Midterm BIOC 308
Lecture 2 (both parts) and Lecture 3 part 1 up to slide 34
Define what drugs are, their classification and their specific action
Chemical applied to physiological systems that affects its function in a specific way, biological substance, synthetic/non synthetic, when taken into organisms body, will in some way alter biological function or organisms, or substances used in prevention diagnosis/ treatment of disease
Classification of drugs:
Pharmacological effect
Chemical structure
Target system
Target molecules
Drug molecules must be found to particular constituents (drug targets) of cells and tissue in order to produce an effect, most drug targets are protein molecules
Protein targets:
Receptors
Enzymes
Carrier molecules (transporters)
Ion channels
Protein receptor and ion channel targets: chemicals can be used to cause activation, blockage or modulation
Enzymes and transporter proteins: main drug effect is inhibition of ongoing basal activity of these drugs
Describe receptors and their classification
Receptors in pharm describe protein molecules whose function is to recongize and respond to endogenous chemical signals (ie. cytokine receptors)
Protein receptors and ion channels targets: chemicals can be used to cause activation, blockade/modulation
Enzyme and transporter proteins: the main drug effect is inhibition of ongoing basal activity of these targets
specificity /selectivity of drug interactions
Selectivity: ability of a drug to discriminate between related targets, showing a increase binding affinity for increase isoform → reciprocal
Ind classes of drugs bind only to certain targets
Ind targets recognize only certain classes of drugs
Specificity: if a drug has increase effect and only increase effect on all biological systems
Explain drug-receptor interaction using the following terms:
Affinity
Ability of drug moelcule to bind to a receptor at any given time
Governs the tendency of a drug to bidn to receptors
Tendency to bind to receptors
Efficacy
(Emax) is max effect that can be expected from this drug
Governs a drugs tendency to activate the receptor once bound
A measure of magnitude of effect once drug is bound
Potency
A measure of quantity of drugs needed to prudce a maximal effect
Drugs with more potency usually have higher affinity for their receptors
Agonist
Drugs that affect receptors in such a way as to elicit a tissue response
Full agonist → activity/response
Partial agonist → partial activity/response
Full agonsits: efficacy of which is sufficient that they can elicit response by occupying all/a fraction of receptors
Partial agonsits: drugs with intermediate levels of efficacy, even when 100% of receptors are occupied the response submaximal
Antagonist
Drugs that anatagonize agonists, stops the effects of drugs, no response/activity
Describe various dose-response curves and the following terms:
Emax: maximal response that a drug can produce (efficacy)
EC50: concentration needed to produce a 50% maximal response, concentration effect surves cant be used to measure affinity of full agonist drug
ED50: dose needed to produce a 50% maximal response
Define and describe 5 types of drug antagonism
Drug antagonism: effect of one drug is diminished/completely abolished in the presence of another
Chemical antagonism: uncommon situation where 2 substances combine in a solution
Effect of drug is lost → chelating agents, neutralizing Abs
Pharmacokinetic antagonism: reduced concentration of active drug at its site of action
Clinically important interactions
Rate of metabolic degradation of active drugs may be increases
Rate of absorption of active drug from GI tact may be decreased
Rate of renal excretion may be increased
Competitive antagonism: both drugs binding to same receptors; antagonism may be reversible/irreversible
Noncompetitive antagonism: opposing action of agonist, does so without competing with it for binding site
Antagonist blocks at some point the chain of events that leads to production of a response by agonist
Binds to allosteric site on receptor to prevent activation of receptor
Differentiate receptor desensitization, tachyphylaxis and tolerance
Receptor desensitization: desensitization = tachyphylaxis, effects of a drug gradually diminished when its given continuously/repeatedly
Tolerance: used to describe a more gradual decrease in responsiveness to a drug, taking days-weeks to develop
Lecture 3 pt 1 (slide 34) to Lecture 4 pt 2
NO QUESTIONS ON ENZYMES - slide 42-58 of lecture 3 pt 3
List 4 main protein targets for drug binding
Receptors
Ion channels
Enzymes
Transporters (carrier molecules)
Define 4 main receptor types (superfamilies) (with examples from the slides) and receptor heterogeneity
Ligand gated ion channels (ionotropic receptors)
Only opens when 1 or more agonist molecules are bound and properly classified as receptors, membrane proteins, and receptor in extracellular domain
Milliseconds
Ex. nicotinic ACh receptors, GABAa receptor domain
G-protein coupled receptors (GPCRs) (metabotropic)
Membrane receptors coupled into intracellular effector systems via a G-protein
Seconds
Ex. muscarinic ACh receptors, adrenoreceptor
kinase -linked receptors
Hours
Ex. cytokine receptors, GFs and insulin
Nuclear receptors
Hours
Ex. oestrogen receptor, steroid receptor
Characterize nicotinic acetylcholine receptor (structure, function and mechanism of action of ACh)
Nicotinic ACh receptor → agonist: acetylcholine and nicotine, antagonists: tubocurrarine, alpha-bungarotoxin
Ligand gated ion channels is an example of nicotinic ACh receptors
Ion channels controlled by the nicotinic ACh receptor is made up of 5 subunits of 4 different types → heteropentameric structure that forms a cluster surrounding a central transmembrane pore
2x alpha, beta, gamma and delta subunits
Two ACH binding sites at the alpha-delta and alpha-gamma subunit interfaces
Lining of transmembrane pore is formed by the M2 helical segment (hydrophobic leucine and valine) of each subunit → contain a preponderance of negatively charged aa’s which makes up the pore cation selective
Permeable mainly to Na+ and K+
When ACHbinds, the twisted alpha helices either straightens out or swing out of the way → opening a channel pore
Receptors have the fastest synaptic events in the NS → NT acts on postsynptic membrane of a nerve/muscle cell and transiently increases its permeability of particular ion
Ligand binding and channel opening occur on a millisecond timescale
Describe the following subtypes of G-proteins
Gs: stimulated adenylyl cyclase, causing increased cAMP formation
