups in critical cellular proteins.
Drug-drug interaction
is the effect of one drug in influenced by the co-administration of another drug, which may be desirable or not. Two or more compounds act at the same receptor or on the same pathway. You can get additive effects (increase the effect) or get an antagonistic effect (cancel each other out) if they work in the same pathway you may get the synergistic effect (more simple than additive).
Anna Nicole Smith
Toxic combination of lots of drugs many that acted on the GABAa receptors that were designed as anti-hallucination or sleep drugs, but dampened her response so much she sedated herself to death
DDI Absorption
Reduced uptake —> decreased effect. For example some antibiotics form insoluble complexes with metals which decrease the absorption of the drug.
DDI Metabolism
reduced clearance, increased plasma concentrations —> too much effect. Inhibition of one pathway, greater clearance through bioactivation pathway —> increase toxicity. Induction of enzymes, decreased plasma concentrations. EG. drug B or chemical can inhibit the enzyme that metabolises drug a. Not used as a strategy to increase drug concentrations unless drug a has a very short half life. Simply give more of drug a or more often. Or e.g. drug b/chemical can induce the enzyme responsible for the metabolism of drug A, therefor plasma concentration of drug a is less than expected (can lead to therapeutic failure). EG st johns wort - a potent inducer of enzymes involved in the metabolism of .70% of all drugs.
DDI distribution
competition fro uptake transporters or protein binding, increased plasma concentrations —> +/- effect depending on target
DDI Excretion
competition for efflux transporters, decreased elimination —> increased prolonged plasma concentrations
Antimalarial therapy DDI
combination kills parasites faster by synergistic action, and reduces the potential for the development of parasite drug resistance.
Paracetamol and Ibuprofen DDI
Combination gives faster and better pain relief, no PK interaction but mat increase chance of adverse effects.
Pharmacokinetics interactions: Mixing
Effects can be mixed, on a single dose there may be inhibition, on chronic therapy, there may be induction. Drug A may have one type of interaction with drug B and a different interaction with drug C.
Drug Food interactions
Food may alter absorption - solid foods ten to delay gastric emptying nut non-nutrient liquids may the the opposite effect. Dairy products containing Ca2+ may produce clinically significant reaction in Cmax of for example some antibiotics. Food may alter metabolism - caffeine metabolism by CYP1A2 inhibits clozapine metabolism, so caffeine consumption may influence effectiveness in schizophrenics. Grape fruit juice has a number of clinically relevant interactions with drugs metabolised by CYP3A4.
Alcohol interactions
Alcohol may alter absorptions - studies have reported gastric emptying may be increased, decrease or not effected, dependent on the dose/concentration of ethanol. Alcohol (acute, high dose) can inhibit drug metabolism - decreased warfarin metabolism, increased anti coagulation, decreases benzodiazepine metabolism. Alcohol (chronic) can induce drug metabolism - increased phenytoin elimination and benzodiazepine metabolism. Alcohol can reduce threshold of drug toxicity - through the depletion of chemical protection. Alcohol can enhance renal elimination - its a diuretic. Alcohol had a pharmacodynamic interaction - particularly through GABA receptors.
Clozapine and smoking:
an atypical antipsychotic metabolised by CYP1A2 - differences in CYP1A2 activity account for ~ >70% of variance in plasma. Smoking increases CYP1A2 —> increased metabolic clearance —> decreased plasma. Doses of clozapine titrated at imitation of therapy - if patient stops smoking, decrease CYP1A2, decreased metabolic clearance —> increased plasma. Can lead to sedation, fatigue and seizure. Needs to be considered if patient stops smoking cessation therapy - clozapine level after smoking cessation equals the baseline level, admission to a smoke-free hospital may be a problem.
(Hyper)polypharmacy:
Polypharmacy coined as a term to refer to taking several medicines at the same time, generally refers to 4 or 5+ medicines per day and is associated with increased risks of advert drug reactions, inappropriate dug use, hospitalisation, morality. Hyperpolypharamcy refers to 10 or more medicines, which has given rise to the concept of deprescribing.
Strategies to counteract poisons:
Using PK and PD approaches to decrease absorption, neutralised the chemical or metabolite so that it cannot react with endogenous targets, enhance elimination, antagonise the effect or replace the activity. Usually used in combination.
