PCTH Lecture Notes on Cannabis and Cannabinoids (P2) - PDF

Day 9 (Cannabis and Cannabinoids) Cannabis Annual herbaceous plant genus grown in temperate and tropical climates Major species: C. indica and C sativa (generally recognized for their pharmacological properties but there are also subspecies that float within the mix as a result...

Day 9 (Cannabis and Cannabinoids) Cannabis Annual herbaceous plant genus grown in temperate and tropical climates Major species: C. indica and C sativa (generally recognized for their pharmacological properties but there are also subspecies that float within the mix as a result of cross breeding, etc.) o Variety of alternate strains and/or hybrid subspecies o Vary in morphology, conditions for best growth and pharmacodynamics Cannabinoids Organic substances originally isolated from Cannabis spp. o Primarily flowers and leaves (can be theoretically obtained from any part of the plant) Subdivision based on origin o Phytocannabinoids: compounds found in cannabis plants (e.g. tetrahydrocannabinol) o Endocannabinoids: endogenous cannabinoids (endo = inside) (anandamide, 2-archidonoglycerol) o Synthetic cannabinoids Phytocannabinoids ~110 known compounds, 3 of note: o ∆9 tetrahydrocannabinol (THC): main Phytocannabinoid o Cannabidiol (CDB) o Cannabinol (CBN) THC is only psychoactive constituent Cannabinol and cannabidiol both have some non-psychoactive physiological effects, to varying degrees Hemp Variety of Cannabis Savita o 10,000-year of use due to its fast-growing plant with vast applications (refer to graphics for applications examples) Stained reputation by association with its more potent cousins Legality varies between countries Often regulated based on [THC] o Canada [THC] < 10 ppm (10mg/g) ▪ 0.3% under current regulations History of Cannabis Use (Not Testable; Context purposes) ~8000 BC (late Stone Age): Hemp fiber use ~2700-2000 BCE: psychotropic and therapeutic properties described in India and China o Relief of cramps, rheumatic and menstrual pain st 1 century CE: Taoists and cannabis seeds o Hallucinations as part of religious practice 16th century CE: Mention of medicinal effects start to appear in Europe W.B. O’Shaughnessy, mid 19th century o Irish scientists and physician o Observed cannabis use in India ▪ Analgesic, anti-convulsant, anti-emetic, hypnotic o Brough back to Ireland Medicinal use expanded, available over the counter o 1850 added to US Pharmacopoeia Sir J.R. Reynolds, Queen Victorias personal physician, 1890 History of Cannabis Use – Decline (Probably not testable) Early 20th century (~1910): Cannabis use declines o Variable potency among preparations (e.g. soil quality or conditions in which plant grows matters) o Poor storage ability (e.g. unknown constituents) o American concern about recreation use (use for psychoactive component rather than its benefits) 1937: Marijuana Tax Act criminalizes drug o Removed from Pharmacopoeia in 1942 History of Cannabis Use – Renaissance (Probably not testable) 1940’s: structure of cannabidiol isolated from Cannabis extracts 1964: THC structure determined 1985: Dronabinol (synthetic THC) approved for cancer patients 1990’s: Endocannabinoid system discovered Cannabinoid Pharmacodynamics First theories: general anesthetic-type mechanism (e.g. lipophilic drugs like THC do their work bc they’re integrating themselves into cell membranes and changing the fluidity by changing position of transmembrane elements and this causes disruption of general cell function) ▪ i.e. altered membrane fluidity to produce effects o based on knowledge of high lipid solubility Was there an endogenous receptor? o Yes – located in 1988 o They are called Cannabinoid Receptors Cannabinoid Receptor 1 (CB1) Transmembrane, G protein-coupled receptors o Generally inhibitory signal transduction (e.g. cannabinoids come in bind to the receptor and inhibit cellular function) ▪ Inhibitions of neurotransmitter release Broad localization throughout the central and peripheral localization o Hippocampus, basal ganglia, cerebellum, cerebrum o Muscle, liver, heart, blood vessels, GI tract, lung, pancreas Limited distribution in brainstem or medulla (important for safety profile of cannabinoids; key areas for survival in brain as they control heart, respiration → even if you go to high levels of cannabis the chances of death are low or even minimal) o Does not lethally affect HR or respiration Cannabinoid Receptor 2 (CB2) Also, G protein-coupled receptors Similar recognition of most cannabinoids, but different affinities when compared to CB1 Predominantly expressed in immune system o B cells, T lymphocytes, Leukocytes, some peripheral nerves Endocannabinoids 2 major endocannabinoids (derived from fatty acids) o 10-arachidonoylethanolmine (aka anandamide) o 2 arachidonoylglycerol Locally acting molecules synthesized on demand, and short acting in durations (autacoids) o Thought to help reduce pain through presynaptic modulation of neurotransmitter release o When they bind to a receptor on a nerve, they will inhibit other processes including synaptic transmission (like a negative feedback loop to turn down cellular signalling) Cannabis Pharmacology is Complicated by… Cannabis horticulture (resulted in increase in THC content in cannabis) o Phytocannabinoid levels in strains has changed over the years ▪ Weakens applicability of much earlier (already sparse) literature Phytocannabinoid stability (a lot of these compounds are not particularly stable bc they are not synthesized in plants at appreciable qualities) o Mostly synthesized in acidic forms, w/limited pharmacological activity ▪ THCA, CBDA, etc. o Must first be decarboxylated (converted) to more active forms ▪ Heat, light and other processes can trigger this conversion ▪ E.g. THC more potent that than acidified for of 9-tetra Multiple plant varieties o Variations in cannabinoid content (particularly THC:CBD ratio) ▪ Cannabis indica [CBD] > [THC] ▪ Cannabis sativa: [THC] > [CBD] o Growing conditions may vary Different preparations o Cultural and/or personal preferences o Solvent extraction methods o Raw vs cooked ▪ Some cannabinoids are not stable in heat Numerous cannabinoid compounds o Of Phytocannabinoids, THC most well known (and well-studied) ▪ Burgeoning research on CBN/CBD compounds as well as others Involvement of non-cannabinoid process o Other receptors involved? Best option: explore each cannabinoid separately o But not all are known, and effects may come from synergy between compounds THC Pharmacology Affinity for both CB1 and CB2 receptors (binds to these receptors, involves G protein, inhibition of cell processes) Effects on mood o Feelings of euphoria/relaxation/well-being o Decreases in anxiety/tension/alertness Effects on perception o Colours/music/emotions/time o Can lead up to hallucinations Cardiovascular o Tachycardia o Vasodilation, particularly in conjunctiva (due to exposure lvls, sensitivity and other processes) THC Pharmacokinetics Absorption o Variable by route of administration ▪ Intravenous/inhalation → tpeak ~ minutes ▪ Oral → tpeak of 1-2 hrs Distribution o Rapidly distributes to highly vascularized areas (e.g. brain) o Very lipid soluble → accumulates in fatty tissue (preferentially accumulates in areas with a lot of body fat) ▪ Important factor in limiting withdrawal symptoms  Metabolism (occurs in liver so THC hiding in fat will not get broken down) o Mainly hepatic o Plasma half life of 4 hours (half life for 2 compartments; hard for fatty compartment to remove THC, but the only reason THC starts to move is bc we start to break down drug in the blood and [gradient] wants to push the drug out of the fat – this process happens slowly) o Multiple metabolites, at least one of which is pharmacologically active o Tissue elimination of THC and metabolites → T1/2 – 7 days Excretion o Primarily excreted through biliary route/feces Cannabidiol (CBD) Pharmacology Can bind to CB1/CB2 receptors but has low affinity for them (most of CBD pharmacology comes from non cannabinoid receptors) Interactions with multiple receptors as both agonist and antagonist o can stimulate certain serotonin (5-HT) receptors which may confer some of its effects as an antidepressant and anxiolytic Some evidence suggesting modulation of THC effects (either through serotonin receptors or some other pathway THC and cannabidiol work to modulate one another’s functions – particularly cannabidiol helping to modulate THC function) o May counteract effects, especially in strains with more equivalent THC:CBD ratio Inhibits the breakdown of endogenous cannabinoids o Can help potentiate the anandamide effects or other effects in the body Cannabinol (CBN) Pharmacology Trace amts within Cannabis spp. Byproduct of THC metabolism (THC breaks down into CBN) o Increased concentrations in older/oxidized product Affinity for both CB1 and CB2 but lower than THC in each case o Likely similar to cannabidiol Likely contributes sedative and analgesic properties of cannabis Nonpsychoactive Cannabis Dangers Dysphoria in some or at higher doses o Anxiety/panic o Paranoia/psychosis Impaired coordination (effects on cerebellum) Lung cancer (attributed to taking cannabis through inhalation) o Similar non-cannabinoid constituents to those found in tobacco (1) Dose Cannabis Affect Learning and/or Memory? Challenges in diagnosis o Reporting bias associated with illicit nature of drug use (e.g. time in which data was taken – in the past cannabis was illegal) o Retrospective studies, already prone to recall bias, and reliance on individuals (2) who – if hypothesis is true – will have memory defects Three areas of interest in answering this question o (1) Under the influence (acute/short-term exposure) o (2) Amidst periods of long-term chronic use (3) ▪ Not necessarily under the influence at the time of interest o (3) Following cessation of use ▪ i.e. are there permanent modifications in ability? Does Cannabis Affect Educational Advancement? Evidence is fairly consistent in demonstrating a link btwn cannabis use and school drop out or poor educational performance However, the nature