Exam 2 Flashcards
1
Q
Pharmaceutical phase
A
Disintegration of a pill or capsule in the GI tract
2
Q
Pharmacokinetic phase
A
absorption from the GI tract into the blood supply
ADME
3
Q
Pharmacodynamic phase
A
mechanism by which a drug interacts with its molecular target
4
Q
oral drug transport: intestine > blood
A
enters GI tract > encounters gastric juices and HCl > enters intestine (if drug has survived) > passes THROUGH the cells lining the gut wall > passes BETWEEN the cells lining the blood vessles > liver
5
Q
Amines: function in drug absorption
A
amines are partially ionized at the slightly acidic and alkaline pHs present in intestine and blood
> can equilibrate between their ionized and non-ioinized forms
can cross cell membranes in non-ionized form
ionized form gives good water solubility and allows good binding interactions with its target binding site
when amine is 50% ionized pH=pKa
6
Q
Oral drugs and their flexibility
A
the more flexible the molecule, the less likely it is to be orally active
> flexibility measured by the number of freely rotatable bonds
7
Q
why will some oral drugs be made purposefully highly polar?
A
so that they are not absorbed from the GI tract, ensuring the drug reaches the site of infection in higher concentration
8
Q
Parameters for predicting acceptable oral activity
A
polar SA </ 140 A and </ 10 rotatable bonds
</ 12 HBDs and acceptors in total and </10 rotatable bonds
9
Q
Drug distribution: those that are confined to the capillaries and do not go to tissues
A
drugs that binds to plasma proteins in the blood
> plasma proteins cannot leave the capillaries, proportion of drug bound to these proteins is also confined to the capillaries and cannot reach its target / tissue = smaller effect
> weak acidic drugs bind to albumin
> basic drugs bind to alpha1-acid glycoprotein
10
Q
what happens to excessively hydrophobic drugs
A
absorbed into fatty tissues and removed from the blood supply
11
Q
Characteristics of drugs entering the CNS
A
they have to cross the BBB
> polar drugs are unable to cross the BBB unless they make use of carrier proteins or are taken across by pinocytosis
12
Q
BBB characteristics
A
blood capillaries feeding the brain are lined with tight-fitting cells which do not contain pores
+
capillaries are coated with a fatty layer
13
Q
Characteristics of drugs crossing the placental barrier
A
fat-soluble drugs will cross the barrier most easily
14
Q
Phase I metabolism
A
lipophilic drug > functionalized drug
reactions of oxidation, reduction, hydrolysis
> enzymes adding a polar functional group to a non-polar drug, making it more polar and water soluble (e.g cytochrome P450)
> or enzymes that reveal a masked polar functional group which is already present in the drug
15
Q
Phase II metabolism
A
functionalized drug > conjugated drug
Conjugation reactions where a polar molecule is attached to a suitable polar ‘handle’ that is already present on the drug or has been introduced by a phase I reaction
> conjugate has even more increased polarity (however some phase II reactions decrease polarity)
> reactions: glucuronidation sulfation, glycine-conjugation / GSH-conjugation, methylation / acetylation
16
Q
hard drugs vs soft drugs
A
hard drugs: resistant to metabolism + remain unchanged in the body
soft drugs: designed to have a predictable, controlled metabolism where they are inactivated to non-toxic metabolites and excreted
17
Q
The first pass effect
A
Impacts bioavailability
drugs taken orally that pass directly to the liver once they enter the blood supply
> exposed to drug metabolism in the liver (via enzymes, like to CP450 family) before they are distributed around the rest of the body
> occurs during lag time
drugs administered differently avoid the first pass effect (are distributed around the body before reaching the liver)
17
Q
Drug excretion mechanisms
A
Volatile / gaseous drugs: via lungs (moving down conc grad from capillaries to alveoli)
Drugs diverted from blood supply back into intestines: via bile
Sweat, saliva, breast milk
Kidneys are the principle route by which drugs / metabolites are excreted (as urine)
Liver & GI tract as faeces and bile
18
Q
Kidneys: drug excretion route
A
blood enters kidneys via renal artery
1) glomerular filtration: creates a plasmalike filtrate of the blood
2) tubular reabsorption: removes useful solutes from the filtrate, returns them to the blood
3) tubular secretion: removes additional wastes from the blood, adds them to the filtrate
4) concentration: removes water from the urine, concentrates wastes
180 L/day is filtered
2 L/day is excreted
non-polar substances are reabsorbed into the blood supply whereas polar substances are retained in the nephrons + excreted in urine
> this is why its important that during metabolism the drug is made more polar
19
Q
Drug administration
A
Oral
Rectally as suppositories
Topically
Inhalation (act directly on respiratory system or some are absorbed into the blood supply to act systemically)
Injecting
> Polar drugs unable to cross cell membranes
> most efficient but also most hazardous (intravenous, intramuscular, subcutaneous, intrathecal)