Activated by cholera toxin, which blocks GTPase activity, thus preventing inactivation
Activation of adenylyl cyclase and increase cAMP level
Gi: inhibits adenylyl cyclase, decreasing cAMP formation
Blocked by pertussis toxin, which prevents dissociation of alpha-beta-gamma complex
Inhibition of adenylyl cyclase and decreases cAMP levels
Go: limited effects of alpha subunits (effects mainly due to beta-gamma subunits)
Blocked by pertussis toxin, occurs mainly in NS
Gq: activated phospholipase C, increasing production of second messengers inositol triphosphate and diacylglycerol thus releasing Ca2+, from intracellular sotres and activating protein kinase C (PKC)
Activation of PLC-P and increases JP2 and Ca2+ levels
Characterize 5 main effector mechanisms for G-protein-coupled receptors (GPCRs). You must know the names of all 5 effectors on slide 17, lecture 3 pt 2. Details of signaling cascades only for adenylate cyclase (cAMP) and phospholipase C (inositol phosphate)
Adenylyl cyclase: enzyme responsible for cAMP formation
Action of G-rptein and generation of secondary messenger explain how a message delivered to the outside of the cells surface can be transmitted ot enzymes within the cells
Avoids difficulties involved in messenger molecule having to cross a hydrophobic cell membrane
Involves a molecular ‘relay runner’ (G-protein) and several different enzymes in signaling cascade, at each stage, the action o fone protein/enzyme activates a much larger number of enzymes
The effect of one NT interacting with one receptor molecule results in a final effect that is several factors larger than one might expect
G-protein can bind to several different types of receptor ligand complexes
Different NTs and hormones interacting with different receptors can switch on the same G-protein activating adenylate cyclase
Phospholipase C: enzyme responsible for inositol phosphate and diacylglycerol (DAG) formation
Phosphatidylinositol (4,5) biphosphate (PIP2) is the substrate for a membrane bound enzyme, phospholipase C-beta (PLCbeta)
PLC splits it into diacylglycerol (DAG) and inositol (1,4,5) triphosphate (IP3), both of which function as second messengers
The activation of PLCbeta by various agonists is mediated through G protein (Gq)
Inositol (1, 4, 5) triphosphate is water soluble mediator that is released into the cytosol and acts on a specific receptor - IP3 receptor - which is ligand gates calcium channel, present on the membrane of the ER
Main role of IP3 is to control the release of Ca2+ from the intracellular stores?
DAG is highly lipophilic and remains within the membrane. It activates protein kinase C (PKC), which catalyzes the phosphorylation of a variety of intracellular proteins
GTP/Gq protein complexes binds to phospholipase C that hydrolyzes PIP2, releasingDAG and IP3
Cytoplasmic IP3 causes the release of cellular calcium stores, while membrane-bound DAG activated protein kinase C, which phosphorylates molecular targets via ATP
Protein kinase C activity is augmented by the presence of calcium
The system is regulated by enzymatic removal of IP3 and DAG, GTPase activity of the Gq protein, and removal of cytoplasmic calcium
Messenger works by mobilizing calcium ions from calcium stores in the ER
It binds to IP3 receptor and opens a calcium ion channel
Once the ion channel is open, calcium ions flood the cell and activate calcium-dependent protein kinases which in turn, phosphorylated and activate cell-specific enzymes
The released calcium ions also bind to a calcium-binding protein called calmodulin, activating calmodulin-dependent protein kinases that phosphorylate and activate other cellular enzymes
3. Ion channels: particularly calcium and potassium channels
4. Rho A/Rho kinase: system that control the activity of many signaling pathways controlling cell growth and proliferation, smooth muscle contraction, etc
5. Mitogen-activated protein kinase (MAP kinase): a system that controls many cell functions, including cell division, and is also a target for several kinase-linked receptors
GPCR desensitization and beta-arrestin pathways (without details of MAP kinase signaling)
Beta-arrestin pathways: desensitization and trafficking, G protein-independent signaling
GPCR desensitization: desensitization and trafficking of G protein coupled receptors
Describe 3 main types of kinase-linked and related receptors. Details of signal transduction mechanisms for
Receptors tyrosine kinases (RTKs)
Receptor tyrosine kinase (RTK): contains intrinsic tyrosine kinase activity (EGFR, VEGFR)
Proving to be highly important targets for novel anticancer drugs (overexpression)
Protein concerned plays the dual role of receptor and enzyme
Receptor protein is embedded within cell membrane and the outer surface contains the binding site for chemical messenger, and the inner surface has an active site that is closed in the resting state
When a chemical messenger binds to receptor, it causes the protein to change shape
This opens up the active site, allowing the protein to act as an enzyme within the cell
Activation mechanism for epidermal growth factor (EGF) receptor
Dimerization and auto-phosphorylation are common themes for receptors in this family some of receptors in this family already exist as dimers or tetramers
Receptor serine/threonine kinase: contains intrinsic serine/threonine kinase activity (TGF-betaR)
Cytokine receptors
Membrane receptors mediate the actions of a wide variety of protein mediators, including growth factors, cytokines and hormones such as insulin and leptin
Most of these receptors are large proteins consisting of a single chain of up to 1000 residues, with a single membrane-spanning helical region, associated with a large extracellular ligand-binding domain and an intracellular domain of variable size and function
Over 100 such receptors have been cloned, and many structural variations exist
Describe two main types of nuclear receptors (Class I and Class II), including structure, function and signaling pathways
Class I: steroid receptors
Maintenance of cellular homeostasis, gene expression regulation in embryogenesis, tissue development, their ability to respond to extracellular signals in an endocrine manner, which allows the cells to adapt to systemic environmental changes
Ex. glucocorticoid and mineralocorticoid receptors (GR and MR), estrogen, progesterone and androgen (ER, PR, and AR)
Generally form homodimers, and translocate to the nucleus, where they can transactivate or transrepress
Class II: RXR heterodimers
Ligands are generally lipids already present to some extent within the cell.