of this relationship is hard to determine o Does cannabis diminish motivation and/or performance? o Does poor motivation and/or performance lead to cannabis use? o Is there a 3rd factor that affects motivation, performance and cannabis consumption? ▪ E.g. other drugs? Childhood experience? Baseline school performance or cognitive ability? Parental educational levels? Socioeconomic status? Identical twin studies? o No difference in school drops out or performance among those discordant for use Cannabis Dependence/Withdrawal Sudden cessation of CB receptor stimulation theoretically unlikely, due to lipophilic nature of THC and slow- release mechanism from fat stores in the body However, abt 50% of daily users will experience withdrawal after daily use o Sleep problems, anger/irritability, dysphoria, nausea Generally, peaks 2-6 days after cessation and lasts up to 2 weeks Is Cannabis Addictive? Animal and clinical models show an increase in dopamine release into nucleus accumbens with chronic cannabis use (more specifically, THC in some studies) → increased reinforcement in humans o Not all animals will self administer cannabis intravenously (limited reinforcement for skinner type experiment) ▪ Species specific differences in reinforcement o More consistent when intracranial administration tested In humans with lifetime exposure to cannabis approximately 9% develop substance use disorder In many of these cases there are other concomitant risk factors o Concurrent tobacco use, onset of use in early adolescence, sex (males > females) From twin studies, abt 55% of risk is attributable to genetics Therapeutic Cannabis Use Multiple possibilities, keeping in mind the “costs” of possible psychoactive side effects (among others) Most present research focus is on potential of non-psychoactive Phytocannabinoids o Emesis, appetite stimulation, pain relief, epilepsy Synthetic Cannabinoids Selective agonists (selective over CB1 and CB2) of cannabinoid receptors Mixed agonists/antagonists (can be agonist of CB1 and antagonist of CB2) of cannabinoid receptors Mostly for research purposes; very few available for clinical use o Dronabinol; synthetic THC o Nabilone: THC analogue, selective for CB1 o Rimonabant: CB1 antagonist o Sativex: 1:1 THC:CBD extract ▪ Cancer/MS patients’ resistance to high dose opioids Emesis Dronabinol (THC analogue): prophylaxis and treatment (oral administration) Particularly effective against CINV o Chemotherapy-induced nausea and vomiting (can come from stomach, brain stimulation…) Complex mechanism of action o THC: peripherally (intestine) and centrally via CB1 o CBD: independent of CB receptors (animal models only) Appetite Stimulation Counteract loss of appetite associated with certain medical treatments/conditions o Cancer and/or treatment o AIDS Significant increase in feeding relative to placebo Most effective at lower doses – why might that be? o At higher doses you gain other effects that are not associated with appetite Can this be exploited in the opposite situation? o Rimonabant (CB1 antagonist) effective in obesity treatment o Briefly approved in Europe (2006-2008) but withdrawn from market due to psychiatric side effects Pain Relief Affects processing of pain, rather than presence of pain? Some systematic evidence for certain types of pain o MS related o Cancer pain o Chronic pain Conflicting evidence for other types of pain o Neuropathic pain o Acute pain (evidence doesn’t suggest that its particularly effective) Strong placebo effect contribution? o Like other analgesics it doesn’t really change the awareness of the pain, but it changes how unpleasant it feels to the patient; aware that the pain is there, but its not bothering them) Epilepsy 30-40% of individuals with epilepsy do not respond to traditional treatments o excess cerebral discharge which is often blocked by (Na+) ion channel blockers, GABAA agonists, etc. designed to reduce neuron excitability Epidiolex (also available in Canada) o CBD extract (99%) with trace amounts (0.1%) of THC o Approved by FDA (2018) for treatment of 2 forms of childhood epilepsy ▪ Lennox-Gastaut syndrome ▪ Dravet syndrome May not work primarily through CB receptors (bc CBD has low affinity for CB receptors); some evidence of broad scale targeting of multiple “traditional” epilepsy targets (as listed above) Contraindications Individuals with (or prone to) severe personality disorders Children/adolescents (under 18) o Pregnant/breastfeeding women Individuals suffering from addiction disorders o Increased risk of substance use disorder (high heritability component) Elderly should be cautioned due to heightened risk of CNS/cardiovascular side effects Marijuana and Legality (Canada) Initially banned in 1923, was “re-legalized” for medical purposes in 2001 o Marihuana for Medical Purposes Regulation (MMPR) Replaced by access to Cannabis for Medical Purposes Regulation (ACMPR) in 2016 o Permits purchase and/or cultivation for personal use ▪ Vague on specific conditions based on physician judgement Bill C-45 (Cannabis Act) o Took effort October 17, 2018 o Decriminalizes personal possession and use (if age of majority) and regulates sale Canada-wide  Day 10 (Cardiovascular Pharmacology) The Cardiovascular System Both left and right heart has an atrium (which receives blood) and a ventricle (pumps out blood) The left ventricle pumps blood to the systemic (peripheral) circulation (consists of arteries, capillaries, and veins) The right ventricle pumps blood to the pulmonary circulation (pulmonary arteries, capillaries and veins) Blood flows from a high to lower pressure Electrical Activity of the Heart The heart beats spontaneously (≈70-100 beats/min) due to spontaneous depolarization of cardiac cells at the SA node (sinoatrial node) Electrical signals (impulses) generated at the SA node pass through the atria to the AV node (atrioventricular node) which slows conduction velocity somewhat The impulses cont. down specialized conduction pathway (His and Purkinje fibers) which rapidly conduct signals to the entire right and left ventricle which pump blood to the pulmonary and systemic circulation, respectively The autonomic nervous system controls heart rate by regulating the depolarization rate of the SA node and conduction velocity of the AV node The Circulatory System (1) The left ventricle (LV) pumps oxygenated blood to the aorta, other arteries, then arterioles and capillaries. Arterioles, with small lumen, offer resistance to blood flow (2) This gives a high blood pressure (BP) in arteries and low pressure in capillaries. Constriction of arterioles increases BP (3) In capillaries, O2/nutrient flow from blood to tissues; CO2/waste products flow from tissue to the blood (4) Deoxygenated blood flows from veins to the right heart then to lungs for oxygenation (5) Oxygenated blood flows via the pulmonary circulation to the left atrium then LV Blood Pressure Blood Pressure (BP): pressure exerted by blood on the wall of blood vessels Systolic BP (SBP): BP during cardiac contraction Diastolic BP (DBP): BP during cardiac relaxation Pulse pressure: difference between SBP and DBP BP (mmHg) = cardiac output (L/min) x total peripheral resistance o Mean Blood pressure (BP; mmHg) = DBP + 1/3(SBP–DBP) ▪ Mean BP is closer to the DBP because diastole lasts longer than systole o Cardiac output (CO; L/min) o Total peripheral resistance (BP/CO; mmHg.min/L) Cardiac Contraction The heart contracts at 70-90 beats/min In systole, contraction of the left ventricle (LV) increases LV pressure and systolic blood pressure (SBP) to ≈125 mmHg In diastole, LV pressure falls to ≈0 mmHg Systolic blood pressure (SBP) in arteries rises to ≈130 mmHg Diastolic blood pressure (DBP) in arteries falls to only ≈75 mmHg because the aortic valve closes (by backward blood flow). This prevents further back flow of blood to the LV  Arterial flow is continuous due to arterial wall distension during systolic, and recoiling during diastole. Blood Pressure (or arterial pressure) Factors that Affect BP Cardiac output (CO): volume of blood pumped out per minute. CO = stroke volume (SV) x HR Total Peripheral resistance (TPR): lumen diameter of arterioles Compliance of arteries. Normal arteries are distended by the systemic BP (SBP) during systole. If arteries are rigid (not compliant), SBP becomes very high Effect of Large Artery Stiffness on BP Elastic arteries allow arterial distension during systole. This reduces systolic BP. During diastole, arteries recoil to allow continuous flow Stiff arteries reduce arterial distension during systole thereby increases systolic blood flow/BP and decreases arterial recoil during diastole. This reduces diastolic blood flow, diastolic BP, capillary transit time and capillary exchange BP Reading in Adults Normal systolic BP (SBP): between 100-140 mmHg BP Normal diastolic blood pressure (DBP): between 60-90 mmHg Hypertension: o 1. ↑SBP (>140 mmHg) o 2. ↑DBP (>90 mmHg) o 3. ↑SBP and ↑DBP Hypertension: General Info About 90% of N. Americans have hypertension, but only ≈60% of hypertensive patients are treated. Only half of treated hypertensive patients have effective control of blood pressure (90 mmHg o 1. ↑SBP only (↑stiffness of large arteries) – Isolated systolic hypertension (wide pulse pressure) o 2. ↑DBP only (narrowing of arterioles) – Isolated diastolic hypertension o 3. ↑SBP/↑DBP – Systolic-diastolic hypertension Essential hypertension (>80% of hypertension): unknown cause  Secondary hypertension: due to a medical condition – e.g., renovascular hypertension (narrowing of kidney arteries), aldosteronism (↑↑ aldosterone release → Na+ retention and ↑ blood volume), pheochromocytoma (↑↑ epinephrine/noradrenaline release due to tumor in adrenal medulla), gestational hypertension Isolated systolic hypertension (ISH) – ↑SBP only (large artery stiffness; wide pulse pressure). Isolated diastolic hypertension (IDH) – ↑DBP only (narrow arterioles; ↑TPR) Systolic-diastolic hypertension – ↑SBP/↑DBP (combination of 1 and 2) Common Causes of Arterial Stiffness in the Elderly Hypertrophy (thickening) of arterial smooth muscle