Implants: providing controlled drug release
20
Q
drug half-life
A
time taken for the conc of drug in blood to fall by half
e.g aspirin: 0.28 hrs, iburprofen: 2hrs
> metabolic stability of a drug determines its half-life
21
Q
what is drug formulation
A
the method by which drugs are prepared for administration (solution, pill, capsule, liposome, or microsphere)
> can protect drugs from particular pharmacokinetic problems
22
Q
Lipinski’s rule of five
A
features important in making a drug orally active
- a molecular weight < 500
- no more than 5 HBD groups (sum of OH and NHs)
- no more than 10 HBA groups (sum of N and O atoms w/o H attached)
- calculated log P value < 5 (measure of hydrophobicity, partition coefficient)
23
Q
what are drug-drug interactions
A
the presence of one drug affects the activity of another
> drugs can interact at level of e.g absorption and metabolism
> e.g can lead to accelerated elimination of one and inhibition of elimination or another
24
What is a multi-target-directed ligand
a single drug that can act selectively at different targets in a controlled manner
25
What are promisucous / dirty drugs
drugs which interct with a large range of targets
26
NMR spectroscopy
testing compounds affinity to a macromolecular target
27
Visual methods of detecting whether ligands bind to macromolecular targets
SPR, SPA, ITC
28
How to study structure-activity relationships
if possible via x-ray crystallography
> identify important binding interactions
> crystallize target with the lead compound bound to the binding site
if crystallography is not possible
- synthesize analogues where one particular functional group of the molecule is removed/altered
> find out which groups are essential
> test biological activity + compare with OG compound
> if analogue shows sig lower activity = group modified must have been important
29
Why might the behavior of an analogue change: consider hydrogen bonding
steric hinderance (swapping an OH with a OCH3)
loss of groups partaking in the intermolecular force
30
Binding role of hydrophibic groups + analogues
aromatic rings are planar + hydrophobic (VdWs)
> analogue w/ cyclohexane ring = less likely to bind well as ring is no longer flat, it is bulkier i.e cannot fit
Alkenes: planar
> analogue: saturated alkyl = bulkier
31
Binding role of amines, alcohols, esters, amides, carboxylic acids, phenols, ketones
involved in H bonding
> depending on which they can act as HBA or as HBD
32
Binding role of quaternary ammonium salts
they are ionized and interact with carboxylate groups via ionic interactions
or induced dipole-dipole with aromatic rings
analogue: turn it into tertiary amine group
33
What are isosteres and what are they used for
atoms or groups of atoms which share the same valency and have chemical or physical similarities e.g SH, NH2, CH3 are isosteres of OH
> can be used to determine whether a particular group is an important binding group (if its involved in H bonding or not)
> groups of atoms that can be used to replace another group, while retaining the desired biological activity
> Used to replace a functional group that is important for
target binding , but is perhaps problematic for e.g. ADME
properties
34
What is the pharmacophore and the importance of it
part of investigating the SAR
pharmacophore summarizes the important binding groups that are required for activity and their relative positions in space respective to one another
35
Hydrophobic pockets: how can we understand the size of them
use different alkyl substituents (varying in length and size) to fill up the hydrophobic pockets
36
What is extension
extra functional groups are added to the lead compound in order to interact with extra binding regions in the binding site
37
Chains linking binding groups together in a drug: how can this be altered for drug optimization
modifying the length of the chain to maximize the interactions of each group with the corresponding binding regions
38
How can ring systems be modified for drug optimization
modified to maximize binding interactions through stategies such as expansion, contraction, variation (replace ring with different size or heteroatom position), or fusion with other rings
39
What happens in drug simplification and why is it done
removing functional groups from the lead compound that are not part of the pharmacophore
> unecessary parts of carbon skeleton or asymmetric centers can also be removed in order to design drugs that are easier + cheaper to synthesize
> oversimplification can result in molecules that are too flexible = decreased activity + selectivity
40
What happens in drug rigidification and why is it done
rigidification is applicable to flexible lead compounds
> Trying to lock a bioactive conformation, if successful better binding energy (affinity)
> aim is to reduce number of conformations available while retaining the active conformation
> via locking rotatable rings into ring structures or introducing rigid functional groups
41
What are conformational blockers
groups which are introduced into a lead compound to reduce number of conformations that the molecule can adopt