Decreased absorption: Ipecac
consists of Cephaeline, which stimulates the central vomiting centre and emetine, which activates sensory receptors in the proximal small intestine. Contradicted for substances that could cause further damage during emesis (dishwashing powder will re-burn on the way up). Never demonstrated to improve clinical outcome. But will affect absorption other antidotes.
Decreased absorption: Activated charcoal
Drug may absorb onto the charcoal and thereby prevent absorption in the first place. It may create a concentration gradient across the mesenteric vasculature such that the drug or metabolite is eliminated taster. Useful for some drugs that undergo substantial enterohepatic recirculation, or small volume of distribution or low preteen binding and are highly absorbed by charcoal. Dose usually 1-2g/kg orally or nasogastric tube.
Neutralise the chemical: Iron
ingestion of a large amount of iron overwhelmed the GI regulatory mechanisms resulting in massive iron absorption (20-60mg/kg - mile toxicity, 60+ mg/kg potentially life threatening). When serum iron levels exceed the capacity of the binding protein, transferrin, severe toxicity may occur secondary to the deposition of iron in soft tissue. Free iron causes toxicity by directly injuring the intestinal mucosa and generating oxygen free radicals . Stage one symptoms include vomiting or diarrhoea, persistent tachycardia, hypotension, and altered mental stat after a relatively short period. Stage 4: 2-4 days post ingestion, characterised by major organ system failure, hepatic necrosis, renal failure, metabolic acidosis, coal and death.
Neutralise the chemical: Paracetamol overdose
In paracetamol overdose, paracetamol is metabolised to a protein-reactive quinonimine that can react with the sulfhydryl groups in proteins. High doses cause liver failure and death.
Enhance Elimination: Salicylate and Urinary Alkalisation
Aspirin is readily hydrolysed to salicylate which stimulates the medullary respiratory centre, production hyperventilation, respiratory alkalosis and eventually metabolite acidosis. Uncouples oxidised phosphorylation increasing gluconeogenesis and lipid metabolism. produces tinnitus, nausea, vomiting, ataxia, come and hyperthermia. Ingestion >300mg/kg is often associated with serious toxic reactions. Ingestion of >500mg/kg is potentially fatal. Sodium bicarbonate is used to raise the urinary pH >7.5, weak aside with pka <7 that undergo significant urinary excretion become trapped in the kidney tubular fluid. Used for salicylate, poorly monitored infusions may produce too high pH, which impairs cardiac contractility plus hypernatremia and fluid over load may occur. Used in conjunction with activated charcoal.
Heroin and Naloxone
overdoses are frequent when used in conjunction with other substances. There are many routes of administration. This drug produces typical narcotic effects because it is converted to morphine, conscious but sedated, shallow but deep breathing, unconsciousness. Is also associated with coma, seizures and delayed encephalopathy in overdoses. A lethal dose is hard to define.
Antidote for heroin
Naloxone is an antagonist at the U, K and S opioid receptors. Drugs may have to be administered via the intralingual route. Naloxone has a shorter half life than herosin so relapse may occur, it may also precipitate withdrawal.
Warfarin and Vitamin K
Warfarin is used one the prophylaxis and treatment of venous thrombosis and pulmonary embolism, It inhibits synthesis of VIT k dependent coagulation factors. Resulting in sequential depression of factors 2, 7, 9, and 10. In overdose appearance of blood in stools or urine, excessive menstrual bleeding, persistent oozing from superficial injuries. Necrosis or gangrene of the skin and death.
Antidote for warfarin
Vitamin K - needed potentially for weeks until blood coagulation returns to normal. Vit K is reduced (at high concentration of warfarin) to the hydroquinone by another warfarin insensitive live reductase. no cycling because Vit K epoxide reductase still inhibited. Normal body stores are depleted after 2-3 hours.
Paracetamol antidote: N-Acetyl Cystine - neutralisation
The antidote acts as a precursor for glutathione synthesis and so boosts the resynthesis of this vital intracellular agent and thus prevents liver damage. The sulfhydryl group of the antidote may bind and detoxify the metabolic directly or may act as an antioxidant and block reactive oxygen species dependent cell death.
Antidote for iron overdose: Deferoxamine - neutralisation
Deferoxamine messmate is an antidote for iron poisoning, it chelates the free iron to form feroxamine, but does not remove iron from proteins (such as transferrin, or haemoglobin). Feroxamine is excreted unchanged in the urine.
In vitro disease models
- test out new drugs (efficacy and toxicity)
- understand disease pathogenesis (better ability to identify new drug targets, we don’t know all drugs mechanism of action, end stage drugs)
- identification of new drug targets.