layer (due to high BP) Disruption of endothelial cells layer (e.g., with diabetes, smoking). This causes build up of WBCs, Ca2+ , fats and smooth muscle cells in arterial wall (i.e., atherosclerosis) Atherosclerosis causes structural change of arterial wall, e.g., Ca++ accumulation and increased collagen content (which provide stiffness to arteries) Atherosclerosis in the Coronary Artery Atherosclerosis is usually due to damage of vascular endothelial cells, resulting in deposit of plaque (due to penetration of white blood cells, fats, smooth muscle cells and Ca++) between the endothelial cell and smooth muscle cell layers of an artery. Non-Pharmacological Management of Hypertension Weight loss for overweight individuals, Physical activity, Low sodium diet, No smoking, Reduce alcohol intake, Reduce glucose intake (for diabetic patients), Increased intake of fruit, vegetables, fibers, and low fat food, Stress management Antihypertensive agents: drugs for reducing BP in Hypertension Diuretics (thiazides): ↑renal Na+ excretion (↓BV→ ↓CO), dilate arterioles (↓TPR) ANG converting enzyme inhibitor (captopril): ↓ANG II (vasoconstrictor, ↓TPR) and ↓ALD (↓BV→ ↓CO) ANG receptor antagonist (losartan): block ANG II receptor (ANG II antagonist) and ↓ALD β-Adrenoceptor antagonist (propranolol, metoprolol; β-blocker) → ↓CO and ↓TPR via blockade of βadrenoceptors in the heart and decrease of activities of sympathetic nerve system and renin-ANG-ALD system α-Adrenoceptor antagonist (prazosin, α-blocker); ↓vasoconstriction (↓TPR) The Kidney Each functional unit of the kidney (nephron) is made of an afferent arteriole (controls rate of inflow), capillaries (glomerular filtration) and an efferent arteriole (controls capillary pressure to enable filtration of ≈20% of plasma to the renal tubules (via a semi-permeable membrane) Na+ (>99% ) in the renal tubule is reabsorbed by another set of capillaries from which it is delivered to the renal vein. Water is reabsorbed with Na+ (Note: As Na+ is the main cation in plasma, it determines blood volume; plasma Na is ≈140 mM) Unabsorbed plasma (glomerular filtrate) and secreted products (wastes) are excreted as urine Thiazide Diuretics A diuretic is a drug that increases urinary excretion of Na+ and water. Thiazide diuretics are commonly used for management of hypertension Diuretics (e.g., Chlorothiazide) decrease BP by: (1) ↓Renal Na+ and water reabsorption → ↑Urine flow → ↓BV (plasma Na is ≈140 mM) → ↓CO (2) Dilation of arterioles (↓TPR) Use: To treat hypertension (↓TPR/CO) and edema (↓BV → ↓ capillary filtration of plasma into tissues) Side effects: ↑HR (reflex tachycardia), ↑urination, electrolyte disturbances (imbalance of ions) Renin-Angiotensin-Aldosterone System Sympathetic Nervous System (SNS) The autonomic nervous system regulates functions of internal organs, and is subdivided into: o 1) sympathetic nervous system (SNS) o 2) parasympathetic nervous system. The SNS mediates fight or flight responses through release of epinephrine (E) and norepinephrine (NE) E and NE cause flight or flight reactions by activating α1-, β1 and β2-adrenoceptors: o 1. Via activation of α 1–adrenoceptors, NE and E constrict arterioles (↑total peripheral resistance) and veins (↑venous return and cardiac output). These actions increase bp o 2. Via activation of β1-adrenoceptors, NE and E directly increase heart rate/contractility as well as increase activities of the sympathetic nervous system and renin-angiotensin system o 3. Via activation of β2-adrenoceptors, E causes bronchodilation Beta-Adrenoceptor Antagonists 1. β1-adrenoceptors mediate epinephrine-induced increase in cardiac rate/contractility, and increase in activities of the sympathetic nervous and renin-angiotensin-aldosterone systems 2. β2-adrenoceptors mediate epinephrine-induced bronchodilation Subtypes of β-adrenoceptor antagonist o 1. Non-selective β-blocker: Propranolol (blocks β1-/β2-adrenoceptors) o 2. Selective β1-blocker: Metoprolol  MOA: via β1-adrenoceptor antagonist (β1-blocker) o (1) ↓ HR and cardiac contractility directly. o (2) ↓ Sympathetic nerve activity (release of norepinephrine and epinephrine) o (3) ↓ Renin-angiotensin-aldosterone system activity Side effects: myocardial depression, ↓exercise tolerance, bronchospasm for propranolol (due to blockade of β2- adrenoceptors). Metoprolol causes less bronchospasm (due to its relative selectivity for β1- adrenoceptors) Use: Treatment of hypertension and prophylaxis of angina (prevent angina attack) → Propanol Angina Coronary arteries supply blood/O2 to the heart. When a coronary artery is blocked by atherosclerosis (plaque buildup), part of the heart muscle is ischemic (↓blood flow) and hypoxic (↓O2). The person may experience angina (pain) When O2 deprivation is excessive, the affected muscle dies, and myocardial infarction (MI, heart attack), heart failure (inadequate contraction) and/or cardiac arrhythmias (irregular heart rate) may occur Beta-blockers (metoprolol and propranolol) directly reduce cardiac function, and decrease activities of the sympathetic nervous and renin-angiotensin-aldosterone systems thereby reducing blood pressure, cardiac contractile function/O2 requirement α-Adrenoceptor antagonists (α-blocker; prazosin) Prazosin is an α-adrenoceptor antagonist. It prevents norepinephrine (NE) from binding to (and activating) α1-adrenoceptors Use: Treatment of hypertension Side effects: reflex tachycardia (↑HR); orthostatic hypotension and syncope. Day 11 (Antimicrobial Drugs – P1) Terminology Antibacterials (e.g. bleach, H2O2, HCl) o Antibiotics: old definition means anything against life; ability to kill any # of organisms (generally bacteria but can also be parasites, fungus..) → new definition now encompasses mostly antibacterial (antibiotics are subclass in antibacterials) o Difference btwn antibiotics and antibacterial: antibacterial kill the bacteria and antibiotic is derived from a living organism (micro-organism) and it can either kill or suppress bacterial growth Antimicrobials o Antivirals o Antiparasitics o Antifungals Antibiotics History Truly revolutionized medicine o Routine scratched and injuries no longer mean amputation or death o Increased survival prognosis post surgery 1st antibiotic discovered? o Penicillin, 1928 o ~14 years from discovery to treatment st 1 antibiotic clinically used? o Prontosil, 1932 o Clinical trials to treatment in >4years! Bacterial Diseases Infections caused by bacteria o Same species of bacteria can colonize a general area in the body Deviations from normal flora (skin, gut, etc.) Can range from minor skin infections to life-threatening conditions o Meningitis, flesh eating disease, bubonic plague Recommended course of action → attack the source o w/o harming our own cells (ideally bc naturally occurring flora are helping us sustain certain processes, so we want to avoid damaging those) Mammalian vs Bacterial Cells Differences? o Specific agents of function ▪ Genes mediating cellular processes DNA replication enzymes Other proteins/enzymes o Macromolecule anabolism ▪ Nucleotide production ▪ Protein production Ribosome subunits o Cell wall Cell Envelope (May or may not contain a cell wall) Mammalian cells have single phospholipid bilayer that circles the cell, keeps all the organelles and constituents within; presents a barrier to drug entry (pharmacologically speaking) In bacterial cells what we have in mammalian cells in expanded into the envelope. There are 2 main structures that subdivide bacteria into 2 types: gram (+) and gram (-) Encompasses the cytoplasmic membrane and structures external o Basis for segregation into gram (+) and gram (-) bacterial strains ▪ Gram (+): known for having large cell wall ▪ Gram (-): have 2nd phospholipid bilayer which allows for hold degradative enzymes for breaking down certain types of antibiotics and prevent them from entering the cell Additional structures on the outside of the cell, present barriers to the entry of antibiotics/drugs and also present mechanisms for selectivity in treatment o Theres going to be differences in drugs in whether they are more effective for gram positive or gram-negative strains (related to MOA, or ability of antibiotic to enter the cell) MOA: Selective Toxicity We are aiming for selective toxicity; we want to target bacterial cells; in a perfect world we would only target harmful bacteria cells, but that is not always possible (if we know the particular bacteria we can sometimes choose the appropriate antibiotic that targets the bacteria) To do this we can exploit differences btwn mammalian (human) and microbiotic biochemistry, anatomy and/or physiology, causing greater harm to the micro-organism Antimicrobials are among the most selective of all pharmacological agents Spectrum of Activity Antimicrobials vary w/respect to their effectiveness against the wide range of micro-organisms within a kingdom (possibilities for whether drugs can treat some or less or all of the known problematic bacteria) o Narrow-spectrum antimicrobial ▪ Drug that will kill (or inhibit) a limited range of micro-organism species  o Broad spectrum antimicrobial ▪ Drug that will kill (or inhibit) a wide range of micro-organism species This concept applies to smaller drug classes as well (e.g. antibiotics) MOA: Inhibition of Cell Wall Synthesis Cell wall is apart of cell envelope; more prevalent in gram (+) bacteria and it forms a small element of the gram (-) bacterial envelope Drugs that inhibit cell wall synthesis interfere with final cross-linking assembly (stabilization/integrity) step o Penicillin binding proteins catalyzes this final step (causes bacteria cell to be unstable) ▪ Inhibited by class Disrupts dynamic EQ of cell wall growth o Renders cell vulnerable to fatal osmotic pressures Beta – lactams (Penicillin found in this class) Major family of antibiotics acting through this mechanism o Named after ubiquitous beta-lactam ring present in each compound Penicillins Cephalosporins Penicillins Naturally derived o Fermentations of penicillin chrysogenum fungus Family members vary in antimicrobial spectrum, gastric