42
what happens to a drug that is too hydrophilic vs too hydrophobic
hydrophilic
> cannot cross cell membranes, prone to plasma protein-binding, phase II conjugation, rapid excretion
hydrophobic
> dissolved in fat globules in the gut, poorly absorbed, poorly soluble in blood, likely to be taken up by fat tissue, low circulating levels
43
how to measure a drugs hydrophobicity
n-octanol/water partition coefficient (P)
> hydrophobic prefer to dissolve in n-octanol layer
> hydrophobic = high P value
> measures relative distribution of un-ionized form
44
characteristics that play an important role in oral bioavailability
flexibility
hydrophobicity/philicity
45
Mechanisms to alter hydrophobicity and hydrophilicity of a drug
masking polar functional groups with alkyl or acyl groups = less polar
adding/removing polar functional groups
varying hydrophobic substituents (increasing/decreasing sizes of alkyl groups)
variation of N-alkyl substituents or aromatic substituents = vary pKa
bioisosteres for polar groups
46
Techniques to make drugs more resistant to chemical and enzymatic degradation
steric shields (addition of bulky alkyl group close to functional group)
electronic effects of bioisosteres (stabilizing functional group, making it less reactive)
metabolic blockers (adding a group that blocks the addition of polar groups so it cannot be metabolized)
removal or replacement of susceptible metabolic groups
group shifts (if the metabolically vulnerable group is important use this strategy, shift the vulnerable group within the skeleton)
ring variation and ring substituents (to improve metabolic stability e.g addition of N into the ring)
47
Techniques to make drugs less resistant to drug metabolism
introduce metabolically susceptible groups
self-destruct drugs (degrades spontaneously under certain conditions)
47
Targeting drugs to exact locations in the body where they are most needed
safer drugs with fewer side effects
- drugs can be linked to amino acids or nucleic acid bases to target them against fast-growing or rapidly-dividing cells
- drugs can be targeted to the GI tract by making them ionized or highly polar (so they cannot cross the gut wall)
- Make drugs more polar so they do not cross BBB (no CNS side effects)
47
What are prodrugs
compounds which are inactive in themselves but which are converted in the body to the active drug, usually by drug metabolism
> esters are commonly used as prodrugs (= make drug less polar = can cross cell membrane easily)
> can cross cell membranes via aid of transport proteins
> can prolong activity of a drug
> can reduce toxic nature of a drug
48
What is a sentry drug
a drug which is administered alongside another drug to enhance the latter's activity
> protect their partner drug by inhibiting an enzyme which acts on the latter
>
49
NTs as drugs
not very effective, short lifetime in body, poor selectivity for the target
50
Hormones as drugs
suitable
some are susceptible to digestive or metabolic enzymes, and show poor absorption when taken orally
51
Peptides and proteins as drugs
suffer from poor absorption or metabolic susceptibility
antibodies
> can identify foreign cells or macromolecules, marking them for destruction, can also be used to carry drugs to specific targets
52
Oligonucleotides as drugs
susceptible to metabolic degradation, but can be stabilized by modifying sugar-phosphate backbone so that they are no longer recognized by relevant enzymes
53
Toxicity testing: what does it test
examine effects on cell reproduction, identify potential carcinogens, acute toxicity, determining safe dose for future clinical trials
54
How is the toxicity of a drug quantified
LD50, ED50, therapeutic index/ratio (LD50:ED50), dose-response curves (ideally the curves should not overlap on the x-axis)
55
What are toxicity tests conducted on
in vitro tests on genetically engineered cell cultures and/or in vivo testing on transgenic mice
56
How are drug metabolism studies conducted
administering a (radio-labelled) labelled drug to a test animal and taking blood and urine samples for analysis to see if any metabolites have been formed
57
Phase I studies
carried out on 20-80 healthy human volunteers taking drug for 1 month, placebo and different dose levels,
Main information
1. Absorption and metabolism
2. Side effects as dosage is increased
58
Phase II studies
~ 2 years, may start before phase I studies are complete, carried out on several hundred patients
Main information
1. Effectiveness in treating disease
2. Short-term side effects in health -
impaired patients
3. Dose range
59
Phase III studies
~ 3 years, may begin before phase II is completed, uses double-blind procedures on a much larger sample of patients (thousands), comparative studies
Main information
1. Benefit/risk relationship of drug
2. Less common and longer term side effects
60
Phase IV studies
drug is placed on the market + can be prescribed but is still monitored for effectiveness and for rare and unexpected side effects
– Pick up long-term and rare side-effects
* can be a reason to take a drug off the market again…..