- We want an ideal disease model (human cells), same disease processes that you see in the patients and have some genetic variability.
- Identify and validate new drug targets, effectiveness of new therapies and prerequisite to clinical testing.
Human embryonic stem cells (hES)
- pluripotent and continue to undergo division (symmetric or asymmetric)
- able to generate all tissues in the body
- obtained cells from the blastocyte, can form any tissue in the body depending on the developmental cue
- Human variability, new ability to look at disease modelling.
- Can we model diseases —> we don’t know what disease this embryo has the potential to develop, but we can model diseases because we can use gene therapy to change disease mutation (induce a mutation), only works with diseases with genetic mutation through genetic manipulation
Primary human cells
- isolated from human tissue, heart, lung, blood, but not all tissue types (can’t culture adult brain cells) however have a limited life span (2-3 weeks).
- more closely mimic the physiological state of cells in vivo and generate more relevant date representing living systems.
- NOTE: Immortalised cell lines may be biologically altered.
- Human cells each time are slightly different. May be disease relevant (tumour cells).
- Difficult to culture.
- To identify a new drug target we need a new disease process, therefore immortalised can’t be but this might be able to.
immortalised cell lines: SH-SY5Y
neuroblastoma, also from a tumour.
- Modelling in Parkinson’s disease = human neuroblastoma —> like to turn into dopamine cells, so often used to test drugs for Parkinson’s.
- However not a good model as it originated from cancer, changed how dopamine cells act
immortalised cell lines: PC12 cell line
- Embryonic neural crest rat
- transformed to continue to undergo division.
- Modelling in Parkinson’s disease = rat cells, also make dopamine cells
immortalised cell lines: HeLa cell line
- polio vaccine developed
- is a natural mutation,
- taken from tumour biopsy from patient lacks
- most widely used cell lines in the world
- Naturally dividing cell line
immortalised cell lines
- most commonly used, (polio vaccine
- population of cells from a tissue source that would not normally proliferate, but due to a mutation they keep dividing.
- Continuous supply of cells, limited variability, and easy to culture and manipulate.
- No human variability, will act exactly the same every time (epigenetics), not a real disease model, don’t really act like “real” human cells (no human variability, not relevant for a clinical situation)
- Not a real disease model
Cell reprogramming (induced pluripotent stem cells):
- able to use skin cells from patients
- pluripotent, continue to undergo division,
- able to generate all tissues in the body.
- Reprogram mature cells (skin cells), that are easy to obtain
- use gene therapy to over-express (oct3/4, sox2, klf4), which induce embryonic development.
- Fibroblasts turn into cells that are the same as embryonic stem cells.
- This means we are close to the gold standard of in vitro cell models.
EXAMPLE: Skin cells from a patient with Parkinson’s, turn them into embryonic stem cells, expose them to chemicals to produce dopamine cells, so we can study dopamine cells in Parkinson’s disease. Advantages - ethics (just using skin tissue), can model disease process —> because you have a clinical history of the patient therefore we are basically looking at the patient themselves. - Non-genetic disorders don’t necessarily show up, so this model looks at genetic disorders and genetic mutations. But can look at some genetic changes that produce the disease phenotype.
- Model human disease - live human cells, variability, investigate pathogenesis, identify new targets and drugs, test new drugs - efficacy and toxicity —> leads to drugs that are more effective when they reach clinical trials, ability to have human variability is important.
- Disadvantages- takes a long time, 3 months to generate mature cells, low yells, variability (different results every time, however, if the drug works it will work regardless of the variability)
Human organoides (ex vivo model in between in vitro and in vivo and is a 3D model)
- Generated from human pluripotent stem cells
- 3D structure
- cells align correctly as would in vivo and allows proper cell connection.
- Organoides generated: gut, brain, liver and pancreas
- no more developed than a 2-3 month gets brain.
Huntington’s:
- Genetic disease, 50% chance that you will get the disease if you parents have already got it.
- Looking at factors that stop cells from dying.
- Movement disorder, selective loss of GABA medium spiny neurons (MSN) in striatum (basal ganglia), also loss in motor cortex and prefrontal cortex, GABA - inhibitory neurotransmitter.
- Due to expanded CAG repeat in the IT50 gene (>36 genes, normal person about 20 repeats),
- Autosomal recessive
- CAG expansion increases with each generation —> age of onset correlates with the length of expansion usually about 40yrs.