stability, lactamase susceptibility Primarily effect against gram (+) o Some gram (-) strains may be susceptible o Not all gram (+) are susceptible ▪ Some strains have natural resistance (ability to demolish penicillin before it can perform damage to the cell) Penicillin is degraded by B-lactamase enzymes Penicillin: Pharmacokinetics Absorption o Oral bioavailability (not all penicillin should be taken orally) ▪ Some have modifications to survive gastric pH (some models are not stable in gastric acid so later models have better ability to survive in gastric pH) Distribution o Generally, antibiotics have good distribution (but key diff btwn antibiotics is whether they can cross blood brain barrier or not) o Penicillin has poor CNS permeation. Unless inflammation has comprised blood-brain barrier Metabolism/Excretion o Majority is excreted unmetabolized in urine o Half life for elimination from the body – 30 minutes to 1 hour, depending on compound Penicillin: Adverse Effects Amongst the safest antibiotic, if not drugs, in use today Hypersensitivity (allergies) o ~5% of population experiences a reaction to penicilloic acid (breakdown product of penicillin) o Rash, edema, anaphylaxis Cation toxicity o Penicillin is given (alongside) sodium/potassium salt (since we are given high amts of penicillin you are also giving high amts of that Na+/K+ with it and that can disrupt the electrical balances in your body to a point of cation toxicity at high doses)  o Large doses may upset concentrations of these crucial ions ▪ Hypernatremia, hyperkalemia (extreme thirst, muscle fatigue, confusion, extreme thirst…) Cephalosporins Structural (still contains beta lactam ring), functional relatives of penicillin o Same MOA Classified by generations o Main differences are (general) improvements in ▪ Effectiveness vs. Gram (-) strains (better at crossing phospholipid bilayer) Typically, at the expense of gram (+) coverage (if you know you’re attacking a gram positive then earlier generations of cephalosporins are better vice versa) ▪ Ability to cross the blood brain barrier ▪ Resistance to B lactamases (more stable drugs in later generations) MOA: Inhibition of Protein Production Interfere with bacterial ribosome activity, by exploiting differences in bacterial ribosomes o Interact with either of the 50s, 30s subunits – not present in mammalian cells ▪ Ribonucleo proteins are structurally different in the mammalian cells compared to the bacterial cell; defined by subunit which is loosely correlated to size Representative members of this family: o Tetracyclines o Aminoglycosides Tetracyclines Block tRNA access (tetracyclines bind to the ribosome and prevent the tRNA from binding to it) Prevents mRNA translation and, as a result, peptide elongation Broad-spectrum antibiotics o Bind and interact with many gram (+) and gram (-) strains (not all ofc, but any that allows the drug to enter the cell – due to the fact that the drug is sterically encumbered with R groups) ▪ Passive diffusion and active transport pathways (transport methods into the cell) Tetracyclines: Pharmacokinetics Absorption o Can be prevented by divalent cation chelation (negative charge on O is capable of binding and chelating other ions that might be in the same environment, e.g. Mg, Ca; prevents tetracycline from being absorbed and hence no pharmacological effect takes place) ▪ Avoid certain foods (e.g. dairy) and medications immediately before/after Distribution o Some CNS distribution – up to 10-25% of plasma concentrations (inefficient permeation) o Binds to tissues undergoing calcification (due to tetracyclines affinity for ions) like bone and teeth ▪ Not so much an issue for a fully developed/grown adult but it can limit where the drug is; can cause issues with younger people taking tetracyclines o Crosses placenta Metabolism o short -, medium- and long acting derivatives (variety based on considerations) ▪ 6-36 hour half lives Tetracyclines; Adverse Effects Gastric discomfort o Significant factor in non-compliance, reduced efficacy if self medicate symptoms (may unintentionally produce a drug interaction btwn tetracycline) ▪ Alkaline pH and/or divalent cations affect absorption Effects on calcified tissue o Deposits in bone/teeth o Avoid use in pregnancy, children under 8 (avoid use in population where bones are still growing bc tetracycline can interact with Ca ions and cause changes in bone growth ) ▪ Permanent discoloration of teeth ▪ Temporary growth stunting Aminoglycosides Family members include streptomycin, neomycin, gentamicin Similar to tetracyclines (bind to smaller subunit it ribosome and prevent protein elongation) Most effective against aerobic, Gram (-) bacteria (due to large structure of these drugs they are more effective against oxygen requiring gram negative bacteria; they have trouble getting through getting through holes in the peptidoglycan layer so if u have a gram-positive bacteria with multiple layers stacked on one another it prevents a physical barrier to the drug) BACTERIA start Mammalian start here o Oxygen dependent transport pumps required to cross inner membrane (due to the fact that the here drug is fairly polar) o Trouble with thick peptidoglycan layer in gram (+) bacteria Side effects (necessitate limiting long term use): o Ototoxicity (accumulation in ear destroying hair cells → hearing loss, vestibular tissues) o Nephrotoxicity/Kidney damage(reversible – short term usage → irreversible – long term usage) MOA: Inhibition of DNA replication Fluroquinolones o Norfloxacin, ciprofloxacin, levofloxacin, etc. Fluoroquinolones Enter bacteria passively through membrane porins (water filled channels) o If they can gain access to nucleus of bacterial cells they bind and interact with 2 different enzymes that are used in bacterial cells for DNA replication) Inhibit bacterial replication by inhibiting 2 crucial enzymes o DNA gyrase (helps to uncoil the compressed DNA, to perform reading and replication) o Topoisomerase IV (facilitate separation of chromosomes) 4 generations o Earlier generations tend to not distribute effectively but they would collect in the urinary tract/bladder bc of how they were getting ready for elimination; often very well used for urinary tract infections o Generally, progression towards agents with… ▪ Greater systemic distribution ▪ Increased effectiveness against anerobic and gram (+) bacteria (less gram negative) Fluoroquinolones: Adverse Effects Generally, very well tolerated Occasionally get GI effects (nausea, diarrhea) Some evidence of arthropathy (joint disorder) o (reversible) erosion of articular cartilage  o Avoid in pregnant/nursing women, children under 18 MOA: alter nucleotide synthesis (closely related to DNA production bc nucleotides are building blocks to DNA) Folate derived cofactors essential for nucleotide production Mammalian cells cannot synthesize folate (aka folic acid) o Required for dietary needs Bacterial cells must synthesize folate de novo, as they lack the ability to transport it into the cell Sulfonamides and Trimethoprim Interfere with synthesis of tetrahydrofolic acid from PABA (sulfanilamide looks very structurally similar to PABA which is precursor to all folate synthesis; when cell produces nucleotides its grabbing whatever looks like PABA to make them; fraction of the time sulfanilamide will be grabbed, and system generates a protein that cannot be used → substrate lock) o Trimethoprim inhibits enzymes Can inhibit certain strains of both gram (+) and gram (-) bacteria Can cross blood brain barrier effectively Generally administered together (as cotrimoxazole) to produce synergistic/complete inhibition of pathway Adverse Effects Sulfonamides (earlier compounds when metabolized can wind up slightly crystallized (e.g. blood, urine) can wind up w/obstruction like kidney stone) o Acetylated metabolite can precipitate in neutral or acid pH ▪ Crystalluria, hematouria, or obstruction ▪ Less prevalent in newer agents ▪ Alkaline urine (increase solubility) and/or hydrate (increase urine flow/excretion) Trimethoprim o Can exacerbate folic acid deficiency (capitalizing on 1 that is already low) ▪ Arises from a small affinity for mammalian dihydrofolate reductase ▪ Limited effect in normal situations Clinical Applications How to select antibiotic How to prescribe (“dose”) an antibiotic How to Select an Antibiotic Many antibiotics are only effective against certain o Classes (e.g. Gram +/-) o Strains (e.g. Streptococcus, Rickettsiae) Therefore, the 1st order of business should be to try and identify the organism (and in doing so, ensure it is bacterial in nature) o Gram stain, culture Culturing allows for a trail run, to gather information on such things as (cultures take time to grow) o Susceptibility ▪ confirms that the bacteria (strain) will be effectively treated by the drug or in other words its not resistant to your intended treatment o Min inhibitory concentration (MIC) – the smallest amt of drug that will actually treat the bacteria, either stall their growth or kill them Situation Critical (Cannot wait to postulate and experimentally determine identity of bacteria) Make an educated guess as to the strain or likely suspects  Based on o Site of infection ▪ Known bacteria in area (diff bacteria tend to the condition of diff parts of the body whether that is the pH levels, humidity or diff [ ] of ions, sugars, salts) ▪ Blood brain barrier, or not? (depending on whether the bacteria is in the CNS) o Patient history ▪ Previous infections ▪ Travel, recent history/events o Known organisms in area Consider broad spectrum antibiotics to buy time o If all else fails Patient Factors Renal/hepatic dysfunction o Consider metabolism/excretion burdens (e.g. if someone is unable to metabolize efficiently then we might consider using penicillin as an options Age o State of renal/hepatic systems o Contraindications in children? (e.g. tetracyclines, also relates to those that may be lactating) Pregnancy/lactation o Teratogenicity o Transfer to child Cost o Coverage, economic situation o If using multiple agents Safety of agent o Allergies o