Heterodimers together with the retinoid X receptor (RXR)
Ligand is usually lipids (eg. fatty acids)
Classification of ion channels in general
Selectivity: for particular ion species, determined by the size of the pore and the nature of its lining
Cation-selective or anion-selective
Cation-selective channels may be selective for Na+, Ca2+ or K+ or non selective and permeable to all 3
Anion channels are mainly permeable to Cl-, although other types also occur
Gating properties: ie. the nature of the stimulus that controls the transition between open and closed states of the channel
Voltage gated channels - open when the cell membrane is depolarised. Most important channels in this group are selective sodium, potassium or calcium channels
Ligand-gated channels (ionotropic receptors): activated by binding of a chemical ligand to a site on the channel molecule, fast NTs such as glutamate, ACh and GABA
Calcium release channels: present on the ER or SR rather than the cell membrane: IP3 and ryanodine receptors, control the release of Ca2+ from intracellular stores
Store operated calcium channels (SOCs): when the intracellular Ca2+ stores are depleted, channels in the cell membrane open to allow Ca2+ entry
Molecular architecture: cation channels, voltage-gated sodium and calcium channels
Describe three main mechanism involved in the regulation of [Ca2+]i
Control of Ca2+ entry
Control of Ca2+ extrusion
Exchange of Ca2+ between cytosol and the intracellular stores
Define excitotoxicity and roles of calmodulin and mitochondria in Ca2+ signaling
Excitotoxicity: activation of NMDA receptor can readily cause so much Ca2+ entry that the cell dies, mainly through activation of Ca2+ dependent proteases but also by triggering apoptosis
Calmodulin: calcium exerts its control over cell functions by virtue of its ability to regulate the activity of many different proteins including
Enzymes (kinases and phosphatases)
Channels
Transporters
TFs
Synaptic vesicle proteins
Ca2+ binding protein serves as an intermediate between Ca2+ and the regulated functional protein, best known of binding proteins being the ubiquitous calmodulin which acts as part of a calcium signal transduction pathway
It regulated at least 40 different functional proteins
Calmodulin is a dimer with 4 Ca2+ binding sites
When all are occupied, it undergoes a conformational change, exposing a ‘sticky’ hydrophobic domain that lures many proteins into association, thereby affecting their functional properties
Describe general mechanisms underlying excitation (‘resting’ cells, action potential), contraction (differences between 3 types of muscles cells), secretion (exocytosis, carrier-mediated transport, diffusion)
Excitation: ability of a cell to show a regenerative all-or-nothing electrical response to depolarization of its membrane, this membrane, this membrane response being known as an action potential
3 factors that are important:
Membrane potential
Permeability of the plasma membrane to different ions
Intracellular ion concentrations
‘Resting’ cell: all cells maintain a negative internal potential between -30mV and -80 mV
Na+ ions are actively extruded from the cell in exchange for K+ ions by an energy-dependent transporter, Na+ pump (Na+-K+ ATPase)
Membrane is relatively impermeable to Na+
Action potential: generated by the interplay of two processes:
rapid , transient increase in Na+ permeability that occurs when the membrane is depolarized beyond about -50 mV
Slower, sustained increase in K+ permeability
Contraction: muscle contraction occurs in response to a rise in [Ca2+]i
Types of muscles cells:
Skeletal - depolarization causes rapid Ca2+ release from the SR
Cardiac - Ca2+ enter through voltage-gated channels, and this initial entry triggers further release from SR
Smooth - Ca2+ signal is due to partly to Ca2+ entry and partly to inositol triphosphate (IP3) mediated release from SR
Describe the regulation of the cell cycle: positive and negative regulators, extracellular matrix, integrins, matrix metalloproteinases and angiogenesis
Positive regulators: cell cycle that control the changes necessary for cell division
Impetus for a cell to start off on the cell cycle (to move from G0 to G1) can be provided by several stimuli, the most important being growth factor action though kinase-linked or GPCRs can also stimulate cell proliferation
Main components of control system that determines process through the cycle are 2 families of proteins:
Cyclins
Cyclin-dependent kinases (CDKs)
Negative regulators: controls the positive regulators
Rb protein that holds the cycle in check while it is hypophosphorylated
Inhibitors of CDKs bind to and inhibit the action of the cyclin/CDK complexes
2 families of inhibitors:
CIP family (CDK inhibitory proteins also termed KIP or kinase inhibitory proteins)
Ink family (inhibitors of kinases)
Extracellular matrix (ECM) and integrins: cells are embedded in the extracellular matrix which is secreted by the cells themselves
ECM forms a store of growth factors by sequestering them
ECM significantly influences the cells through the cells ‘integrins’
Integrins: transmembrane receptors that on interaction with elements of the ECM, mediate growth factor signaling pathways and also mediate cytoskeletal adjustments within the cell
ECM is secreted by the cells and provides a supportive framework → main protein is collagen
Profoundly influences cell behaviour by signaling through its integrins
ECM expression by cells is regulated by growth factors and cytokines
Activity of some growth factors, is in turn, determined by ECM because they are sequestered by its components and released by proteases
Integrins are proteins on a cells extracellular surface that sense the ECM and signal to the cell what environment it is in/which cell neighbours it has
Matrix metalloproteinases (MMPs): MMPs belong to a family of endopeptidases that contains 23 members, contains Zn, are dependent on Ca and can degrade and remodel the proteins that form the ECM
Degradation of ECM by MMPs is necessary for tissue growth, repair and remodeling
When growth factors stimulate a cell to enter the cell cycle, they also stimulate the secretion of MMPs (as inactive precursors) which then sculpt the matric, producing the local changes necessary to accommodate the increase cell numbers
Metalloproteinases also release growth factors from the ECM and can activate some that are present in precursor form
Angiogenesis: formation of new capillaries from existing small blood vessels, important stimulus is vascular endothelial growth factor (VEGF)
Sequence of events:
The basement membrane is degraded locally by proteases (MMPs)
Endothelial cells migrate out, forming a sprout
Other endothelial cells following the leading cell proliferate under the influence of VEGF
Matrix is laid down around the new capillary
Characterize apoptosis (morphology, two main pathways, p53 as the ‘guardian of the genome’)
Apoptosis: cell suicide by a built-in self-destruct mechanims consisting of a genetically programmed sequence of biochemical events
Different from necrosis which is organized disintegration of damaged cells resulting in products that trigger the inflammatory response
Involved in numerous physiological events:
Shedding of intestinal lining
Death of time-expired neutrophils (type of leukocytes)
It is implicated in the pathophysiology of many conditions, from cancer, when there is insufficient apoptosis, to neurodegenerative conditions, which may involve increased neuronal apoptosis
Continuous active signaling to tissue-specific trophic factors, cytokines and hormones, and cell-to-cell contact factors is required for cell survival and viability
Main signaling pathways in apoptosis:
Cysteine ASparatase proteases
Caspases 8,9 = initiator caspases
Caspase 3 = effector caspase, it initiates cleavage of cell constituents
Morphology: cell “rounds up” (cytoskeletal changes and loss of adhesion)
Chromatin in the nucleus condenses into dense masses, cytoplasm shrinks
Followed by blebbing the plasma memrabne
Transformation of the cell into a cluster of membrane bound entitled
Displays ‘eat me’ signals → surface exposure of phosphatidylserine, changes in surface sugars
Macrophages recognize these signals and rapidly phagocytize the remains
The fact that the remains are membrane bound is important because the release of the internal constituents into the cells surroundings could trigger an unwanted inflammatory reaction
Cell proliferation and apoptosis as targets for drug development
Growth of tissues and organs in the embryo and later during development
Replenishment of lost/time expired cells such as leukocytes, gut epithelium, uterine endometrium
Development of immunological tolerance to host proteins
Repair and healing after injury or inflammation
Hyperplasia (increase in cell number and in connective tissue) associated with chronic inflammatory, hypersensitivity and autoimmune diseases
growth , invasion metastasis of tumor
Regeneration of tissues (stem cells)
Examples of over-exuberant apoptosis with increase in cell death:
Neurodegenerative diseases → Alzheimers, MS and parkinson's disease
Conditions with tissue damage/cell loss, such as myocardial infarction, stroke and spinal cord injury
Depletion of T cells in HIV infection
Osteoarthritis
Hematological diseases such as aplastic anemia
Examples of defective apoptosis:
Cancer - evasion of the immune response and resistance to cancer chemotherapy
autoimmune/inflammatory disease such as myasthenia gravis, rheumatoid arthritis and bronchial asthma
Viral infections with ineffective eradication of virus-infected cells
Possible new drug targets:
Drugs that promote apoptosis by various mechanisms a potential new approach to cancer treatment but none has yet been approved for clinical use
Inhibiting apoptosis might prevent or treat a wide range of common degenerative disorders
Angiogenesis and metalloproteinases inhibitors: only bevacizumab (mAb that neutralizes VEGF) approved fro use in cancer treatment
Cell cycle regulation for a new anti-cancer drugs
Several small molecules that inhibit CDKs by targeting the ATP-binding sites of these kinases have been developed; they arrest the cell cycle
Kinase inhibitors targeting various components of the growth factor signaling pathway
Describe bioassays and the following principles:
The use of standards:
Scientists aren't able to agree on absolute values due to variability in techniques, therefore biological assays are designed to measure the relative potency of two preparations, using a standard and unknown
Best standard → pure substance, even though, 100% purity usually isnt achieved
Lab samples can be calibrated
Variability in techniques means that absolute values may differ between labs. Bioassays measure relative potency between a standard and unknonw sample to ensure consistency
The design of bioassays:
Main problem with all types of bioassays is biological variation and design of bioassays
Minimizing variation
Avoiding systematic errors resulting from variation
Estimation of the limits of error of the assay result
Graded and quantal responses
Example comparison of potency of unknown and standard in bioassay: comparing the magnitude of responses prejudiced by the same does (volume) of standard and unknown gives no quantitative estimate of their relative potency
Parallel lines assays
Aim to minimize biological variation, avoid systemic errors and estimate error limits. Potency is compared by measuring equieffectvive doses rather than simply compared responses at same dose. Common design, 2+2 parallel line assay, uses 2 doses of standard and 2 doses of unknown in random order, allowing statistical analysis to determine confidence limits
Identify the main advantages and disadvantages of animals models of human disease
Should resemble the human disease
Similar pathophysiological phenotype (face validity)
Similar causation (construct validity)
Similar response to treatment (predictive validity)
Disadvantage: difficult/impossible to observe in animals (the disease), ‘cause’ of many humna diseases is complex/unknown and drugs acting by novel mechanisms could be missed
Advantage: biological similarities, controlled studies and longer life spans
Distinguish two main categories of bioassays in humans
Experimental pharmacodynamic/pharmacokinetic investigations - use human subjects (healthy volunteers/patients) as experimental models to confirm mechanisms observed in animals also apply to humans. Ethical and safety considerations are paramount, overseen by research ethics committees
Clinical trials: these trials measure therapeutic efficacy and safety by comparing a new treatment (A) with a standard treatment/control (B), which may induce existing therapies, a placebo/no treatment if no standard exists. Clinical trials use a control group to provide a valid comparison ensuring that any therapeutic claims are based on objective testing rather than anecdotal observations
Recognize 3 main sources of bias in clinical trials
Ways to reduce bias/avoidance of bias
Randomization: essential to avoid bias, randomization assign patients to treatment/control groups without bias