- Symptoms: uncontrolled movements , depression, psychiatric issues, speech impairments.
- Huntington protein is found in every cell in your body, why is it the GABAnergic selective loss in the striatum only?? —> can’t figure this out
BDNF and huntingtons disease
transport if BDNF impaired in HD (post mortem tissue—> reduction of BDNF) due to mutant Htt. In the mature brain BDNF supports and protects the medium spiny neurons. BDNF comes from cell in th cortex, and is transported by corticospinal projections, Htt is involved in transporting BDNF from cortex to striatum and promote its release. Mutant Htt —> impairs/prevents transport —> low levels of BDNF in striatum. Loss of BDNF causes susceptibility of MSN’s to excitotoxic injury. Increased activity of NMDA receptors, normally BDNF would protect against this toxicity. TARGET: BDNF, want to replace BNDF to try and prevent further cell loss?
Neuroprotective therapy:
In vitro disease models. hiPSC cells from HD patients, differentiated to MSNs, human HD in vitro disease model. Removed BDNF from culture media during MSN differentiation of HD hiPSCs. Removal of BDNF increased the rate of HD cell death in culture. Only a 2D culture don’t have the connection between cortex and striatum. BDNF maintains and protects cells. Spiny neurons loose projections in the absence of BDNF
In vivo disease model:
Quinolinic acid (QA) lesion model of HD, QA selectively kills MSNs. Patients with HD have QA in tissues. We deliver BDNF to the brain by: does not cross BBB because it is too BIG, there fore you have to inject it into the brain, limited spread from the pump. —> Gene therapy
Test using transgenic mouse rat models
This rat line carries a truncated Htt cDNA fragment with 51 CAG repeats. Progressive sensorimotor symptoms and cell loss.
- Clinical symptoms start at 6 months of age.
- MNS cell loss at about 12 months.
- Behaviour impairment comes before cell loss. Administered AAV-BDNF into striatum at 3 months (pre-symptomatic). Tested sensorimotor function every month until 18 months of age.
- Another test: On rotation pipe, normal rat will just run on, if motor impairment they fall off. BDNF prevent and reduced the loss of MSN in thus rats after 18 months. Also significantly reduced sensorimotor function.
- Connor et al Gene therapy 2016. BDNF potential to treat HD, but shouldn’t rule out other targets yet. But is one potential tgHD has better validity than the QA test.
- Next stage before clinical trail —> safety profiles, toxicity testing (IPS), want to out it in non-human primates. HD is an orphan disease —> nothing to treat the disease.
Gene Therapy:
using vectors that have been modified that removes their disease component. Get cell to make therapeutic protein. One off injection, In hd use viral vector known as AAV-BDNF, therefore the cell itself starts making BDNF. Expression of BDNF into surrounding environment. - - Administered AAV-BDNF into rat striatum. 3 weeks later injects with QA in striatum.
- Tested sensorimotor function every week for 8 weeks, want to see if we have prevented or reduced cell loss and or clinical symptoms.
- Test shows on rat that it doesn’t want to use paw on opposite side of the lesion, cylindrical experiment —> normal rat will have 50/50 use of the paws, however if there is a QA lesion on one side, you will see that they won’t use a particular paw.
- Can look at impairment and improvement. Corridor test —> Dishes with coco pops, rat without lesion will randomly eat/look t food, QA lesion will ignore all coco pops on once side —> sensory neglect.
- AAV-BDNF protects MSNs from QA-induced cell death, AAV-BDNF prevented/reduced QA-induced sensorimotor function impairment compared to untreated.
How do Clozapine and Risperidome work?
- primary cultures of macrophages, modulates the immune system, produce pro inflammatory cytokines. Significant reduction in interleukin 12 (anti-inflammatory mediators).
- Based on in vitro and involve results clozapine has entered into phase2 clinical trail.
- Beneficial used of using drugs already in circulation—> can avoid toxicity testing because someone else has already done it.
EAS model of MS:
- progressive degeneration of white matter tracts, paralysis.
- Clozapine reduced disease progression and reduced the number of lesions.
- Significant increase of IL10 (pro-inflammatory mediators)
Anti-psychotic Agents to treat MS
MS is an auto immune disease.
- AP’s are dopamine d2 receptors but also affect a lot of other receptors.
- Clozapine and Risperidome have anti inflammatory properties.