Side effects Antibiotic Dose Scheduling If you give an antibiotic orally, we have a general time-concentration relationship where there is absorption phase and there is a delay to peak concentration, then there a dissipation of drug over time as it distributes throughout the body and eliminated via metabolism. When faced with a concentration time profile we are interested in the peak concentration and how long it exists above that minimum level required for an effect Concentration Dependent Killing (Drugs) Efficacy link to Cmax relative to MIC (drug is more effective in higher doses with a short period of time) o Typically due to prolonged post antibiotic effect (severe long term consequences of recovery ▪ Significant disruption in cell function? ▪ Slow time course of cell rebound? o Time above MIC less relevant Once/day therapy preferred Examples o Aminoglycosides (secondary effects cause significant dmg to cell membrane and it takes time for the cell to recover from that dmg even though the drug may be long gone) o Rifampin ▪ ~10mins to enter cell, bind to RNA polymerase Prolonged effect Time Dependent Killing Efficacy relies on plasma concentrations being greater than MIC for prolonged periods (persistent effect – not as concerned with concentration achieved as long as above MIC for as long as possible) o t > MIC o Cmax not important, so long as greater than MIC Dosing should be more frequently to increase t > MIC o Multiple times/day o Continuous infusion Examples o Beta lactams (if given once they would just cause some issues with peptidoglycan being produced at that moment in time, but the rest of the peptidoglycan is fine; if you continuously infuse then every single peptidoglycan layer produced will have an issue of not being linked to its neighbour and then you have the potential for cell lysis to take their toll) Take Home Points Antibiotics try to exploit inherent differences in bacterial and mammalian cells o Broadly speaking, these fall into a few different mechanisms ▪ Plus, others not covered today Bacterial strain dictates choice of antibiotic more than anything else, but patient factors need also be considered o Gram (+) vs (-), resistance? o Age, physiology, etc. Choice of antibiotic dictates treatment strategy o Concentration of time dependent effectiveness Day 12 (Antimicrobial Drugs – P2) Antifungals Mammalian vs Fungal Cells Both are eukaryote but there are a few major differences in cell composition o Fungal cell membrane contains ergosterol, rather than cholesterol o Fungal cell wall composed of chitin and beta-glucan Antifungals: MOA Disruption of (integrity) cell membrane through various mechanisms o Interference with ergosterol and/or ergosterol synthesis o Destabilization of the cell membrane Both mechanisms are fungicidal 2 major classifications o Polyenes o Azoles Polyenes (somewhat hydrophilic but mainly lipophilic chemicals) Integrates into fungal cell membrane, forming a complex with ergosterol that produces pores within (salts n ions can move through) o Net results is leakage of intracellular components and cell death Representative drug: nystatin o Used topically, due to systemic toxicity (bc of affinity for mammalian cells, but has lower bioavailability when given orally bc very little of it actually gets into the body – if given intravenously then you would see side effects since its in systemic circulation) Main clinical uses  o Treatment of candida infections ▪ Mouth (“thrush”), esophagus skin, vagina as common sites Azoles Most widely used antifungals 2 major subclasses o Imidazole’s (e.g. ketoconazole, miconazole, clotrimazole) ▪ Lower selectivity for fungal (vs. mammalian) enzymes ▪ Higher selectivity for drug interactions and side effects o Triazoles (e.g. fluconazole, itraconazole) Inhibit 14-alpha-demthylase (a CYP 450 enzyme) → inhibiting ergosterol o Reduced ergosterol synthesis Main clinical uses: o Candida infections (mouth, intestine, vagina) o Fungal infections in the skin, nails ▪ E.g. tinea pedis (“Athletes foot”) tinea corporis (“ringworm”) o Systemic fungal infections (e.g. cryptococcosis) Antimicrobial Resistance HA(I) vs. CA(I) HA: healthcare-associated (infection) o Aka hospital-acquired or nosocomial infections o Related to delivery of healthcare o Often acquired from a facility secondary to treatment of some other primary condition o Due to high prevalence of bacterial strains that have experienced previous/alternate forms of treatment, multidrug resistance more common CA: community-acquired (infection) o Unrelated to a hospital or healthcare delivery o Multidrug resistant strains (that were previously only HA) can and do spread to community over time ▪ Rightward shift in bacterial resistance curve MRSA Methicillin-resistant Staphylococcus aureus (resistant to penicillin; cannot be treated by any beta lactams) o Beta-lactam (penicillin & cephalosporin) resistance Not necessarily any more virulent (better at causing disease) than methicillin sensitive S. aureus (MSSA), but far more difficult to treat → more dangerous Major concern in hospitals o Open wounds o Invasive devices o Immunocompromised Why should We Care? Increased rates of treatment failure Poor patient outcomes o Increased mortality Increased need for combination therapy o Leads to further resistance (bacteria gains resistance to both drugs) ▪ Through division and propagation, we get increase multidrug resistant (MDR) strains aka “super bugs” o Increased cost of treatment Resistance is occurring at a much greater rate than new antibiotics are being developed Antibiotic Resistance Antibiotic Resistance: means that bacteria is completely immune to antibiotic, but it means that we cannot treat that bacterial infection w/o going to such a high dose of drug that we would risk harming the host Bacterial growth is not inhibited by a drug, at the max tolerable dose for a given host 2 major mechanisms of development of resistance o Evolution (changes in genome) ▪ Natural ▪ Acquired o Clinical/environmental practices Note that specific mechanisms of antibacterial resistance are not specific to any one of these mechanisms of development Mechanisms of Resistance From a strategic standpoint (i.e. basic principles – no specifics), how might a bacteria go about gaining resistance to an antibiotic? o Alter antibiotic affinity for substrate o Express inactivation enzyme o Express efflux pumps o Alter cellular permeability o Not an exclusive list Altered Substrate Affinity Binding site for antibiotic altered, changing drug affinity (bacteria gain resistance naturally or through acquisition by altering affinity for specific substrates → if you have a ribosomal subunit (bacteriaand a aminoglycoside that typically binds to the ribosome and prevents protein production, small changes in the structure of that ribosome might be sufficient to significantly decrease aminoglycoside affinity w/o adversely affecting the production of protein) o Fluoroquinolone resistance through altered affinity for enzymes o Sulfonamide resistance through decreased enzyme activity ▪ Alternatively, some strains also overproduce PABA, conferring some resistance to class o Aminoglycoside resistance due to altered ribosomal proteins Alternate target in particular bacterial strain (bacteria with natural resistance; e.g. bacteria having a DNA gyrase enzyme that others don’t use so even if fluoroquinolone enters the cell and binds there wont be any effect) Ability to Inactivate the Drug Expression of an inactivation enzyme o Beta-lactams ▪ Beta-lactamases o Aminoglycosides ▪ Multiple enzymes (acetyl, adenyl- and phosphotransferases) o Tetracycline ▪ Similar pathway though some other enzymes Expression of Efflux Pumps Concerning kicking out the drug once it has gotten into the cell Extrude antibiotic as it enters (resistance through prevention of drug accumulation inside the cell) o Maintain low intracellular concentrations Tetracyclines and Tet efflux pumps Altered Cell Permeability Prevent drug access to interior of cell  Low membrane permeability due to lack of expression of porin proteins Almost all classes where membrane diffusion alone is insufficient o E. coli resistance to beta-lactams ▪ Loss of OmpC/OmpF porins (or diminished function) prevents intracellular accumulation Mutations in which protein does not exist → porin loss No drug accumulates in structure of porin Decrease regulation Natural Resistance Intrinsic/Inherent Resistance MOA does not impact bacterial livelihood o E.g. we are seeking to destroy the cell wall but in gram (-) bacteria that cell wall isn’t as important to the livelihood of bacteria so we have a bit of natural resistance o E.g. anerobic bacteria that have no reason to use oxygen as a way of powering their ion transport across cell membranes have a natural resistance to aminoglycoside bc there is no oxygen dependence Acquired Resistance Spontaneous mutation in genome (sometimes mutations can be bad too) DNA/plasmid transfer of gene expressing relevant protein – relating to resistance o Beta-lactamase o Efflux pump o Less sensitive enzyme Rational Antibiotic Use Intelligent therapeutics/adherence Limit use in non-medical situations Intelligent Therapeutics/Adherence Only use if infection is bacterial Do not underdose o “weakest” bacteria are the ones that succumb at lower concentrations o May facilitate selective breeding by using insufficient drug ▪ Surviving bacteria have access to resources for division and space to do so o Drugs may need time or higher concentration to neutralize “stronger” (partially resistant) bacteria ▪ Regular/prolonged exposure to access cell, lower affinity for substrates, etc. Finish course of treatment o Conventional wisdom, based on similar logic to underdosing with antibiotics ▪ Drug may need time to neutralize “stronger” (partially resistant) bacteria Regular/prolonged exposure to access cell, lower affinity for substrates, etc. ▪ Some studies show no evidence of increased resistance with shorter treatment durations The opposite may be true (increased chance of resistance) o Combined with arguments that prescription