Binding: outcomes are assessed without patients/investigators know in which treatment was given to avoid bias (though this isnt always feasible, such as with surgeries)
Rigorous follow-ups: collecting comprehensive data completeness and minimizes dropouts
Adherence to specific interventions
Describe the following the terms associated with clinical trials:
Type I and type II errors
Type I (false positive) occurs when a different between treatments A and B is found, even though more exists
Type II (false negative) occurs if no difference is found although A and B do actually differ
Meta-analysis
Pools data from multiple randomized trials, enhancing the statistical power and significance of findings, helping to draw more reliable conclusions
Subject to publication bias - studies with positive results are more likely to be published than those with negative results
Clinical outcomes measures
Increasingly assessed by improvements in quality and length of life, alongside social and economical benefits, rather than by only objective indicators (eg. lowered BP)
Health related quality of life is often measured using scales combined with life expectancy to calculate quality adjusted life years (QALYS), integrating survival and suffering relief
Placebo
Placebo effects are believed to provide notable benefits (powerful therapeutic effect), especially in pain/nausea trials, however, evidence of placebo efficacy varies by condition and trial design
Therapeutic index and how it is calculated (there may be one problem)
Ratio between average maximum tolerated (non-toxic) dose and average minimum effective dose in a group of subjects
Reflects a drugs safety margin, can also be defined as LD50/ED50 (lethal dose/effective dose)
Larger TI indicated a safer drug, while a smaller TI reflects narrow safety margins (eg. heparin)
TI is limited since it doesnt account for individual sensitivity/idiosyncratic reactions
TI = max non toxic dose/max effective dose or TD50/ED50
TI window (window between therapeutic and toxic effect)
Wide window = good
Narrow window = bad
Define the pharmacokinetic processes of ADME
Successful drug must be able to cross the physiological barriers that exist to limit the access of foreign substances from the body
Drug absorption → may occur by several mechanisms designed either to exploit/breach these barriers
Distribution → systems within the body, such as the blood/lymphatic vessels, to reach its target organ in an appropriate concentration
Metabolism → body inactivates the drug through enzymatic degradation (liver)
Excretion → drug is eliminated from body (kidneys, liver and by feces)
Pharmacokinetics: what the body does to the drug
Administration → ADME
Describe the translocation of drugs and the “compartmental model” of drug distribution
Describe lipophilicity and how it is measured/calculated
Correlates to solubility and permeability, metabolism, toxicity, protein binding and distribution
Ability of a chemical compound to dissolve in fats, oils lipids and nonpolar solvents
Partition coefficient between water and immiscible solvent (log P) in two non-miscible solvents (eg. water n octanol)
Equation: log P = log([compoundorganic]/[compoundaqueous])
Log P is affected by structural properties of the compound: MW, dipolarity and hydrogen bond acidity and basicity
Lipophilicity changes with conditions of the phases: partitioning solvents/phases, pH, buffer, and cosolutes/co solvents
Recognize the main mechanisms for drug crossing of cell membranes
By diffusing directly through the lipid
By diffusing through aqueous pores formed by special proteins that transverse the lipid
By combo with transmembrane carrier protein that binds a molecule on one side of the membrane
By pinocytosis (“cell-drinking”)
Describe factors governing the diffusion of drugs across membranes
Nonpolar molecules (hydrophobic) dissolve freely in membrane lipids and therefore diffuse freely across cell membranes
Number of molecules crossing the membrane:
Flux = number/area x sec → total amount lost = p x (cin-cout) x A
Define and explain pH trapping
Phenomenon where weak acids, like aspirin (pka=4), gets trapped in a specific body compartment due to pH difference
In acidic stomach compartment (pH = 1) aspirin mostly exists in its protonated, neutral form, allowing it to diffuse through the gastric mucosal barrier into the bloodstream
Once in the blood (pH = 7), the drug becomes deprotonated and negatively charged, preventing it from corssing back through cell membranes and effectively trapping in the plasma
Weak acids - pH differences across membranes determine where they accumulate. In acidic environment, weak acids remain neutral and can easily cross membranes, but in more basic conditions. They ionize and becomes “trapped” due to their inability to diffuse back
Doesnt fully determine drug absorption sites - other factors life gastric emptying rates and large absorptive areas of ileum, play significant roles
Extent of drug trapping on one side of the membrane is determined by the drugs acid dissociation constant (pKa) and by the pH gradient across the membrane
Review carrier-mediated transport
SLC transporters (solute carrier): predominantly facilitative (passive)/secondary active; rely on electrochemical gradient/ion gradients generated by ATP dependent pumps → membrane proteins that facilitate the movement of solutes across cell membranes and play crucial roles in various physiological processes
Uniporters: transports one solute molecule at at time, down its concentration gradient (passive)
Symporter: transport of two molecule together in the same direction
Antiporter: exchange of one molecule for another in opposite directions
Organic cation transporters (OCTs): transport molecules like dopamine and certain drugs and OCT2, foynd in kidneys, can concentrate nephrotoxic drugs like cisplatin
Organic anion transporters (OATs): handle secretion of urate, prostaglandins and various drugs, such as antibiotics and anti-inflammatory agents
ATP binding cassette (ABC) transporters: active pumped fueled by ATP
ABC transporters: P-glycoprotein is most widely known ABC transporter
170 kDA proteins with 1280 amino acids and 12 transmembrane segments
P-glycoproteins: interest in transporters such as Pgp by pharmaceutical industries, limits oral absorption of compounds counteracts access to CNS
Describe plasma protein binding and partitioning into body fat and other tissues
Binding to plasma proteins: fraction of drug that is unbound and pharmacologically active in plasma can be less than 1%, the remainder being associated with plasma proteins, it is the unbound drug that is pharmacologically active!