– Learn more about use and discover new applications
61
Use and coverage of a patent
patents are taken out as soon as a useful drug has been identified
they cover a structural class of compounds rather than a single structure
a significant period of the patent is lost as a result of the time taken to get a drug to the market place
patents can cover structures, their medicinal use, and their method of synthesis
62
Which type of drug research may be fast-tracked
drugs that show promise in a field which is devoid of a current therapy
63
What are orphan drugs and how is their development prompted
They are drugs that are effective in rare diseases and special incentives are given to companies to develop them
64
What is prioritized in chemical development
a synthetic route that is safe, cheap, efficient, and has the minimum number of synthetic steps, and will provide a consistently good yield of high-quality product that meets the predetermined purity specifications
65
What is the aim of process development
develop a production process which is safe, efficient, economic, environmentally friendly
66
ADMET
Absorption, distribution, metabolism, excretion, toxicology
67
MEC
minimal effective conc
68
Why does drug conc in the body go down with time
Due to
Breakdown: metabolism
Elimination: excretion
69
Intravenous administration graph characteristics
Starts high goes low
From administration until curve crosses MEC = biologically active part
area-under-curve indicates total amount of drug in circulation
70
Oral administration graph
starts low goes up fast and then goes back down slow
Peak: Cmax
AUC0 > infinity
Section of curve (peak) where it crosses MEC (twice) is the pharmacologically active part
> there is a lag time i.e when uptake (absorption) occurs
Therapeutic window is section between MEC and conc where adverse side effects occur
71
Oral bioavailability F value
F = (AUC0>infinity oral) / (AUC0>infinity i.v.)
at equal doses
> when F = 1
AUCoral = AUCi.v.
> when F = 0.3
AUCoral = 0.3*AUCi.v.
72
Intramuscular and subcutaneous graphs
also start at 0 with lag time and then have a peak and slow decline
Intramuscular reaches peak faster > subcutaneous > oral
i.m has higher peak, falls sooner > sc > oral
73
What information does the drugs half-life and oral bioavailability give us
oral bioavailability: A & M, and how much drug is needed
drug half-life: M & E, and how often to take drug
74
First hurdle of an oral drug: absorption
depends on the drugs solubility in H2O
> Uncharged drug: HBD, HBA groups
> Charged: dipoles H2O
- Ionized form is important for water solubility
- Neutral species is important for membrane permeability
- Acid: solubility is high at higher pH
- Base: solubility is high at low pH
- Ampholyte: U-shaped curve, low solubility at neutral pH
There is a large variability between chemicals
<10 ug/mL: no absorptioin
>65 ug/mL: solubility is not limiting absorption
75
Absorption and molecular size
large molecule needs enough functional groups to be soluble
functional groups form H bonds w/ water molecules = solvation shell
To penetrate lipid membrane, solvation shell needs to be disrupted
Low rate of penetration of membranes
- molecular weight > 500 = incompletely absorbed
76
Second hurdle of an oral drug absorption
Intestinal transport mechanisms
1) Paracellular transport
- drugs pass between cells (through tight junctions)
- limited to small molecules w/ MW <200
2) transcellular transport
- drugs pass through cells
- requires appropriate pKa (Lipinski Ro5)
- passive diffusion
Via active transport
3) Carrier-mediated transport
- via specific transport proteins
4) P-glycoprotein mediated efflux
- pumps drugs OUT of cells & back into lumen, limiting absorption, reducing bioavailability of some drugs
77
Examples of transporters in carrier-mediated transport
a.a transporters (for drugs like, gabapentin, methyldopa, L-dopa, D-cycloserine, baclofen)
oligopeptide transporters (cefadroxil, cephradine, cefixime, ceftibuten, cephalexin, caprtopril, lisoprinil)
phosphate transporters (fostomycin)
glucose transporters (p-nitropheyl-beta-D-glucopyranoside)
78
How can drug-drug interactions alter bioavailability + e.g
Taking a drug that is a strong P-gp inhibitor
e.g taxol (5% bioavail.) + cyclosporin A (strong p-gp inhibitor drug) = taxol 50% biovail.