durations are often “standard” on convenience, some professionals suggest stopping treatment when symptoms improve (or at minimum, consulting prescriber) Try to treat infection with a single agent where possible o Limit exposure to newer antibiotics o However complete eradication better than partial and some antibiotics synergize so well to be better than sum of drugs alone Avoid prophylaxis treatment, unless warranted (avoid protective treatment) Antibiotic Awareness  Ineffective against viral, fungal infections (giving an antibiotic to someone w/o an bacterial infection is simply exposing bacteria in the body at sub-damaging levels; exposing them to drug and possibly propagating resistance Patient pressure for antibiotics? (patient getting mad at doc for not prescribing antibiotics for their non bacterial related problems…) Ad campaigns to targe education Limit Non-Medical Use Selection for drug resistant bacteria occurs anywhere antibiotics are used o Agriculture o Industry o Other Particularly problematic when low levels are present, as is typical o Agriculture – low levels used to stimulate growth in farm animals o Agriculture/industry – low levels present in water waste/runoff (e.g. animal wastes) Future of Antibacterials? Growing problem of resistant cannot be ignored Preserve present available options through careful, judicious use o Rational antibiotic use New antibiotic development Alternate options for antibacterial therapy New Antibiotic Development Problem – not a sound investment, in “business-speak” (expensive, takes a long time and issues since bacteria is able to develop resistance through the course of development) Result – significant decline in new antibiotic treatment How to incentivize development? → longer patent Teixobactin: New Antibiotic Class? Harvesting of uncultured bacteria (up to 99% of all species) for alternate antibiotic compounds o Refinement of laboratory methods and or cultivation in situ Discovery of Texiobactin as a new class o Inhibition of cell wall synthesis through lipid binding ▪ Peptidoglycan and teichoic acid precursors Elements of gram (+) cell walls ▪ Ineffective against most gram (-) bacteria by MOA ▪ No evidence of resistance in S. aureus or other Alternate Antibacterial Options Micreos (Holland Biotech Company) o Utilizes bacteriophages and endolysins specific to certain bacterial strains (based on surface protein expression) → (phages nonspecific for bacteria containing endolysin that can eat away at bacteria) ▪ E.g. Staphefekt, specific for S.aureus in treatment of eczema o Resistance unlikely based on MOA? → phages are nonspecific for bacteria  o Recently announced 30 million of Euro funding deal to further development of other endolysins, enter NA markets Day 13 (Anti Cancer Pharmacology) What is Cancer? Uncontrolled proliferation of cells, capable of spreading throughout the body (our cells are constantly dividing, with cancer something has gone wrong with the regular turn over of cells where we wind up getting uncontrolled proliferation usually is in a specific locus of the body which is capable of spreading through the body) o Manifests as an abnormal mass of cells called neoplasm (or tumor) → mass can only be sensed at a certain size (prevents challenges for detection and prevention) ▪ Benign tumors remain in a confined area and can frequently be treated through surgery; they are considered harmless (non-cancerous) ▪ Malignant tumors are capable of invading (via breaking apart) nearby tissue and distant tissue through blood lymph, through a process termed metastasis Cancer is derived from the Greek Karkinos, meaning “crab” so named by Hippocrates ~400 BC based on the physical appearance of a tumor and supporting neovascular growth Hallmarks of Cancer Originally proposed as 6 key features or capabilities of neoplastic cells Important framework for understanding tumor pathogenesis Updated in 2011 and again in 2022, to reflect further progress in cancer research and understanding of pathogenesis o Additional hallmarks o Emerging hallmarks and enabling characteristics Theory of Treatment Stop malignant cells from surviving, growing or spreading How can we do this o What is needed for cell survival? → ability to evade apoptosis (cells can undergo self-destruction if not needed) o What is needed for cell growth? → sufficiency in growth signals and insensitivity to anti growth signals (those are what prevents our cells from growing uncontrollably) o What is needed for cell spread? → metastasis (limitless ability to replicate or reproduce and the ability to potentially invade and travel to other sites of the body) Cancer – Therapeutic Approaches Surgery (major form) o Most effective if cancer is found early, prior to metastasis Radiation o X-rays or other high energy targeted to tumor, to kill cancerous cells o Radiation and pharmacological therapy are typically used in conjunction with surgery (not always true, sometimes surgery is not feasible) Pharmacological therapy o Cytotoxic agents o Hormonal therapy o Molecular targeting therapy o … not an exhaustive list!  Note: it is hard to 100% remove these cancerous cells without risking the side effects – while these mechanisms relate to the hallmarks of cancer, they often relate the hallmarks of normal cell life; therefore the treatments we use do target cancer cells but they also interfere with normal cells too Side Effects Based on therapeutic mechanisms, frequently seen in rapidly dividing tissues/cell types Can vary among different drug classes but most drugs have risk of one or more of the following: o Gastrointestinal upset (e.g. nausea and vomiting) → lethargy and/or weight loss (due to rapid turnover of cells in GI tract) ▪ Can sometimes be treated with anti-emetics such as ondansetron o Bone marrow suppression ▪ Anemia → fatigue and/or weakness ▪ Neutropenia → increased risk of infections ▪ Thrombocytopenia → increased risk of bleeding/bruising o Hair loss o Infertility Cytotoxic Agents Generally impact one key area of cell division, by impairing a particular process necessary for that step (generally impacts the cell cycle → ability of cells to divide → cell div involves several different phases where there are diff checkpoints ensuring that certain thresholds are met) o Cell cycle checkpoint fails and division is halted (mechanism) Examples include o Alkylating agents o Antimetabolites o Antimitotic agents o Not an exhaustive list! Major issue: regulated cell division is a normal part of healthy physiology (no way to preferentially targe DNA replication in a cell) o While many mechanisms in this class share parallels with bacterial antibiotics, there is no bacterial/mammalian cell divide to leverage o Often have narrow therapeutic indices and/or significant side effects Alkylating Agents Form covalent bonds with nucleophilic macromolecule in the cell, such as DNA Many alkylating agents have 2 such active sites (can be somewhat nucleophilic which means that they will attract positively charged parts of DNA → locks 2 parts of DNA together → in doing so during DNA replication they cannot bc of the covalent bonds in addition to the other existing interactions; either prevents DNA unravelling or dmg to DNA) allowing for DNA cross linking Predisposes strand to breakage during separation prior to DNA replication As a class, depress bone marrow function and cause hair loss, with infertility common with prolonged use Representative drugs: cyclophosphamide, cisplatin Cyclophosphamide Requires activation (metabolism) by CYP P450 enzymes into nucleophilic structure o As a neutral pro-drug, this allows for oral administration (if given as a permanently negative charged drug then it will have trouble crossing membranes) Permanently cross links guanine nucleotides within or across strands Inability to replication DNA leads to programmed cell death Antimetabolites DNA synthesis is needed to replicate genetic material prior to cell division; this requires free nucleotide “building blocks”  Antimetabolites inhibit or impair key processes involved in nucleotide synthesis Similar mechanism to trimethoprim and sulfonamide antibiotics, but no mechanism of selectivity for cancerous vs non-cancerous cells based on target enzyme alone Representative drugs: methotrexate, fluorouracil Methotrexate Competes for occupancy of dihydrofolate reductase enzyme o Prevents synthesis of the tetrahydrofolate cofactor required for purine (adenine, guanine) synthesis o DNA, RNA and protein synthesis stall o Cells arrest in 5 phase of cell cycle, leading to apoptosis At lower doses, can be used as an immunosuppressant in rheumatoid arthritis or Crohn’s disease Teratogenic potential based on mechanism; avoid in pregnancy Microtubule – Influencing Agents Origins as plant-based alkaloids Beyond constitutive role in cellular structure, motility, and intracellular transport... microtubules are key elements in chromosome separation during mitosis o Disruption of this key process can cause cell division to arrest in Metaphase o Such blockages in cell division lead to apoptosis Disruptions in microtubule assembly and disassembly have similar outcomes as far as cell viability Representative drugs: vincristine, paclitaxel Vincristine One of the vinca alkaloids (along with vinblastine) o Originally isolated from periwinkle plant (Catharanthus roseus) o One of the earliest leukemia treatments Binds to one of two key microtubule elements (β tubulin, dimerized with α tubulin), preventing polymerization o May also destabilize already-assembled microtubules Side effects may relate to disruption in other key areas of the body e.g. brain → neurotoxicity Hormonal Treatment Many cancers have growth regulated through endogenous hormones o e.g. Hormone receptor-positive breast cancer Aim of treatment: reduce hormone signalling cascade o Inhibit/reduce activation of receptor o Inhibit/reduce hormone production Only useful in hormone-responsive cancers o i.e. presence of hormone receptors on tumor tissue Representative drugs: tamoxifen, anastrozole Glucocorticoids, with less-selective hormonal suppression, may