- Re-directed use of current agents. Fetal syndrome inflammation —> inflammation in the brain predisposes baby to schizophrenia, in post mortome brains there is inflammatory properties.
Male vs female rats
Had both male and female rats, 99% of preclinical testing done in male rats because female rats had oestrogen cycle that causes variation. Female rats never got as “sick” as male rats, simnifically better improvement with BDNF.
Biologics therapies:
peptides/re-cmobinant proteins/ gene therapy, cell therapy, monoclonal antibodies.
Gene therapy:
delivery of genetic sequences that alter the instruction set of a cell for therapeutic purpose.
- Genetic sequences are synthetic copies of genes that encode a specific protein, short genetic sequence that knockdown the expression of a specific gene. Should be high specific leading to reduce side effects, and produce long term therapeutic effect following a single application. - Aim: to deliver therapeutic gene to the organ cell of interest.
- Problem: mammalian cells are vey efficient at getting rid of foreign DNA.
- Solution: therapeutic genes are delivered using gene delivery vehicles.
Somatic cell gene therapy:
therapy that is restricted to the patient and genetic material is not passed onto their offspring
Germline therapy
sperm and eggs of an individual passed onto offspring
Viral Vectors:
exploit the natural life cycle of a virus, viruses bind to membranes bound receptors and have evolved efficient ways to gain access to the nuclear transcriptional machinery. Viruses are “gutted” and engineered to carry a therapeutic transgene. Viral genes involved in virus replication removed, so ability of the wild type virus to replicate is lost. Lets generation of viral vectors are devoid of many viral genes and are relatively safe.
Example: Parkinson’s disease: Biologice
Caused by progressive loss of Dopamine neurons in the SNc. Strategy one: to increase SA synthesis locally in striatum, by transferring genes involved in DA synthesis to the neurones in the stream, Does not prevent disease progression. Strategy 2: boost production of growth factors that promote DA cell survival and axonal regeneration (GDNF), by introducing GDNF genes into the brain.
Gene delivery vehicles
- non viral vectors: include plasmids, siena and liposomes,
- viral vectors: include genetically modified viruses.
Testing biologics - ex vivo
Ex vivo - genetic modification preformed on cells in a dish before transplantation into human.
Testing biologics - in vivo
In vivo - performed directly on the patient.
Cell therapies
involve the injection of living cells into the patients (bone marrow). Replace damages tissue, stem cells or progenitor cells are differentiated into a specific cell type in the lab, then grafted onto the site of injury. Release therapeutic proteins, transplant cells that have the capacity to release soluble factors such as cytokines and growth factors.
Autologous - cell therapies
cells are removes from patient, modified and re-transplanted into the same person.
Syngeneic - cell therapies
cell donor is genetically identical (identical twins)
Allogenic - cell therapies
cell donor is a different person to the recipient go the cells
Some limitations: Immunotherapies
Monoclonal antibodies are large proteins (150kDA), so penetration into a tumour can be an issue, long serum half lives. Immortalised cells (hybridima) are genetically engineered to produce the therapeutic anti body. Late does often requires to achieve clinical efficacy (6-12g per patient —> high production costs)
Modes of action for Cancer immunotherapies
Antibody binds to a soluble ligand involved in cancer growth, interferes with its activity and interaction with binding partners. Antibody bind to cell surface receptor on a tutor cell, blocks its interaction with the ligand, interferes with signalling pathways import mat in maintaining tumour growth, triggers receptor internalisation or apoptosis of tumour cells. Antibody binds to tumour antigen and induces human immune effector responses.
Antibody therapies:
antibodies recognise and bind to specific antigens. Monoclonal antibodies for human therapies, are specific for one antigen, typically IgG subtype, have been humanised or are chimeras antibodies, and are currently commercial antibodies therapies in use for cancer and immune disorder treatment.
Target based screening
advantages - application of molecular and chemical knowledge, small molecule screening strategy, biologic development (antibodies). Disadvantages - target might not be relevant to disease, does not provide a useful therapeutic index.
Phenotypic screening
advantages - do not require prior understanding of the mechanism of action, and more effective translation from assay to therapeutic index. Disadvantages - optimising properties without knowledge of mechanism and lower through put than target based.
Inhibitory mechanisms:
- Target sites include - catalytic sites, ligand binding sits, allosteric site, protein-protein interface and protein- membrane interactions.
- You want to make molecules related to known ligands - analogues, and screen for new molecules.