Extent of drug binding to proteins depends on concentration of free drug, protein concentration and drugs affinity for proteins binding site. Binding is dynamic, with association and dissociation rates affecting how quickly a drug can act
Binding capacity and kinetics: plasma album has a binding capacity of approx 1.2 mmol/L and the extent and rate of binding affect drug distribution, high bound drugs with slow association rates may have limited distribution, while with fast dissociation rates may distribute more freely, regardless of binding
Partition into body fat and other tissues: fat represents a large, nonpolar compartment of the body
Only important for a few drugs:
Effective fat/water partition coefficient
Fat tissues have a very low blood supply - less than 2%
More important when lipid soluble drugs are given chronically
Make a distinction between absorption and administration and describe common routes of drug administration
Absorption: passage of a drug from its site of administration into plasma, drug must enter plasma before reaching its site of action
Administration: giving a drug by one of several means (routes)
Routes of drug admin:
Enteral: (eg. aspirin) simple and painless, first pass metabolism and slow delivery
Parenteral: (eg. morphine) not subject to first pass metabolism, and irreversible
Mucous membrane: eg beclomethasone
Transdermal: eg nicotine
Or.. oral, sublingual, rectal, application to other epithelila surfaces, inhalation and injection
Identify factors affecting GI absorption
75% of drug given orally is absorbed in 1-3 hrs
Altered by physiological and formulation of drug
Main factors:
Gut content - food intake affects both gut content and blood dlow to the digestive organs, for some drugs, food can delay absorption, while for other food may enhance absorption due to increased blood flow
GI motility - conditions like mirgaines/diabetic neuropathy can slow gastric emptying, delaying drug absorption
Splanchnic blood flow
Particle size and formulation - drugs are often formulated to control absorption rates, capsules and tablets amy include a combo of slow and fast release, extending the dose interval and minute
Physicochemical factors eg drug interactions - chemical interactions in the gut can impact drug absorption
Genetic polymorphisms in transporters
Drug-drug competition for transporters
Define bioavailability
(F) fraction of an orally administered does that reaches the systemic circulation as an intact drug
F = quantity of drug reaching systemic circulation/quantity of drug admin
Describe the distribution of drugs in the body: different body compartments, body fluid compartments
Distribution of drugs in the body: drug distributuon to different body compartments
Total body water: small water soluble molecules eg/ ethanol
Extracellular water: larger water m=soluble molecules eg. mannitol
Blood plasma: highly plasma protein bound molecules, very large molecules, highly charged molecules eg. heparin
Fat: highly lipid soluble molecules eg. diazepam
Bone and teeth: certain ions eg. fluoride and strontium
Drug distribution: drug molecules exist in bound/free form in each compartment, but only the free drug is able to move between compartments → body fluid compartments
Total body water → 50-70%
Plasma blood → 4.5%
Interstitial fluid → 16%
Lymph 1.2%
Intracellular fluid → 30-40%
Transcellular fluid → 2.5%
4 compartment model: after IV admin, drugs are distributed sequentially
Vessel rich group: tissues with increased blood flow (eg. brain, liver, kidneys, heart) receive drugs first
Define and explain the volume of distribution of a drug (Vd) and its dependence on the drugs distribution pattern
Volume of distribution: volume of plasma that would contain totally content of drug (Q) at a concentration equal to that in plasma (Cp)
Vd= Q/Cp
Low Vd = drugs mostly confined to vascular compartment (eg. highly protein bound drugs)
High Vd = drugs extensively distributed into nonvascular compartments (eg. muscle, adipose)
Interpreting Vd: Vd isnt a physical volume but a reflection of distribution pattern, drugs with high Vd odten indicate significant tissue binding/sequestration outside the plasma
Ex. chloroquine (235 L/kg)
Define “loading dose”
If a drug takes a long time to reach therapeutic levels, then a higher dose (loading dose) may be given initially before dropping down to a lower maintenance dose (use in case of eg. digoxin - helps to treat heart failure)
Characterize the blood-brain barrier
BBB: regulate “brain penetration”, ensuring that compounds reach therapeutic targets in the brain
Structure: continuous layers of endothelial cells with tight junctions, reinforced by pericytes around capillary walls, covers over 400 miles of brain capillaries with surface area = 12m^2
Mechanisms of affecting BBB permeation:
Restrictive physicochemical characteristics - limits passive diffusion based on molecular size, lipophilicity and polarity
Efflux activity - increases expression of efflux transporters (eg. Pgp) pumps drug back into circulation
No paracellular/fenestrated permeation - tight junctions eliminate “leaky: access points
Limited pinocytosis - minimizes vesicle based transport
Endothelial cell metabolism: drugs may be metabolized before crossing
Uptake transporters: facilitate entry of certain compounds
Review examples of special drug delivery system
Prodrugs: inactive precursor that are metabolized into active forms within body
Ex aspirin (converted into salicylate (active metabolite) for anti inflammatory and analgesic effect), Levodopa (absorbed in GI tract, crosses BBB via aa transport mechanism and converted to dopamine in brian for Parkinsons disease)
Ab-drug conjugates: mechanism - drugs are chemically attached to Abs targeting tumor specific Ags, this ensures selective delivery of cytotoxic agents to tumor cells, minimizing off-target effects
Packaging in liposomes: structure - tiny vesicles formed by sonication of phospholipids in aqueous suspension