79
P-gp
> drug efflux, back into the lumen
> keeps foreign molecules out of the brain
p-gp-like proteins are also upregulated in some cancers
80
Omeprazole
gastric H+ pump inhibitor
has several metabolites (present in urine & feces
81
Drug metabolism specificity
drugs are metabolized by special enzymes in order to make them more water-soluble and ready to excrete via the kidneys
the liver in the main site of metabolism for most drugs (also a bit in GI, lungs)
82
Epoxidation
oxygenation of double bonds
> oxidation of alkenes and aromatic rings: break double bond to add O via triangle shaped bond, then break the 2 single bonds on O, turning one into an OH and adding another OH
phase I metabolism
83
Oxygenation of hetero atoms
(nitrogen, sulphur, oxygen)
> by P450
X > X+ - O-
X-H > X-OH
phase I metabolism
84
Oxygenation of carbon-hydrogen bond
C-H > C-OH
> by P450
methyl > alcohol > aldehyde/ketone > carboxylic acid
phase I metabolism
85
CYP450 family
a haem-enzyme:
- has a cys-bound heme group
- Fe3+ binds O2 (iron is bound by N, cys, and O)
- "bottom" of substrate binding site
- drugs bind to them via interactions (mainly apolar a.a)
- heme group binds N-containing groups, that can act as CYP inhibitors
57 of them
certain ones for:
> metabolism of endogenous compounds
> drug metabolism
- major isoforms: 2C9, 2D6, 3A4
86
examples of drugs binding to CYP enzymes
Cimetidine:
inhibits gastric acid secretion
> CYP inhibitor, first-in-class H2 antagonist
> lower pKi
Ranitidine
> No CYP inhibitor, best-in-class H2 antagonist
> higher pKi
Changes in ring side chain
87
Excretion routes
via bile,
88
Examples of strong albumin binding proteins + the effects of this
Warfine
- anti-coagulant
- v. low therapeutic window
Sulfonamide antibiotic
- competes with warfarine, leading to bleeding by high levels of warfarine
affected by drug-drug interactions
89
how can grape fruit juice affect a drug conc
by inhibiting CYP450 (specifically CYP3A4)
> thus you have higher levels of the drug in your body as it is not metabolised by CYP450
90
St. John's wort
herbal antidepressant
induces intestinal p-gp and intenstinal & liver CYP3A4 = efflux of drug back into intestine (limiting absorption of other drug (D&D interaction)) and increasing level of CYP3A4 (stronger metabolism of other drug)
91
NT drug-drug interactions: side effects
histamine antagonist is present but also acetylcholine antagonist is present = dry mouth
acetylcholine antagonist but also histamine antagonist = sedation
92
Metabolites and side effects / toxicity
active drug is converted into various metabolites
- they can be inactive or active
> active:
they can bind to the same target w/ longer action
they can bind to a different target inducing a side effect
93
Torsades des pointes
most common cause of withdrawl or restriction of drugs
> e,g terfenadine removed from market
prolongation of QT-interval via interaction with the HERG K+ channel = irregular heart beats
> acute cardiac arrest, leading to sudden death
94
Terfenadine
CYP450 inhibitor = low therapeutic index of terfenadine giving cardiac side-effects (torsades des pointes)
Its active metabolite does not interact w/ HERG channel = marketed on its own
> a commercial 'rescue'
95
Bioactivation
active drug is converted into a reactive intermediate which then either:
> is converted into an inactive metabolite
> or reacts with cellular macromolecules resulting in toxicity (bioactivation)
can happen with very high doses (6grams) of paracetamol
> via killing liver cells
96
Why is there variability in drug response across patients
drug response depends on:
- compliance (how well the patient adheres to the prescription / treatment)
- patient status (one size fits all is not always right) e.g NMB in children has a shorter duration of action compared to elderly or those with renal / hepatic failure
- genetic make-up (variation in enzymes etc)
97
Personalized medicine
population of patients with given disease is treated w/ different drugs according to their genotype
98
SNP's in drug discovery
SNP variant can be a cause of disease
SNP variant may be less or not sensitive to drug
SNP variant may modulate drug response indirectly
99
Genetic variations and drug action
Drug transporters (e.g p-gp)
- variability in drug absorption, distribution and elimination
Drug metabolizing enzymes (e.g P450s)