- Library design, related to know active molecules, diverse chemistry to discover unrelated molecules, and building drug-like compounds up from smaller fragments.
Classical pharmacology
Receptors were unknown molecular entities, they were classified based on their ligand response profile, classification could lead directly to new therapeutics.
Analogues based on known ligands:
- adrenaline - endogenous agonists for adrenoceptors
- antagonism for angina - pain from low oxygen supply to the heart
- Agonism for asthma - pulmonary obstruction.
Discovery of propanol:
adrenaline increases angina pectoris, angina is traded by nitroglycerin. Effects are attributes to coronary artery vasodilators were clinically ineffective. Hyperbaric O2 reduced ventricular fibrillation associated with coronary artery occlusion, ie increased O2 levels. What about decreasing myocardial O2 demand? This is controlled by the work of the heart and is a function of arterial blood pressure and heart rate. Wanted to find a b-receptor antagonist to reduce heart rate. Critical evaluation of disease data to generate a hypothesis about what is required from a drug
From agonist to antagonist:
Start point : adrenaline analogue isoprenaline (b-AR agonist). Promoted only the tachycardia, vasodilation and bronchodilation adrenaline responses, which were also features not inhibited by anti-adrenaline drugs available around 1958 (a-AR blockers). Can a selective antagonist be developed from an agonist that is only affecting the biological response of interest? —> Target phenotype is affected by the compound (drug) “Hit discovery”.
Development pathways:
Unsuccessful discovery path: symmetrical analogues of isoprenaline. Successful discovery path: DCI stimulates heart rate, but not contractile effects, and it antagonised the effects of adrenaline and isoprenaline on the latter. Dichloroisoprenaline (DCI) Bronchodilation antagonist, Pronethalol Propanolol. DCI is an antagonist of adrenaline, so it is a useful starting point for antagonist development —> “Lead development”
Early asthma treatment:
From the early 1900’s adrenaline was used as a reliever because of its relaxing effects on airways smooth muscle. Administered initially by injection, then by aerosol. Highly efficient for the desired outcome, but…caused hypertension, tachycardia, muscle tremor, short acting due to metabolic instability. Search for adrenaline analogues with improved bronchodilatory profile.
Isoprenaline:
selective b-adrenoceptor agonist. More selective than adrenaline, Administered by inhalation, but… cardiac side effects, rapidly metabolised, poor oral bioavailability. Toxicity and metabolism profile needs to be improved.
Improving on isoprenaline
Longer acting derivative made by disrupting the catechol ring. More metabolically stable. Retained the cardiac side effects because of b1 activity orciprenaline, longer acting
Discovery of salbutamol:
Looking for longer acting bronchodilators. b2 AR selective fast onset longer acting than isoprenaline weaker binding Best results by inhalation, orciprenaline, longer acting, terbutaline,b2 AR selective longer acting
Adrenaline:
HBI, All polar groups interact with the protein, Hydrogen bonds and ionic bonds are used. The catechol hydroxyl groups interact with orthosteric site amino acids known to be important for agonist binding and receptor activation.
Chemical space requires refining
filtering for desired properties, Chemical properties useful in discovery, Lipinski’s Rule-of-5 • Developed from the 90th percentile of 2245 orally active drugs at phase II or higher. Violation of more than 1 and the drug is unlikely to be orally active.
Non-catechol binding in the activated b2A
Catechol and non-catechol ligands bind similarly. Proteins change conformation to fit a ligand: A conformational change propagates from TM6 through to the extracellular loop 3 (ECL3). Ligand dependent changes in the b2AR orthosteric site.
Unwanted properties:
Assay results can be clouded by interfering compounds, Optical interference, Reactive, Aggregators,Known problem compounds can be filtered out based on substructure. The final properties of a library will be context dependent e.g. Anti-neoplastic agents can be classed as “reactive” but might need to be included in a library for an oncology setting. Intended route of administration will need different properties.
High through our screening (HTS)
Use robotics to screen hundreds of thousands of molecules for an effect against a target protein. Needs a large library of chemical compounds and a robust assay. Robotics – mix library of compounds with protein in many-welled plates (96, 384, 1536!), incubate and then measure some effect. Performs liquid handling and moves plates around for storage or measurement on inline instruments. Screen at single dose, dose response performed for molecules that inhibit over a specified level (active above a defined cutoff). Any hits require validation of chemical structure and activity. Hits from the same chemical family can form a lead series. Virtual HTS —> either receptor based or ligand based. Receptor based - Use atoms from the protein structure 3D. Ligand-based - compound structure 2D, 3D
Discovering new ligands using protein structure:
Molecular docking predicts the binding modes of small organic molecules based on optimising the complementarity between the physicochemical properties of the target site and the ligand.