Mechanism: encapsulate drugs (nonlipid soluble) to protec them until liposome breaks down, liposomes are taken up by reticuloendothelial cells (eg. in liver)/penetrate malignant tumor via enhanced permeability and retention
Coated implantable devices: designed for localized drug release from implants
IUDs
Stends
Tumor therapy
Describe the main routes of drug elimination
Urinary tract
GI tract
Skin
Lungs
Biliary tract
Describe the main types of drug metabolism (biotransformation)
Metabolism: enzymatic conversion of one chemical entity to another within body
Drug metabolism: essential process that modifies drugs for easier elimination from body, primarily converting lipophilic drugs into more polar forms
Biotransformation: metabolic process that chemically modifies substances in the body to make them easier to excrete
Phase I reactions: general characteristics, P450 monooxygenases (including the mechanism), other reactions
Catabolic → involved oxidation, reduction/hydrolysis
introduce/expose functional groups, prepare the drug for phase II conjugation reactions
Products: more water soluble than parent compounds, can be more chemically reactive, toxic/carcinogenic than original drug (eg. acetaldehyde from ethanol)
Prodrug activation: some drugs are converted to active forms through phase I metabolism, enhancing bioavailability and therapeutic effects
Cytochrome P450 (CYP) enzymes: heme proteins responsible for drug metabolism, P450 family includes a large, diverse group of enzymes differing in aa sequences
Primarily reaction introduces a hydroxyl group by adding one more O2 atom to drug, NADPH and cytochrome P450 reductase facilitate electron transfer for this process
Phase II reactions, general characteristics
anabolic/synthetic
Conjugation with endogenous molecules such as glucuronic acid sulfate/glycine
Increase water solubility significantly facilitate renate and biliary erection
Products: increased water soluble compounds, typically inactive and excreted as primary metabolic products
Mechanism: involves attaching a substituent group to a drug/its phase I metabolite, forming a pharmacologically inactive and water soluble conjugate for excretion
Requires a functional group like hydroxyl/amino
Conjugates are excreted in urine/bile
Drug-drug interactions (inhibition of induction of CYP450, mechanism-based (suicide) inactivation)
Inhibition of CYP450: many drugs interactions results from CYP enzyme inhibition
Types of inhibition: competitive inhibition (drugs like quinidine compete for active sites without being substrates), non competitive inhibition (ketoconazole based inhibition (“suicide inhibitors”), eg. reactive oxidation products) irreversibly inactivates P450 enzymes
Modulation of CYP activity (induction/inhibition) by one drug (“perpatrator”) alters the metabolism of a co administered drug (“victim”) protentially causing:
Reduced efficacy and adverse effects
Secondary pharmacological implications of metabolism:
Bioactive metabolites
Routes that result inactive metabolites - detoxification process
Metabolites with a similar activity to drug: metabolic can exhibit a different potency/duration of action with respect to original drug
Activity with metabolite has no relationship tot hat of parent drug
Acetaminophen toxicity: caused by its CYP metabolite NAP21
Prodrugs (and why they are used)
Drugs become active only after they have been metabolized
Prodrugs used to
Increase lipid/water solubility
Improve taste (more patient compatible)
Alleviate pain when is administered by injection
Decrease toxicity
Increase chemical and bio stability
Change in length of time of duration of action
Deliver drug to specific body site
Bioprecurose prodrugs - already contain embryo of active spp. With their structure (metabolized into active drug)
Carrier prodrugs: link between carreri and active spp. Mus tbe a group easily metabolized once absorption/drug reached target
Ie lipophilic carrier to imporve transport through membranes
ADEPT/anitbody: directed enzyme prodrug therapy
Microsomal enzyme induction
Enzyme induction can increase drug toxicity and carcingenciity because phase I metabolites = toxic/carcinogenic
Enzyme induction is exploited therapeutically by admin phenobarbital to premature babies
Describe the stereospecificity of drugs and their metabolites, first pass (presystemic) metabolism and enterohepatic recirculation of drugs
Stereoisomers in drugs: many drugs (eg. warfarin, ibuprofen) are mixtures of stereoisomers, stereoisomers differ in pharmacological effects and metabolism pathways
Toxicity concerns: toxic effects may be linked to one stereoisomer, which may not be the active one
Regulatory standards: new drugs are encouraged to be pure stereoisomers for consistent effects and reduced risks
Metabolism and stereospecificity: enantiomers can underdog different metabolic routes, producing distinct metabolites and racemic mixtures are treated as combos of 2 drugs, each with unique PK and PD
Characterize and compare three fundamental processes that account for renal drug excretion
Renal excrection: drug clearance by kidneys
Glomerular filtration: drugs < 20,000 Da freely diffuse into glomerular filtrate, highly protein bound-drugs (eg. warfarin) show limited filtration due to albumin binding
Tubular secretion: drug transfer into proximal tube via active transporters:
OATs - transport acid drugs
OCTS
Protein binding doesnt limit secretion since transporters act on free drugs
Passive reabsorption: lipid soluble drugs are poorly excreted due to reabsorption, polar drugs remain in the tubular lumen and are concentrated as water is reabsorbed
Renal excretion and pH
Routes of excretion: kidneys (primary route), hepatobiliary systems, lungs and other routes (minor)
Factors influencing renal excretion:
pH of urine: acidic urine enhances excretion of basic drugs (eg. amphetamines), alkaline urine enhances excretion of acidic drugs, pH adjustments can enhance drug clearance in case of toxicity