- variability in drug metabolism
- side effects / toxicity
Drug targets (e.g receptors)
- variability in drug effects
100
Genetic variations and drug transport
Multi-drug resistance gene (MDR-1)
> P-glycoprotein
- cancer cells may overexpress PGP
- intestine may limit uptake of drug molecules
- BBB, protects us against toxicity but is a hurdle for CNS-acting drugs
> pumps substance out of the cell
- physiology: protects against toxic metabolites
> many drugs are P-gp substrates
- cancer resistance
- limits oral availability
- limits brain penetration
prominant is western africans but not so much in white people or japanese people
101
genetic variation of P450-enzymes
deletion in gene = no enzyme
duplicate / multiple copies of gene = higher enzyme level
SNPs in gene = dysfunctional or normal enzyme
e.g effect of nortriptyline
> genetic variation in P450-enzymes can lead to poor metabolizers
102
Multicopy P450-2D6 isoenzyme
affects plasma kinetics
> extensive metabolizers
103
drug discovery steps
disease > putative therapy > target selection & validation > hit finding & optimalization > lead optimalization
104
drug development and patent process
pre-clinical development > clinical development > drug registration > drug (production, marketing, logistics)
105
how long does drug discovery and drug development take and how much money
10-15 years
1000-1500 million euros
106
which factors are taken into consideration when deciding which disease to tackle
Priority for the pharmaceutical industry
> can the profits outweigh the cost of developing
questions to be addressed
> is the disease widespread?
> does the disease affect the 1st world?
> are there drugs already on the market?
> what can be the competitive advantage?
> what about exploiting new markets?
107
What criteria MUST new drugs meet?
- drugs must address a new need
> tackling new diseases or use a new approach to treating a disease
- or provide a significant "added benefit" over an existing medicine - limited room for "me-too's"
> improved formulation of existing drugs
drugs must also meet five criteria:
- must be safe and effective
- cost effective
- affordable
- really affordable
108
what are me-too's
new medicine that looks like existing medicine
> similar compound with same mechanism of action
> same target protein
> very similar chemical structure
market competition
Not necesarily innovative, unless
> better activity profile
> better side-effect profile
> active in population which is resistant to existing medicine
> easier to use by patient population
109
techniques for looking for a new drug
+ e.gs
serendipity
> e.g penicillin (beta-lactam from fungus inhibits bacterial cell wall synthesis), viagra (PDE5 inhibitor involved in regulation of blood vessel muscle tone)
start from natural ligands
> e.g from histamine to H2R antagonists (4 GPCRs: allergy & itch H1R and H4R, gastric acid secretion H2R, CNS function H3R)
me-too's
110
H2R blocker
cimetidine
> 1st H2R antagonist on the market
> inhibits excessive secretion of gatric acid, heals ulcers
> worlds 1st blockbuster drug
> inhibits CYP450
111
More innovative approaches to looking for a new drug
go for unmet medical need
try a new approach to target a disease better
111
Cimetidine to 'me too's'
Imidasols bind iron in heme group (prevent in CYP450)
> drug-drug interactions
Ranitidine does not interact with CYP450 and thus is a better H2R blocker
> no drug-drug interactions
> 2nd H2 blocker on the market
112
"old" drug discovery using phenotypic animal screens
In vivo studies with
> no knowledge on target, but includes also some ADMET
> living animals or humans
> measure an observed physiological effect
> pharmacodynamics & pharmacokinetics
> can identify possible side effects
> transgenic animals: genetically modified animals (e.g humanized mice that express human variant of a target)
113
Target-centric screening: + & -
Advantages:
> no animals needed
> high throughput possible
disadvantages
> efficacy is not certain
> molecular target =/= disease
114
Phenotypic screening: + & -
Advantages
> efficacy guaranteed
DIsadvantages
> disease model difficult to define
> animals needed
> complicated lead optimization
> low throughput
115
How is a hypothesis generated
from either in-house experimentation, or from external published material, or just the eureka moment