also be used Tamoxifen Selective Estrogen-Receptor Modulator (SERM) o Anti-estrogenic in breast tissue (effective against breast cancer) o Partial estrogenic activity in non-breast tissue Differential effects related to particulars of estrogen receptor expression and assembly Complex metabolism, involving many active metabolites Side effects related to agonist effects elsewhere in body o Menstrual irregularities o Thromboembolic events o Endometrial cancer (2x to 3x risk, time-dependent) Molecular Targeting Agents Vague classification – most drugs have a structure that “targets” a receptor Refers to chemotherapeutics selective for specialized receptors modulating key elements of cell growth  Two major families of compounds: o Protein kinase inhibitors o Monoclonal antibodies Protein Kinase Inhibitors Theory of treatment: o Growth factors often make use of tyrosine kinase receptors for their signal transduction o In cancerous cells, regulation of receptor and/or transduction activity is often impaired Understanding the ‘phosphorylation cascade’ in pathogenesis of any cancer is very important o Common kinases may frequently appear early in the cascades of multiple cancers o Downstream kinases are often more specific to a tissue or type of cancer ▪ Targeting these increase chance of selective effects and/or minimized side effects Representative drugs: imatinib, erlotinib Imatinib Small-molecule inhibitor of several key cytoplasmic kinases involved in transduction of various types of cancer Indicated in treatment of o Chronic Myeloid Leukemia (CML) ▪ Bcr/Abl oncogenic kinase o GastroIntestinal Stromal Tumors (GISTs) Safe for oral administration Half-life of ~18 hours, BUT key metabolite is also biologically active Biologics Relatively newer therapeutic options compared to cytotoxic and anti-metabolite agents Monoclonal antibodies that generally act in one of two ways o Stimulate immune response against the cancerous cells o Inhibit receptor activation of cancerous cells, preventing proliferation and/or promoting apoptosis Very selective targeting for specific macromolecules Representative drugs: bevacizumab, trastuzumab Bevacizumab Monoclonal antibody targeting VEGF ligand o Vascular Endothelial Growth Factor VEGF is involved in angiogenesis, creation of new blood vessels o Key importance in nourishing a growing tumor Not suitable for oral administration o Intravenous infusion o Often in combination with other drugs Initially approved in 2004 (US) for colorectal cancers o Further approvals have expanded use Resistance Lower-than-expected response to chemotherapeutic treatment... o...immediately upon initiation of therapy → Primary resistance o...during the prescribed time course of therapy → Acquired resistance Adaptation or mutation of cancer cells provides a selective survival advantage compared to sensitive cells o e.g. mutation in target protein, decreasing drug affinity Mechanisms of Resistance Differing prominence/likelihood depending on drug class/MOA Pharmacokinetic o Alterations in drug accumulation in cell ▪ Reduced uptake of drug into cell ▪ Increased expression of transport proteins (e.g. P-glycoprotein) that actively expel drug  o Changes in rate of drug inactivation Pharmacodynamic o Increased utilization of alternate biochemical pathways ▪ Common with antimetabolites o Improved proficiency in DNA repair ▪ Common with alkylating agents o Alterations in target molecule expression or structure ▪ e.g. an enzyme mutation causing reduced affinity Combination Therapy Better outcomes? o Tumors are often heterogeneous May allow for a lower dose of each individual agent o Minimize likelihood or severity of concentration-dependent toxicities More well-rounded attack against cancerous cells o Benefits with heterogenous population of cells with degrees of resistance to treatment Summary Theory of treatment centered around the key hallmarks of cancer, which in and of itself is a rapidly progressing list o Based on an abbreviated list of hallmarks, possible treatment avenues (and example compounds) include: ▪ Cytotoxic agents Alkylating agents (cyclophosphamide) Anti-metabolites (methotrexate) Microtubule-influencing agents (vincristine) ▪ Hormonal treatments (tamoxifen) ▪ Molecular targeting agents Protein kinase inhibitors (imatinib) Biologics/Monoclonal antibodies (bevacizumab) Side effects can occur, as can treatment resistance (primary or acquired) o Combination therapy can be useful in mitigating both Day 14 (Reproductive Pharmacology) Endocrine Reproductive Function Sex Steroids Subclass of steroid hormones o Estrogens o Progesterone  o Androgens Sex difference exist in prevalence, not species Menstrual Cycle (Take Home Points) Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) stimulates follicle growth o As follicles grows, it releases estrogen that first inhibits (negative feedback), then stimulates (at higher levels, positive feedback) LH/FSH production LH surge stimulation ovulation at approx. day 14 of menstrual cycle Follicle, post ovulation, turns into corpus luteum o Source of estrogen/progesterone secretion that promotes endometrial growth, and supresses LH/FSH levels through negative feedback Degeneration of corpus luteum results in loss of progesterone production, making endometrium unsustainable and resulting in menstruation Female Contraception Hormonal o Synthetic estrogen + synthetic progesterone (aka progestins) ▪ Oral only o Synthetic progesterone alone ▪ Oral, injectable, implantable Non-Hormonal o Copper intrauterine device ▪ Inhibit sperm movement and fertilization Combined Oral Contraceptives Combination of synthetic estrogen (usually ethinyl estradiol) and progestin (usually one of levonorgestrel or norethindrone) MOA o Inhibition of FSH and LH release (estrogen and progestin formulations) through negative feedback ▪ Inhibition of ovulation ▪ Inhibition of endogenous hormone release Standard prescription o 21 combination hormone tablets 7 placebo tablets ▪ Withdrawal bleeding period Two variation o Monophasic formulations ▪ Identical estrogen/progestin does throughout o Biphasic/triphasic formulations ▪ 2/3 different combination doses ▪ Designed to more closely approximate natural hormone variations Extended cycle formulations possible o E.g. seasonale ▪ 84 combination hormone tablets ▪ 7 placebo tablets o E.g. Lybrel/Amethyst ▪ Only combination hormone tablets (no placebo interval) Progestin – Only Contraceptives a.k.a. the “minipill: o Typically a lower dose than in combined oral contraceptive ▪ Reduced probability of negative feedback on pituitary gland MOA: o Decreased sperm motility through thickening of uterine mucus lining o May suppress ovulation in some cases Useful where estrogen is inadvisable o Estrogen-responsive cancers Daily administration; no placebo period Sources of potential anxiety o Withdrawal bleeding may or may not occur that limit patient acceptance  Requirement of more consistent dosing o Take at same time every day Long-Term Contraceptives Same hormones, different routes of administration Injectable treatments o e.g. Depo-provera® (medroxyprogesterone acetate) ▪ Intramuscularly or subcutaneously every 3 months Implantable devices o Intrauterine devices (IUD) ▪ Progestin-releasing IUD → 3-5 years of contraception ▪ Copper IUDs → ~10 years of contraception Adverse Effects – Mild/Moderate Menstrual irregularities o Breakthrough (mid-cycle) bleeding o Lack of withdrawal bleeding Weight gain Acne/Hirsutism o Some progestins are more androgenic than others ▪ i.e. are more/less capable of stimulating testosterone receptors Many effects will respond to a change in pill formulation o Different synthetic hormone and/or change in dose Adverse Effects – Severe Depression Thromboembolic disease o 3-fold increase (1 to 3 events per 1000 women years) Cardiovascular event/stroke o Increased risk over age 35 Hormonal Contraception and Cancer Growth stimulating effects of estrogen Greater concern for combined oral contraceptives compared to progestin only contraceptives Emergency Contraception High-dose progestins o >> prophylactic contraception doses o Aim to inhibit ovulation Antiprogestins o Ulipristal, RU-486 (mifepristone) ▪ Partial agonist/antagonist respectively ▪ Prevent fertilization by delaying ovulation as primary mechanism Copper IUD o Toxicity to sperm and ova, local inflammatory response o Most effective method, with pro/con of ongoing contraception Male Pharmacological Contraception Testosterone preparations (with or without progestins) have been shown to be effective in most, but not all males o 2-3 months to achieve “severe oligozoospermia” ( change in depression = > Serotonin Norepinephrine Reuptake Inhibitors (SNRIs) the effect Prevent serotonin and norepinephrine reuptake by selectively inhibiting SERT and ➔ Placebo can lead to NET improved results o Increase [neurotransmitter] in synaptic cleft Side effects similar to SSRIs Note: antidepressants have slow onset of effects and should only be Bipolar Disorder Characterized by drastic mood changes – alternating used in those w/severe depression between extreme “highs” (mania) and extreme “lows” (depression) Affects ~1% of the population Average age of onset ~25 yr old Symptoms of Bipolar Disorder Manic phase: higher energy, higher self esteem, racing thoughts, very quick talking, impulsive behaviour, irritability, reduced need for sleep (four required) Depressed phase: lack of [ ], low self esteem, suicidal thoughts, helplessness, loss of interest, low energy levels Mood Stabilizers Indicated for the treatment of bipolar disorder Mechanism of action not clearly understood In general more effective in treating mania than depression, therefore often used in combination with antidepressants o Antidepressant monotherapy should be avoided in patients with bipolar disorder Lithium (always used in combination w/other drugs) A small monovalent cation (+1 charge) Relatively slow onset of action (1-3 weeks) Mechanism of action unclear o May alter signal transduction pathways o Possibly influence neurotransmitters and receptors Not metabolised; excreted