Renal function: lipid soluble drugs like digoxin on renal clearance, making dosage critical in renal impairment (eg. elderly and renal disease)
Define and explain the following terms:
Drug clearance
Rate of elimination of drug (mass/unit time) volume of palsma cleared of drug per unit time (in mL/min or L/hr)
Overall clearance of a drug by all routes (CLtot)
Rate of drug elimination = Cp x CLtot
First-order kinetics
Occur when a constant proportion of a drug is eliminated per unit time
Rate of elimination is directly proportional to drug concentration
Rate of elimination = -kel x Cp
Zero-order kinetics
Occurs when processes responsible for drug elimination become saturated at/ear therapeutic drug concentration
Rate of drug elimination is constant and does not depend on plasma concentration of drug
Same amount of drug eliminated per unit time
Saturation kinetics
Elimination of drug does not follow simple first order kinetics, elimination kinetics is non linear
Drug half-life
Time it takes for a drugs concentration to fall (be eliminated) to half of its original value
t1/2 = (0.693 x Vd) / CLtot
Describe the single-compartment model used to describe the pharmacokinetics
Assumptions
Body behaves as a single, uniform compartment (only one compartment)
Drugs distribute instantly and homogenously after administration
Drug elimination follows first order kinetics, where rate of elimination is proportional to plasma concentration
C0 = Q/Vd
Describe the two-compartment model used to describe the pharmacokinetics
Assumptions:
Instantaneous mixing with compartments
Input and output via central compartment, drug admin and elimination through central compartment
Slower inter-compartment mixing
Distribution phase (alpha-phase)
Initial, rapid decline in plasma drug concentration represents drug transfer from central compartment to peripheral (tissue) compartment, phase ends when drug reaches distribution equilibrium → where drug rates entry and exit from tissue are equal
Elimination phase (beta-phase)
Slower, parallel decline in plasma and tissue concentration, begins after distribution elimination, system behaves like single compartment model
Identify factors affecting drug elimination half-life
Metabolic enzyme activity: induction (increases clearance and decreases half life) and inhibition (decreases clearance, prolongs half life)
Organ function: renal failure (decreases renal clearance, prolongs half life), liver dysfunction (decreases clearance, prolongs half life)
Cardiac output: decrease blood flow to clearance organs reduces drug elimination
Discuss repeated dosage in terms of half life and saturation kinetics, loading dose, and maintenance dose
Steady state concentration (Css): achieved when rate of drug administration = rate of elimination, takes ~ 3-5 half lives for drug to reach ss during repeasted dose
Loading dose: administered to rapidly achieve therapeutic drug levels, particularly for drug with long half lives
Maintenance dose: given to maintain ss of plasma concentration once equilibrium is reached
Repeated dosing patterns: smaller; more frequent doses → reduce oscillations and mimic continuous infusion
Larger infrequent → increased peak-through differences
Formulas to solve problems: slide 13 (half life), 22 (loading dose), 23 (maintenance dose). Some problems will require just basic arithmetic, there may be questions asking to explain other formulas from Lecture 8
See know quiz
Drugs
Dimenhydrinate: brand name for Gravol/Dramamine, diphenhydramine (primary constituent of dimenhydrinate) is a competitive H1 receptor agonist - antiemetic effect → top structure is diphenhydramine (DPH) - antihistamine, bottom structure: 1,3-methyl-8-chloro xanthine - stimulant drug
Epinephrine (adrenaline): first to bind to receptor protein (beta adrenoceptor), recognition site, when epinephrine binds to receptor, chain of reactions lead to:
Increase in force of heartbeat
Increase in rate of heartbeat
Buprenorphine: (partial agonist) exhibits high affinity binding to a mu-opioid receptors, differs from other full opioid agonist such as morphine and fentanyl
Reduces effect of most other opioid agonists
Oliceridne: (biased agonist) act on biased agonism at mu-opioid receptor by activating G protein pathway with minimal receptor phosphorylation and recruitment of beta-arrestin
Omeprazole: selective and irreversible proton pump inhibitor, suppressed stomach acid secretion, treats heartburn
Pimavanserin: inverse agonist, combo of inverse agonist and antagonist activity at serotonin 2A recetprs (5-HT2A), no appreciable binding affinity for dopamine, histamine, muscarinic/adrenergic receptors, atypical antipsychotic
Venetoclax: primary biological target: Bcl-2 (B cell lymphoma 2) → promotes apoptosis
Function: inhibits Bcl-2, an antiapoptotic protein, thereby promoting apoptosis in cancer cells (chronic lymphocytic leukemia (CLL))
Abemaciclib (verzenio): drugs arresting the cell cycle
Selectively inhibit CDK4 and CDK6, which are crucial for the G1 to S phase transition in the cell cycle.
By inhibiting these kinases, they prevent the phosphorylation of the retinoblastoma (Rb) protein (checkpoint 1), thereby blockign cell cycle progression and proliferation
Used for treatment of certain types of breast cancer
Prevents the progression of cancer cells from G1 phase to S phase of the cell cyle, effectively halting their proliferation
Tubocurarine: nondepolarizing neuromuscular blockers are competitive ACh antagonists that bind directly to nicotinic receptors on the postsynaptic membrane, thus blocking the binding of ACh so the motor endplate cannot depolarize (antidote is an acetylcholinesterase (AChE) inhibitor (anti-cholinesterase)
Muscle relaxant
Blocks the transmission of nerve impulse to muscles, causing paralysis
Doesnt directly depolarize the muscle membrane
Can cause histamine release, leading to hypotension and bronchospasm in some patients excreted primarily through kidneys and minimally metabolized in the body