> link a fundamental biological process to pathology
> modifying the pathway should be expected to be curative
important step: does a target translate from animal to human
116
Considerations in targets and drug action
selectivity between species to be considered
> antibacterial and antiviral agents
- identify unique targets to the invading pathogen
- identify shared targets but ~different in structure
selectivity within the body to be considered
- selectivity between different enzymes, receptors etc
- selectivity between receptor subtypes or isoenzymes
- organ selectivity via delivery or selective expression of target
117
Target specificity: unique target in pathogens
cell division in bacteria is v different than in humans
FtsZ and FtsQ are involved in bacterial cell division
FtsZ and FtsQ are specific for bacteria
118
What type of ligand selectivity is difficult to control
Kinase inhibition
> not a single kinase inhibitor is active at ONLY one kinase
> kinase inhibitors are active at several kinases
> difficult to translate a kinase to disease modulation
> first generation inhibitors all target ATP site, which is common to all kinases
119
H4R and the immune system
validation & optimization
new GPCR discovered in the genome
aerosolized histamine recruits mast cells in trachea via H4R
> high HA levels = hay fever / allergic asthma
pharmacological validation
> use of H4R-specific antagonist
genetic validation
> knockout of H4R receptor
> generate inflammation in airway, look at number of inflammatory cells infiltrating the airway
optimization by SAR
- addition of a HBD is essential
- Cl/Br optimal on 5-position
- polar group on 5-position tolerated
- OCH3 on 5-position too big
120
What is validation and when does it take place
after target selection
target validation is a form of risk assessment
> the better the validation, the lower the risk in advancing an expensive project
121
How to validate a target
demonstrate that target is relevant to disease mechanism using genetics, animal models, tool compounds (compounds acting on targets but arent drugs), antibodies
122
What happens after target validation
1) compound screening
> HTS and selective library screens
> improving compound properties
2) secondary assays
> in vitro and ex vivo
> selectivity and liability assays
3) in vivo analysis
> compound pharmacology
> early safety and toxicity studies
4) candidate
> preclinical safety etc etc
123
how to hit your target: which is better, pros and cons
- low molecular weight compounds
> specificity is an issue
> easier to administer
> can enter a cell
> often cheaper
- by using biologicals e.g antibodies
> very specific
> not easy to administer
> can not enter a cell
> complex production
> very expensive
- by using peptides
124
What is a hit
a compound that has activity at a predetermined level against a target. Validated after first identification
Often active at micromolar levels; no selectivity needed, no real other useful properties, yet! (comes in hit-2-lead phase)
125
How to find hits?
Experimental screening
– High-throughput screening (HTS)
– Focussed libraries (e.g. known GPCR binders on a new GPCR)
– Fragment libraries; smaller libraries with smaller compounds!
– Natural products
Virtual screening
– Computer-based screening to identify “potential hits”
– Ultra-HTS screens in silico need experimental validation thereafter
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How does HTS screening compound libraries work?
HTS
> starts with the large sample pool and identifies the Hits (smaller sample pool)
Hit-to-lead phase
> Confirmed hits undergo further characterization (e.g studying of SAR relationships)
> further reducing the sample pool from hits to leads
Lead optimization
> systematically modify lead to improve its drug-like properties
> goal is to turn a lead into a drug candidate
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Virtual screening vs HTS
HTS
> automatized, but expensive and time-consuming
Virtual screening
> high-performance computing (simulates interactions between molecules and proteins)
> analyze large databases of chemical compounds in order to identify hits by e.g. docking in x-ray structures, structure-based design, or ligand-based design
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What to screen in virtual screening?