through the kidneys Very low therapeutic index Ineffective at blood levels 1.5 mmol/L Must monitor blood levels during treatment Over 80% of patients experience side effects: Tremor, Weight gain, Polyuria (↑ urine), polydipsia (↑ thirst), Hypothyroidism, GI symptoms (vomiting, diarrhea) Anticonvulsants Only a few anticonvulsants are approved for the treatment of bipolar disorder Specific mechanism unknown o Possibly act by reducing neuronal excitability Schizophrenia Characterized by deficits in thought processes, perceptions, and emotional responsiveness Affects ~1% of the population Typical onset in early adulthood  o Late teens to early 30s Symptoms classified as “positive” or “negative” o Positive (presence of problematic behaviours): ▪ Hallucinations (illusory perceptions), especially auditory ▪ Delusions (illusory beliefs), especially persecutory ▪ Disorganized thought and nonsensical speech ▪ Bizarre behaviours o Negative (absence of healthy behaviours) 2 of these required; at least ▪ Flat affect (no emotion showing in the face) one must be a positive ▪ Reduced social interaction symptom ▪ Anhedonia (no feeling of enjoyment) ▪ Avolition (less motivation, initiative, focus on tasks) ▪ Alogia (speaking less) ▪ Catatonia (moving less) Antipsychotics Indicated for the treatment of schizophrenia and the manic phase of bipolar disorder Affect dopamine and/or serotonin neurotransmission Theory of treatment → Dopamine Hypothesis o “Positive symptoms of schizophrenia are associated with excess dopamine activity in the brain” MOA of Antipsychotics Positive symptoms of schizophrenia are associated with excessive dopamine (DA) activity in the mesolimbic system. All antipsychotics (AP) act by blocking dopamine D2 receptors to reduce dopamine activity Typical Antipsychotics Also called “first generation antipsychotics” Dopamine D2 receptor antagonists → block dopamine activity Effective against positive symptoms Higher risk of extrapyramidal (motor) side effects o Abnormal, involuntary movements Atypical Antipsychotics Also called “second generation antipsychotics” Dopamine D2 and serotonin 5-HT2A receptor antagonists → block serotonin and to a lesser degree, dopamine activity Effective against positive and negative symptoms Higher risk of metabolic side effects o Weight gain and/or insulin resistance Anxiety Disorders Characterized by excessive, exaggerated, irrational fear and dread Multiple variations and subclassifications o Panic disorder o Generalized anxiety disorder o Various phobias Affects ~12% of the population Frequently co-occur with depression Anxiolytics (antidepressants can also be used!) Indicated for the treatment of anxiety disorders Some affect GABA neurotransmission o GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the brain o Theory of treatment → Increase GABA neurotransmission, since anxiety is associated with low levels of GABA in the brain Benzodiazepines (BZDs) Those used as anxiolytics have medium or long durations of action (>6 hours)  Fast onset of action ( excretion/metabolism o...in species occupying higher positions on the food chain (biomagnification) Implications for exposure in human consumption Mechanisms of Toxicity 4 major mechanisms that may or may not apply based on the particular poison MOA → nonspecific macromolecule dmg → enzyme and/or receptor mediated toxicity → inflammatory or immune mediated mechanisms → production of reactive species Nonspecific Macromolecular Damage Hydrolysis, oxidation or reduction reactions o Altered structure ▪ e.g. protein denaturation Tends to affect areas of direct contact o e.g. skin, eyes, GI/respiratory mucus membranes Production of Reactive Species Acting as nucleophiles, electrophile or free radicals Reactive Oxygen Species Reduction of molecular oxygen to superoxide anion O2 Defensive enzymatic scavenging via superoxide dismutase (SOD), but hydrogen peroxide byproduct is also cytotoxic Interaction with cysteine residues in proteins, with downstream effects on cell signaling Reaction with iron to produce reactive hydroxide radicals Production of Reactive Species Macromolecular damage altering function, but at specific sites  o i.e. certain residues and/or specific hydrocarbon side chains ▪ Sulfhydryl modification of enzyme proteins a key MOA Often leads to rigid disulfide bonds, decreasing protein flexibility (and function) More systemic locations of activity o Often absorbed as inert compound, and converted to active product within body at site distant to initial exposure ▪ e.g. liver, kidneys Inflammatory Or Immune Mediated Immune response triggered to point of pathology by substances that themselves do not produce significant damage Hypersensitivity response o Requires prior exposure to sensitize immune response Autoimmunity Enzyme and/or Receptor Mediated Alteration of key metabolic pathways, or interaction with critically functional receptors Due to their crucial physiological roles, production of acute toxicity usually results from altered functional processes in one or more of the following: o Neurotransmission, Cardiac rhythm, Oxygen delivery, ATP generation, Intracellular calcium homeostasis Recovery Prospectus, Post-Exposure Chiefly dependent on regenerative capacity of affected tissue/organ... o Heart, central nervous system → limited regenerative capacity ▪ Importance of blood-brain-barrier in protecting against polar compounds o Lungs → medium regenerative capacity ▪ Loss of gas exchange membranes Fibrosis: the deposit of collagen/ECM o Liver → large regenerative capacity, but gradual fibrosis proteins...and concentrations experienced in and around cells as consequence of o e.g. liver, kidneys recovery process Assessing Toxicity Hazards “scar tissue” No-observed-adverse-effect-level (NOAEL) o Highest dose absent of any toxic/adverse effect o Generally conducted in animal models, and applied to humans with a “fudge factor” indicative of uncertainty ▪ Generally, one log unit reduction for each element of uncertainty ▪ Always includes individual variation, as well as any other degrees of separation from humans e.g. 100 mg in rats? 1 mg in humans (individual variation, species variation) Treatment Usually supportive measures… o Remove source of exposure, artificial ventilation, limit (further) absorption/distribution, facilitate excretion …though some specific agents indicated for certain exposures o Heavy metal chelators, antivenom, antagonists/inhibitors (e.g. atropine, for organophosphate exposure), competitive agents (e.g. ethanol, for methanol poisoning) Animal Toxicology Venoms/toxins usually polypeptide in nature Functional uses related to hunting, defence and/or deterrent to predation Some venoms have been adapted into therapeutic agents (e.g. ancrod, teprotide) Others get in the way of a once in a lifetime gustatory experience  Many animal toxins target voltage gated sodium or potassium ions channels o Altered nerve conduction o Often high specificity for certain subtypes Plant Toxicology Toxins/venoms primarily for defensive purposes Alkaloid: nitrogen containing organic compounds of plant origin o Many alkaloids produce quite potent pharmacological effects in humans Many “toxic alkaloids”, in dilute forms, have useful therapeutic (or social) indications Bacterial Toxicology: Botulinum Toxin Protein produced from Clostridium botulinum Characterized as a neurotoxin for its ability to cause muscle paralysis Enters into presynaptic nerve terminals and through a multi step complex procedure, prevents acetylcholine release by blocking vesicular fusion with membrane Can also block release of other neurotransmitters, if it can gain entry to nerve terminal (specific proteins required) Can cause botulinism poisoning associated with eating improperly canned food At lower, non lethal levels, several medical applications o Strabismus/eye movement disorders o Chronic pain 1987: first used cosmetically to prevent appearance of facial wrinkles Relatively safe but can produce unintended paralysis Antitoxins are available and can provide rapid treatment by blocking botulinum interaction with vesicular proteins Air Pollutants Major pollutants include carbon monoxide, sulfur oxides, nitrogen oxides and hydrocarbons o Honorable mention to particulate matter ▪ Solids/liquids suspended in air Penetration into lungs dependent on size o Only smallest particles compounds (10%) can result in nausea, dizziness or weakness and, if persistent, eventually death Treatment: Remove from exposure, Supplemental oxygen, Artificial respiration Heavy Metals Four major heavy metals known for deleterious effects (Cadmium, Mercury, Lead and Arsenic) Mechanism of toxicity typically involves reaction with oxygen/sulfur residues on critical enzymes inactivating them through complexation Effects vary by metal, but generally include: o Nausea, abdominal pain and diarrhea (oral ROA) o Neurological deficiencies o Renal damage (due to accumulation) Lead o Extensive distribution, with deposition in bone, hair and teeth Mercury o Variance in absorption (vapor/elemental) and resulting effects Arsenic  o Most lethal of heavy metals; deposits in hair, a useful fact for forensic testing Cadmium o Can cause softening of bones and associated joint pain Chelation Therapy Major strategy of treatment for heavy metal toxicity Commonly used chelators: o Dimercaprol (intramuscular) o Succimer (oral) “Mop up” heavy metals through covalent binding o Prevents further interaction with endogenous macromolecules Must be administered with care o Most chelating agents have their own potential toxicity Pesticides Insecticides, fungicides, herbicides, rodenticides Low level exposure through food contamination Heavy contamination, or exposure during application (e.g. spraying) can produce acute toxicity Organophosphates Effective, commonly used insecticides (malathion) o Many are selectively banned due to human toxicity (e.g. parathion) Unfortunate history of applications in chemical warfare o e.g. sarin nerve gas (1938, IG Farbenindustrie) MOA o Irreversible inhibition of acetylcholinesterase ▪ Enzyme responsible for terminatio