Selecting a focussed library:
Based on relationship to known active molecules, Substructure, Similarity
Structure guided selection:
Pharmacophore, bAR in activated state with catechol and non-catechol agonists bound
Sampling and Scoring:
During the docking calculation, compounds are posed in the binding site thousands to millions of times and scored. Fitness functions score the poses. They consist of multiple terms that describe different types of interactions between the ligand and protein eg. VdW and electrostatic interactions. The ligand is treated as flexible, while the receptor is considered rigid. Sampling – is the native binding mode found amongst the poses calculated ? Ranking – can the native binding mode be given the top score ? Some methods try to account for protein flexibility (induced fit), and the role of water in ligand binding.
A- Assignment:
The assignment process is used to determine which subjects get which treatment.
A- Assignment: first come first served
this process covers a range of possibilities that will typically introduce bias. The main source of bias from this process is the loss of blinding. As the investigator can guess which subjects are getting different treatments even if he/she is blinded to the actual assignment
A- Assignment: randomised assignment process
is considered the best method of deciding which subjects get which treatment. In order to ensure balanced allocation of treatments the number of subjects to be randomised is decided a head of time and a balanced list of treatment is drawn up. this list is then randomly permuted and subjects drawn from will be permuted. This ensures a desired balance. If different subgroups might have different responses it is common to strait the randomisation sequence. Separate sequences are drawn up for each sub-group.
B - Blinding:
Blinding is used to reduce bias. Bias can arise from both the investigator and the subjects influence and their expectation of the experiment. Blinded trials often become unblinded if the treatment has very obvious beneficial affects.
C - Comparison: Active treatment - Concentration
Concentration control can be used to reduce the influence between random subject differences in pharmacokinetics. By measuring concentration and individualising the dose to reach the desires target concentrations, then the concentration effect relationship can be discovered (RCCT).
C - Comparison: Active treatment - Biomarker BRCT
If there is a bio marker that reflects the effect of the drug it can be used to control the intensity of the treatment and reduce both pharmacokinetic and pharmacodynamic variability. Subjects are randomised to one or more biomarker target levels and the dose adjusted to reach the target biomarker effect
C - Comparison: Placebo
if there is a genuine uncertainty about the effect of the active treatment then it is usually considered ethical to randomise the placebo.
C - Comparison: Standard treatment
investigation of a new treatment in comparison to a previous standard treatment. This trial is designed to show that the new treatment is no worse than the standard treatment - a non-inferiority trial. If the standard treatment is given to all subjects then is would be an add-on trial. It looks for the effects of the new treatment in addition to the standard treatment. A placebo trial group would receive the standard treatment.
C - Comparison
good experimental science uses a control group to account for factors that might influence the outcome that are not experimentally assigned.
C - Comparison: Active treatment
Active treatment is usually desirable to learn about the relationship between the intensity of treatment and outcome.
C - Comparison: Active treatment - Dose
The within active treatment control that is widely used is dose control (RDCT).
B - Blinding: open trials
are unblinded. they are still commonly used for marketing purposes but have little scientific merit.
B - Blinding: Double blind
trails mean that neither the researcher or the subject know the treatment.
B - Blinding: Single blind
trials mean the investigator will know the treatment but the subject will not.
B - Blinding: Triple blind
trials may occur when the sequence is lost - this means no one ever will know the treatment that was given.
B - Blinding: Double Dummy trials
are used when two physically different treatment are compared. EG a tablet and inhaler. So the subjects are either given a active tablet and an inactive inhaler, or inactive tablet and active inhaler, so that they are not influenced by the treatment.
B - Blinding: Double Dummy trials
are used when two physically different treatment are compared. EG a tablet and inhaler. So the subjects are either given a active tablet and an inactive inhaler, or inactive tablet and active inhaler, so that they are not influenced by the treatment.
S - Sequence
The sequence of treatments can influence what is learned from a trial and the kind of bias that can arise.