Ligand databases
* Own database
* ZINC – online database with
230 M(!!) purchasable compounds – ready to dock!
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Testing hits in assays
Test hits in the primary assay at more conc’s
– Use freshly prepared solutions
* Storage of libraries in DMSO means that degradation of compounds can happen
– Concentration response curve
– Compare hits, cluster chemical families
Curve characteristics:
Biological activity
> often S-shaped relationship with 10Log[ligand]
EC50/IC50/Ki
> to quantify biological activity and compare ligands
Like Michaelis-Menten kinetics for enzyme-substrate interaction
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Hit validation
‘Hits’ are screened against an alternative assay (this could be a
functional assay instead of a binding assay or a different assay
format) to rule out false positives.
> false positive: a compound that pops up as hit in the assay, but is not active at target
> false negative: a compound that does not pop up as hit in the assay, but is active at target
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Why would you have a false positive during hit validation?
many assay interference mechanisms
> impurities, aggregation, redox, absorbance, auto-flourescence, light scattering
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Hit-to-lead phase
Identify those hits that have
properties (other than just activity against the target) that would indicate that they have potential for being
developed as drugs (e.g. pharmacokinetic (PK) properties).
> Some hit exploration (chemistry) is key at this phase to see the potential of the chemical scaffold for improving on-target action and getting an idea on some ADMET properties
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hit-2-lead-2-candidate iterative cycles
a cycle of: make > test > analyse > design > make etc etc
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Working with racemates
bio-isosteres can be used to remove asymetrical carbons
> we dont wanna work with racemic mixtures
> chiral synthesis or separation into an enantiomerically pure mixture can be hard
Replace carbon w/ e.g N or O
Create symmetry in molecule
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Working with peptide bonds
bio-isosteres can be used to remove peptide bonds
> dont want peptide bonds as they are easily cleaved via ADMET
> peptides to peptidomimetics
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Via the hit-to-lead phase, validated hits would have been optimized and tested
to determine which factors
- Selectivity versus a panel of other receptors (off-targets)
– Activity against relevant animal species
– Idea of off-target effects
* E.g. hERG ion channel giving cardiotoxicity after blockade
* Ames test for mutagenicity
– CYP450 interaction
* get an idea of metabolism & potential for drug-drug interactions
– Drug-like properties, like
* metabolic stability in liver microsomes or hepatocytes (liver cells)
to estimate half life
* Solubility, etc
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what is a lead compound
molecule that has therapeutic potential; i.e. prospect to be optimized for potency, ADMET (pharmacokinetics, safety) and to be tested in vivo models of disease
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Which adverse effects are checked for
- CYP450 interaction
- HERG channel interaction has been moved from late stage to early discovery efforts
- Ames test to test for genotoxicity
> Drug (or metabolites made by liver) rescue his- strain, that caries mutations in genes involved in essential histidine synthesis
> The Ames test uses bacteria that can’t grow without histidine because of a mutation. If a drug (or its metabolite) causes new mutations that fix that problem, the bacteria regain the ability to grow. That means the chemical can damage DNA — a red flag for genotoxicity
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What is IND and when does it first occur
Investigational New Drug application
> first occurs in clinical development stage
sponsor submits an IND application to FDA based on the initial testing
> FDA checks for safety based on
animal tox and clinical study plan before a company can start a Phase 1 clinical trial
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difference between and IND and NDA filing and when one can actually sell a drug commercially.
IND
Purpose: Request to start clinical trials in humans
When: Before Phase I trials
NDA: New Drug Application
Purpose: Request to market and sell the drug
When: After clinical trials (Phases I–III)
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Clinical development phase
first in man
determine safety & efficacy of product (in that order!!)
At the end: approval by government for commercial use
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Which phase costs the most
clinical development phase and approval phase
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Clinical attrition
failure or dropout of drug candidates during the clinical trial process
Attrition at late clinical trial phase is very expensive and can be disastrous for smaller companies.
– Happens often because of toxicity and lack of efficacy
high risk, high costs, a lot of failure; even after approval sometimes (that is why there is also Phase 4)