S - Sequence: The parallel design
has different treatments assigned to different groups of subjects, to see if the drug actually works. But it won’t tell you what dose is needed of the dose response relationship
S - Sequence: A cross over design
uses two or more treatments in each subject. This allows individual dose response curves to be observed and the true she of the dose response relationship can be determined. There are some disadvantages, there may be a carryover effect (drugs with long half lives). This would bias the response seen in a placebo treatment period, If there is some systematic differences between period, (summer and winter) there may be a period effect that influences the response. Because each subject is asked to take several treatments there is a higher risk of drop out.
S - Sequence: Titration design
is a special kind of crossover design. These involve given a fixed sequence of doses (forced titration) to each subject to learn about the dose response relationship. A more realistic titration design (flexible titration) involves starting with a low dose and if the subject responds the dose is keep constant. The does is only increased if the desired response is not reached,
Analysis:
you want to know if the drug works fro the desired treatment, and how well does it work. Analysis procedures, if the endpoint of the trial is an event such as death then a survival analysis is performed and might use the log-rank (survival) test to evaluate the hypothesis the survival can be changed by treatment (ANOVA test - several groups) or the t-test for two groups. The application of pharmacological principles can be used to determine the answer to ho much question. The 4 basic properties of drug, clearance, volume of distribution, maximum effect and does production 50% pf the maximum can be used to predict the how much questions. The trial is designed to estimate the values of these parameters using PK and PD parameters.
model based analysis
procedure can be more informative and can be used to account for unplanned trial execution differences. The influence of subject demographic features can also be used to learn about why treatment differences exist between subjects and the identify ways to individualise drug treatment.
power based analysis
is used to determine the likelihood that a clinical trial will detect a drug effect when the drug effect really exists. Because of the random variability in trial a drug may recall work but the effect may be lost in noise. A power analysis takes into account the size of the expected treatment effect, the expected size in variability in response an the statical criterion used to judge if the null hypothesis should be rejected. This criterion is usually expressed the probability of accepting that the drug works when in fact it does not. A target power is usually greater than 0.8, so the null can be rejected with 80% confidence.
intention to treat - analysis
only considers the treatment assignment. It does not take into account information about a subject. This means the size of treatment effect will be underestimated if some subjects do not take the active treatment they were assigned, of if a placebo patient takes the active drug. It is useful for making pharmacoeconomic decision where the cost of the drug has to be paid whether it is taken or not.
As treated - analysis
will take into account information about what the subject actually took for their treatment. It will be less likely to have the underestimation bias that is associated with the inattention to treat approach. More suitable for scientific decisions.
Disease progression model:
a symbol to describe disease as a progress is “S” the disease status. Disease status is expected to vary with time, S(t). Disease status may be defined in terms of clinical outcomes such as survival and symptoms or interns of a biomarker, Biomarkers are also know as clinical signs when used by clinicians as a diagnostic or prognostic variables. The simplest model to describe changing disease status with time is linear. in general if the change is relatively small in relation to the time scale of observation then any disease progress curve will reasonably described by a linear function.
The action of cholinesterase inhibitors in AD
is very similar for all drugs in this class. There is a delayed onset of benefit taking 2-3 months to reach its peak followed by continuing progression of the disease at the same rate as expected. This is a clear example of an offset type of dug action. It there is a disease modifying effect it is small and hard to detect without withdrawal of treatment.
Parkinsons study group:
the DATATOP study was performed over 2 year period but the patients that enrolled are followed up 8 years after. The time course of disease status of PD and the effects of treatment were described by a disease progress model. The patterns were quite variable from patient to patient, The first patient were used to build a disease progression model and drug action. The initial rate seems to slow with treatment. There is a marked symptomatic effect attributed to levodopa. Out is not obvious if the relationship should be linear. The treatment of levodopa and deprenyl show that both have offset effects and protective effects. The trial was designed but rested on a key assumption that symptomatic effects of levodopa would wash out after 2 weeks after stopping the treatment. The marked difference form placebo could be due to a true disease modifying effect or a very slow loss of symptomatic effect.
Drug effects on the slope of a linear model
lead to permanent changes in the disease status which are not reversed when treatment is stopped. The persistent change after stopping treatment is the hallmark of a disease modifying the action if the natural history is linear.
disease progress model
it is possible to imagine a drug action that is equivalent to a change in the base line parameter of the model. This kind of effect on disease produces a temporary offset. When treatment is stopped the response to the drug washes out and the status returns to base line. In many cases it is reasonable to suppose that the processes governing a deli in onset of the drug effect will also affect the loss of effect but the offset effects of levodopa treatment in Parkinson’s disease are one exception to this assumption.
